Links to Exploration step
Le document en format XML
<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Discovery of Hydrocarbon-Stapled
Short α-Helical
Peptides as Promising Middle East Respiratory Syndrome Coronavirus
(MERS-CoV) Fusion Inhibitors</title>
<author><name sortKey="Wang, Chao" sort="Wang, Chao" uniqKey="Wang C" first="Chao" last="Wang">Chao Wang</name>
<affiliation><nlm:aff id="aff1">State Key Laboratory of Toxicology and Medical Countermeasures,<institution>Beijing Institute of Pharmacology and Toxicology</institution>
, 27 Tai-Ping Road, Beijing 100850,<country>China</country>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Xia, Shuai" sort="Xia, Shuai" uniqKey="Xia S" first="Shuai" last="Xia">Shuai Xia</name>
<affiliation><nlm:aff id="aff2">Key Laboratory of Medical Molecular Virology of MOE/MOH, School of Basic Medical Sciences and Shanghai Public Health Clinical Center,<institution>Fudan University</institution>
, 130 Dong An Road, Shanghai 200032,<country>China</country>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Zhang, Peiyu" sort="Zhang, Peiyu" uniqKey="Zhang P" first="Peiyu" last="Zhang">Peiyu Zhang</name>
<affiliation><nlm:aff id="aff3">Key Laboratory of Structure-Based Drug Design and Discovery of the Ministry of Education,<institution>Shenyang Pharmaceutical University</institution>
, Shenyang 110016,<country>China</country>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Zhang, Tianhong" sort="Zhang, Tianhong" uniqKey="Zhang T" first="Tianhong" last="Zhang">Tianhong Zhang</name>
<affiliation><nlm:aff id="aff1">State Key Laboratory of Toxicology and Medical Countermeasures,<institution>Beijing Institute of Pharmacology and Toxicology</institution>
, 27 Tai-Ping Road, Beijing 100850,<country>China</country>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Wang, Weicong" sort="Wang, Weicong" uniqKey="Wang W" first="Weicong" last="Wang">Weicong Wang</name>
<affiliation><nlm:aff id="aff5">Pharmaceutical Preparation Section, Plastic Surgery Hospital,<institution>Chinese Academy of Medical Sciences and Peking Union Medical College</institution>
, Beijing 100144,<country>China</country>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Tian, Yangli" sort="Tian, Yangli" uniqKey="Tian Y" first="Yangli" last="Tian">Yangli Tian</name>
<affiliation><nlm:aff id="aff1">State Key Laboratory of Toxicology and Medical Countermeasures,<institution>Beijing Institute of Pharmacology and Toxicology</institution>
, 27 Tai-Ping Road, Beijing 100850,<country>China</country>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Meng, Guangpeng" sort="Meng, Guangpeng" uniqKey="Meng G" first="Guangpeng" last="Meng">Guangpeng Meng</name>
<affiliation><nlm:aff id="aff3">Key Laboratory of Structure-Based Drug Design and Discovery of the Ministry of Education,<institution>Shenyang Pharmaceutical University</institution>
, Shenyang 110016,<country>China</country>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Jiang, Shibo" sort="Jiang, Shibo" uniqKey="Jiang S" first="Shibo" last="Jiang">Shibo Jiang</name>
<affiliation><nlm:aff id="aff2">Key Laboratory of Medical Molecular Virology of MOE/MOH, School of Basic Medical Sciences and Shanghai Public Health Clinical Center,<institution>Fudan University</institution>
, 130 Dong An Road, Shanghai 200032,<country>China</country>
</nlm:aff>
</affiliation>
<affiliation><nlm:aff id="aff4">Lindsley F. Kimball Research Institute,<institution>New York Blood Center</institution>
, New York, New York 10065,<country>United States</country>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Liu, Keliang" sort="Liu, Keliang" uniqKey="Liu K" first="Keliang" last="Liu">Keliang Liu</name>
<affiliation><nlm:aff id="aff1">State Key Laboratory of Toxicology and Medical Countermeasures,<institution>Beijing Institute of Pharmacology and Toxicology</institution>
, 27 Tai-Ping Road, Beijing 100850,<country>China</country>
</nlm:aff>
</affiliation>
<affiliation><nlm:aff id="aff3">Key Laboratory of Structure-Based Drug Design and Discovery of the Ministry of Education,<institution>Shenyang Pharmaceutical University</institution>
, Shenyang 110016,<country>China</country>
</nlm:aff>
</affiliation>
</author>
</titleStmt>
<publicationStmt><idno type="wicri:source">PMC</idno>
<idno type="pmid">29442512</idno>
<idno type="pmc">7075646</idno>
<idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7075646</idno>
<idno type="RBID">PMC:7075646</idno>
<idno type="doi">10.1021/acs.jmedchem.7b01732</idno>
<date when="2018">2018</date>
<idno type="wicri:Area/Pmc/Corpus">000130</idno>
<idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000130</idno>
</publicationStmt>
<sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Discovery of Hydrocarbon-Stapled
Short α-Helical
Peptides as Promising Middle East Respiratory Syndrome Coronavirus
(MERS-CoV) Fusion Inhibitors</title>
<author><name sortKey="Wang, Chao" sort="Wang, Chao" uniqKey="Wang C" first="Chao" last="Wang">Chao Wang</name>
<affiliation><nlm:aff id="aff1">State Key Laboratory of Toxicology and Medical Countermeasures,<institution>Beijing Institute of Pharmacology and Toxicology</institution>
, 27 Tai-Ping Road, Beijing 100850,<country>China</country>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Xia, Shuai" sort="Xia, Shuai" uniqKey="Xia S" first="Shuai" last="Xia">Shuai Xia</name>
<affiliation><nlm:aff id="aff2">Key Laboratory of Medical Molecular Virology of MOE/MOH, School of Basic Medical Sciences and Shanghai Public Health Clinical Center,<institution>Fudan University</institution>
, 130 Dong An Road, Shanghai 200032,<country>China</country>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Zhang, Peiyu" sort="Zhang, Peiyu" uniqKey="Zhang P" first="Peiyu" last="Zhang">Peiyu Zhang</name>
<affiliation><nlm:aff id="aff3">Key Laboratory of Structure-Based Drug Design and Discovery of the Ministry of Education,<institution>Shenyang Pharmaceutical University</institution>
, Shenyang 110016,<country>China</country>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Zhang, Tianhong" sort="Zhang, Tianhong" uniqKey="Zhang T" first="Tianhong" last="Zhang">Tianhong Zhang</name>
<affiliation><nlm:aff id="aff1">State Key Laboratory of Toxicology and Medical Countermeasures,<institution>Beijing Institute of Pharmacology and Toxicology</institution>
, 27 Tai-Ping Road, Beijing 100850,<country>China</country>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Wang, Weicong" sort="Wang, Weicong" uniqKey="Wang W" first="Weicong" last="Wang">Weicong Wang</name>
<affiliation><nlm:aff id="aff5">Pharmaceutical Preparation Section, Plastic Surgery Hospital,<institution>Chinese Academy of Medical Sciences and Peking Union Medical College</institution>
, Beijing 100144,<country>China</country>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Tian, Yangli" sort="Tian, Yangli" uniqKey="Tian Y" first="Yangli" last="Tian">Yangli Tian</name>
<affiliation><nlm:aff id="aff1">State Key Laboratory of Toxicology and Medical Countermeasures,<institution>Beijing Institute of Pharmacology and Toxicology</institution>
, 27 Tai-Ping Road, Beijing 100850,<country>China</country>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Meng, Guangpeng" sort="Meng, Guangpeng" uniqKey="Meng G" first="Guangpeng" last="Meng">Guangpeng Meng</name>
<affiliation><nlm:aff id="aff3">Key Laboratory of Structure-Based Drug Design and Discovery of the Ministry of Education,<institution>Shenyang Pharmaceutical University</institution>
, Shenyang 110016,<country>China</country>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Jiang, Shibo" sort="Jiang, Shibo" uniqKey="Jiang S" first="Shibo" last="Jiang">Shibo Jiang</name>
<affiliation><nlm:aff id="aff2">Key Laboratory of Medical Molecular Virology of MOE/MOH, School of Basic Medical Sciences and Shanghai Public Health Clinical Center,<institution>Fudan University</institution>
, 130 Dong An Road, Shanghai 200032,<country>China</country>
</nlm:aff>
</affiliation>
<affiliation><nlm:aff id="aff4">Lindsley F. Kimball Research Institute,<institution>New York Blood Center</institution>
, New York, New York 10065,<country>United States</country>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Liu, Keliang" sort="Liu, Keliang" uniqKey="Liu K" first="Keliang" last="Liu">Keliang Liu</name>
<affiliation><nlm:aff id="aff1">State Key Laboratory of Toxicology and Medical Countermeasures,<institution>Beijing Institute of Pharmacology and Toxicology</institution>
, 27 Tai-Ping Road, Beijing 100850,<country>China</country>
</nlm:aff>
</affiliation>
<affiliation><nlm:aff id="aff3">Key Laboratory of Structure-Based Drug Design and Discovery of the Ministry of Education,<institution>Shenyang Pharmaceutical University</institution>
, Shenyang 110016,<country>China</country>
</nlm:aff>
</affiliation>
</author>
</analytic>
<series><title level="j">Journal of Medicinal Chemistry</title>
<idno type="ISSN">0022-2623</idno>
<idno type="eISSN">1520-4804</idno>
<imprint><date when="2018">2018</date>
</imprint>
</series>
</biblStruct>
</sourceDesc>
</fileDesc>
<profileDesc><textClass></textClass>
</profileDesc>
</teiHeader>
<front><div type="abstract" xml:lang="en"><p content-type="toc-graphic"><graphic xlink:href="jm7b01732_0006" id="ab-tgr1"></graphic>
</p>
<p>The hexameric α-helical coiled-coil
formed between the C-terminal
and N-terminal heptad repeat (CHR and NHR) regions of class I viral
fusion proteins plays an important role in mediating the fusion of
the viral and cellular membranes and provides a clear starting point
for molecular mimicry that drives viral fusion inhibitor design. Unfortunately,
such peptide mimicry of the short α-helical region in the CHR
of Middle East respiratory syndrome coronavirus (MERS-CoV) spike protein
has been thwarted by the loss of the peptide’s native α-helical
conformation when taken out of the parent protein structure. Here,
we describe that appropriate all-hydrocarbon stapling of the short
helical portion-based peptide to reinforce its bioactive secondary
structure remarkably improves antiviral potency. The resultant stapled
peptide P21S10 could effectively inhibit infection by MERS-CoV pseudovirus
and its spike protein-mediated cell–cell fusion; additionally,
P21S10 exhibits improved pharmacokinetic properties than HR2P-M2,
suggesting strong potential for development as an anti-MERS-CoV therapeutic.</p>
</div>
</front>
<back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Harrison, S C" uniqKey="Harrison S">S. C. Harrison</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Dimitrov, D S" uniqKey="Dimitrov D">D. S. Dimitrov</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Eckert, D M" uniqKey="Eckert D">D. M. Eckert</name>
</author>
<author><name sortKey="Kim, P S" uniqKey="Kim P">P. S. Kim</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Vigant, F" uniqKey="Vigant F">F. Vigant</name>
</author>
<author><name sortKey="Santos, N C" uniqKey="Santos N">N. C. Santos</name>
</author>
<author><name sortKey="Lee, B" uniqKey="Lee B">B. Lee</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Lu, M" uniqKey="Lu M">M. Lu</name>
</author>
<author><name sortKey="Blacklow, S C" uniqKey="Blacklow S">S. C. Blacklow</name>
</author>
<author><name sortKey="Kim, P S A" uniqKey="Kim P">P. S. A. Kim</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Lai, W" uniqKey="Lai W">W. Lai</name>
</author>
<author><name sortKey="Wang, C" uniqKey="Wang C">C. Wang</name>
</author>
<author><name sortKey="Yu, F" uniqKey="Yu F">F. Yu</name>
</author>
<author><name sortKey="Lu, L" uniqKey="Lu L">L. Lu</name>
</author>
<author><name sortKey="Wang, Q" uniqKey="Wang Q">Q. Wang</name>
</author>
<author><name sortKey="Jiang, X" uniqKey="Jiang X">X. Jiang</name>
</author>
<author><name sortKey="Xu, X" uniqKey="Xu X">X. Xu</name>
</author>
<author><name sortKey="Zhang, T" uniqKey="Zhang T">T. Zhang</name>
</author>
<author><name sortKey="Wu, S" uniqKey="Wu S">S. Wu</name>
</author>
<author><name sortKey="Zheng, X" uniqKey="Zheng X">X. Zheng</name>
</author>
<author><name sortKey="Zhang, Z" uniqKey="Zhang Z">Z. Zhang</name>
</author>
<author><name sortKey="Dong, F" uniqKey="Dong F">F. Dong</name>
</author>
<author><name sortKey="Jiang, S" uniqKey="Jiang S">S. Jiang</name>
</author>
<author><name sortKey="Liu, K" uniqKey="Liu K">K. Liu</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Follis, K E" uniqKey="Follis K">K. E. Follis</name>
</author>
<author><name sortKey="York, J" uniqKey="York J">J. York</name>
</author>
<author><name sortKey="Nunberg, J H" uniqKey="Nunberg J">J. H. Nunberg</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Bullough, P A" uniqKey="Bullough P">P. A. Bullough</name>
</author>
<author><name sortKey="Hughson, F M" uniqKey="Hughson F">F. M. Hughson</name>
</author>
<author><name sortKey="Skehel, J J" uniqKey="Skehel J">J. J. Skehel</name>
</author>
<author><name sortKey="Wiley, D C" uniqKey="Wiley D">D. C. Wiley</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Liu, S" uniqKey="Liu S">S. Liu</name>
</author>
<author><name sortKey="Xiao, G" uniqKey="Xiao G">G. Xiao</name>
</author>
<author><name sortKey="Chen, Y" uniqKey="Chen Y">Y. Chen</name>
</author>
<author><name sortKey="He, Y X" uniqKey="He Y">Y. X. He</name>
</author>
<author><name sortKey="Niu, J K" uniqKey="Niu J">J. K. Niu</name>
</author>
<author><name sortKey="Escalante, C R" uniqKey="Escalante C">C. R. Escalante</name>
</author>
<author><name sortKey="Xiong, H" uniqKey="Xiong H">H. Xiong</name>
</author>
<author><name sortKey="Farmar, J" uniqKey="Farmar J">J. Farmar</name>
</author>
<author><name sortKey="Debnath, A K" uniqKey="Debnath A">A. K. Debnath</name>
</author>
<author><name sortKey="Tien, P" uniqKey="Tien P">P. Tien</name>
</author>
<author><name sortKey="Jiang, S" uniqKey="Jiang S">S. Jiang</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Miller, E H" uniqKey="Miller E">E. H. Miller</name>
</author>
<author><name sortKey="Harrison, J S" uniqKey="Harrison J">J. S. Harrison</name>
</author>
<author><name sortKey="Radoshitzky, S R" uniqKey="Radoshitzky S">S. R. Radoshitzky</name>
</author>
<author><name sortKey="Higgins, C D" uniqKey="Higgins C">C. D. Higgins</name>
</author>
<author><name sortKey="Chi, X" uniqKey="Chi X">X. Chi</name>
</author>
<author><name sortKey="Dong, L" uniqKey="Dong L">L. Dong</name>
</author>
<author><name sortKey="Kuhn, J H" uniqKey="Kuhn J">J. H. Kuhn</name>
</author>
<author><name sortKey="Bavari, S" uniqKey="Bavari S">S. Bavari</name>
</author>
<author><name sortKey="Lai, J" uniqKey="Lai J">J. Lai</name>
</author>
<author><name sortKey="Chandran, K" uniqKey="Chandran K">K. Chandran</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Lu, L" uniqKey="Lu L">L. Lu</name>
</author>
<author><name sortKey="Liu, Q" uniqKey="Liu Q">Q. Liu</name>
</author>
<author><name sortKey="Zhu, Y" uniqKey="Zhu Y">Y. Zhu</name>
</author>
<author><name sortKey="Chan, K" uniqKey="Chan K">K. Chan</name>
</author>
<author><name sortKey="Qin, L" uniqKey="Qin L">L. Qin</name>
</author>
<author><name sortKey="Li, Y" uniqKey="Li Y">Y. Li</name>
</author>
<author><name sortKey="Wang, Q" uniqKey="Wang Q">Q. Wang</name>
</author>
<author><name sortKey="Chan, J F" uniqKey="Chan J">J. F. Chan</name>
</author>
<author><name sortKey="Du, L" uniqKey="Du L">L. Du</name>
</author>
<author><name sortKey="Yu, F" uniqKey="Yu F">F. Yu</name>
</author>
<author><name sortKey="Ma, C" uniqKey="Ma C">C. Ma</name>
</author>
<author><name sortKey="Ye, S" uniqKey="Ye S">S. Ye</name>
</author>
<author><name sortKey="Yuen, K" uniqKey="Yuen K">K. Yuen</name>
</author>
<author><name sortKey="Zhang, R" uniqKey="Zhang R">R. Zhang</name>
</author>
<author><name sortKey="Jiang, S" uniqKey="Jiang S">S. Jiang</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Berkhout, B" uniqKey="Berkhout B">B. Berkhout</name>
</author>
<author><name sortKey="Eggink, D" uniqKey="Eggink D">D. Eggink</name>
</author>
<author><name sortKey="Sanders, R W" uniqKey="Sanders R">R. W. Sanders</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Azzarito, V" uniqKey="Azzarito V">V. Azzarito</name>
</author>
<author><name sortKey="Long, K" uniqKey="Long K">K. Long</name>
</author>
<author><name sortKey="Murphy, N S" uniqKey="Murphy N">N. S. Murphy</name>
</author>
<author><name sortKey="Wilson, A J" uniqKey="Wilson A">A. J. Wilson</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Cai, L" uniqKey="Cai L">L. Cai</name>
</author>
<author><name sortKey="Jiang, S" uniqKey="Jiang S">S. Jiang</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Pan, C" uniqKey="Pan C">C. Pan</name>
</author>
<author><name sortKey="Liu, S" uniqKey="Liu S">S. Liu</name>
</author>
<author><name sortKey="Jiang, S" uniqKey="Jiang S">S. Jiang</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Gao, J" uniqKey="Gao J">J. Gao</name>
</author>
<author><name sortKey="Lu, G" uniqKey="Lu G">G. Lu</name>
</author>
<author><name sortKey="Qi, J" uniqKey="Qi J">J. Qi</name>
</author>
<author><name sortKey="Li, Y" uniqKey="Li Y">Y. Li</name>
</author>
<author><name sortKey="Wu, Y" uniqKey="Wu Y">Y. Wu</name>
</author>
<author><name sortKey="Deng, Y" uniqKey="Deng Y">Y. Deng</name>
</author>
<author><name sortKey="Geng, H" uniqKey="Geng H">H. Geng</name>
</author>
<author><name sortKey="Li, H" uniqKey="Li H">H. Li</name>
</author>
<author><name sortKey="Wang, Q" uniqKey="Wang Q">Q. Wang</name>
</author>
<author><name sortKey="Xiao, H" uniqKey="Xiao H">H. Xiao</name>
</author>
<author><name sortKey="Tan, W" uniqKey="Tan W">W. Tan</name>
</author>
<author><name sortKey="Yan, J" uniqKey="Yan J">J. Yan</name>
</author>
<author><name sortKey="Gao, G" uniqKey="Gao G">G. Gao</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Lau, Y" uniqKey="Lau Y">Y. Lau</name>
</author>
<author><name sortKey="De Andrade, P" uniqKey="De Andrade P">P. De Andrade</name>
</author>
<author><name sortKey="Wu, Y" uniqKey="Wu Y">Y. Wu</name>
</author>
<author><name sortKey="Spring, D R" uniqKey="Spring D">D. R. Spring</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Walensky, L D" uniqKey="Walensky L">L. D. Walensky</name>
</author>
<author><name sortKey="Bird, G H" uniqKey="Bird G">G. H. Bird</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Tan, Y" uniqKey="Tan Y">Y. Tan</name>
</author>
<author><name sortKey="Lane, D P" uniqKey="Lane D">D. P. Lane</name>
</author>
<author><name sortKey="Verma, C S" uniqKey="Verma C">C. S. Verma</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Kawamoto, S A" uniqKey="Kawamoto S">S. A. Kawamoto</name>
</author>
<author><name sortKey="Coleska, A" uniqKey="Coleska A">A. Coleska</name>
</author>
<author><name sortKey="Ran, X" uniqKey="Ran X">X. Ran</name>
</author>
<author><name sortKey="Yi, H" uniqKey="Yi H">H. Yi</name>
</author>
<author><name sortKey="Yang, C" uniqKey="Yang C">C. Yang</name>
</author>
<author><name sortKey="Wang, S" uniqKey="Wang S">S. Wang</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Hojo, K" uniqKey="Hojo K">K. Hojo</name>
</author>
<author><name sortKey="Hossain, M A" uniqKey="Hossain M">M. A. Hossain</name>
</author>
<author><name sortKey="Tailhades, J" uniqKey="Tailhades J">J. Tailhades</name>
</author>
<author><name sortKey="Shabanpoor, F" uniqKey="Shabanpoor F">F. Shabanpoor</name>
</author>
<author><name sortKey="Wong, L L L" uniqKey="Wong L">L. L.L. Wong</name>
</author>
<author><name sortKey="Ong Palsson, E E K" uniqKey="Ong Palsson E">E. E. K. Ong-Palsson</name>
</author>
<author><name sortKey="Kastman, H E" uniqKey="Kastman H">H. E. Kastman</name>
</author>
<author><name sortKey="Ma, S" uniqKey="Ma S">S. Ma</name>
</author>
<author><name sortKey="Gundlach, A L" uniqKey="Gundlach A">A. L. Gundlach</name>
</author>
<author><name sortKey="Rosengren, K J" uniqKey="Rosengren K">K. J. Rosengren</name>
</author>
<author><name sortKey="Wade, J D" uniqKey="Wade J">J. D. Wade</name>
</author>
<author><name sortKey="Bathgate, R A D" uniqKey="Bathgate R">R. A. D. Bathgate</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Frank, A O" uniqKey="Frank A">A. O. Frank</name>
</author>
<author><name sortKey="Vangamudi, B" uniqKey="Vangamudi B">B. Vangamudi</name>
</author>
<author><name sortKey="Feldkamp, M D" uniqKey="Feldkamp M">M. D. Feldkamp</name>
</author>
<author><name sortKey="Souza Fagundes, E M" uniqKey="Souza Fagundes E">E. M. Souza-Fagundes</name>
</author>
<author><name sortKey="Luzwick, J W" uniqKey="Luzwick J">J. W. Luzwick</name>
</author>
<author><name sortKey="Cortez, D" uniqKey="Cortez D">D. Cortez</name>
</author>
<author><name sortKey="Olejniczak, E T" uniqKey="Olejniczak E">E. T. Olejniczak</name>
</author>
<author><name sortKey="Waterson, A G" uniqKey="Waterson A">A. G. Waterson</name>
</author>
<author><name sortKey="Rossanese, O W" uniqKey="Rossanese O">O. W. Rossanese</name>
</author>
<author><name sortKey="Chazin, W J" uniqKey="Chazin W">W. J. Chazin</name>
</author>
<author><name sortKey="Fesik, S W" uniqKey="Fesik S">S. W. Fesik</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Hilinski, G J" uniqKey="Hilinski G">G. J. Hilinski</name>
</author>
<author><name sortKey="Kim, Y W" uniqKey="Kim Y">Y. W. Kim</name>
</author>
<author><name sortKey="Hong, J" uniqKey="Hong J">J. Hong</name>
</author>
<author><name sortKey="Kutchukian, P S" uniqKey="Kutchukian P">P. S. Kutchukian</name>
</author>
<author><name sortKey="Crenshaw, C M" uniqKey="Crenshaw C">C. M. Crenshaw</name>
</author>
<author><name sortKey="Berkovitch, S S" uniqKey="Berkovitch S">S. S. Berkovitch</name>
</author>
<author><name sortKey="Chang, A" uniqKey="Chang A">A. Chang</name>
</author>
<author><name sortKey="Ham, S" uniqKey="Ham S">S. Ham</name>
</author>
<author><name sortKey="Verdine, G L" uniqKey="Verdine G">G. L. Verdine</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Chang, Y" uniqKey="Chang Y">Y. Chang</name>
</author>
<author><name sortKey="Graves, B" uniqKey="Graves B">B. Graves</name>
</author>
<author><name sortKey="Guerlavais, V" uniqKey="Guerlavais V">V. Guerlavais</name>
</author>
<author><name sortKey="Tovar, C" uniqKey="Tovar C">C. Tovar</name>
</author>
<author><name sortKey="Packman, K" uniqKey="Packman K">K. Packman</name>
</author>
<author><name sortKey="To, K H" uniqKey="To K">K. H. To</name>
</author>
<author><name sortKey="Olson, K A" uniqKey="Olson K">K. A. Olson</name>
</author>
<author><name sortKey="Kesavan, K" uniqKey="Kesavan K">K. Kesavan</name>
</author>
<author><name sortKey="Gangurde, P" uniqKey="Gangurde P">P. Gangurde</name>
</author>
<author><name sortKey="Mukherjee, A" uniqKey="Mukherjee A">A. Mukherjee</name>
</author>
<author><name sortKey="Baker, T" uniqKey="Baker T">T. Baker</name>
</author>
<author><name sortKey="Darlak, K" uniqKey="Darlak K">K. Darlak</name>
</author>
<author><name sortKey="Elkin, C" uniqKey="Elkin C">C. Elkin</name>
</author>
<author><name sortKey="Filipovic, Z" uniqKey="Filipovic Z">Z. Filipovic</name>
</author>
<author><name sortKey="Qureshi, F Z" uniqKey="Qureshi F">F. Z. Qureshi</name>
</author>
<author><name sortKey="Cai, H" uniqKey="Cai H">H. Cai</name>
</author>
<author><name sortKey="Berry, P" uniqKey="Berry P">P. Berry</name>
</author>
<author><name sortKey="Feyfant, E" uniqKey="Feyfant E">E. Feyfant</name>
</author>
<author><name sortKey="Shi, X E" uniqKey="Shi X">X. E. Shi</name>
</author>
<author><name sortKey="Horstick, J" uniqKey="Horstick J">J. Horstick</name>
</author>
<author><name sortKey="Annis, D A" uniqKey="Annis D">D. A. Annis</name>
</author>
<author><name sortKey="Manning, A M" uniqKey="Manning A">A. M. Manning</name>
</author>
<author><name sortKey="Fotouhi, N" uniqKey="Fotouhi N">N. Fotouhi</name>
</author>
<author><name sortKey="Nash, H" uniqKey="Nash H">H. Nash</name>
</author>
<author><name sortKey="Vassilev, L T" uniqKey="Vassilev L">L. T. Vassilev</name>
</author>
<author><name sortKey="Sawyer, T K" uniqKey="Sawyer T">T. K. Sawyer</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Wang, C" uniqKey="Wang C">C. Wang</name>
</author>
<author><name sortKey="Shi, W" uniqKey="Shi W">W. Shi</name>
</author>
<author><name sortKey="Cai, L" uniqKey="Cai L">L. Cai</name>
</author>
<author><name sortKey="Lu, L" uniqKey="Lu L">L. Lu</name>
</author>
<author><name sortKey="Wang, Q" uniqKey="Wang Q">Q. Wang</name>
</author>
<author><name sortKey="Zhang, T" uniqKey="Zhang T">T. Zhang</name>
</author>
<author><name sortKey="Li, J" uniqKey="Li J">J. Li</name>
</author>
<author><name sortKey="Zhang, Z" uniqKey="Zhang Z">Z. Zhang</name>
</author>
<author><name sortKey="Wang, K" uniqKey="Wang K">K. Wang</name>
</author>
<author><name sortKey="Xu, L" uniqKey="Xu L">L. Xu</name>
</author>
<author><name sortKey="Jiang, X" uniqKey="Jiang X">X. Jiang</name>
</author>
<author><name sortKey="Jiang, S" uniqKey="Jiang S">S. Jiang</name>
</author>
<author><name sortKey="Liu, K" uniqKey="Liu K">K. Liu</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Wang, C" uniqKey="Wang C">C. Wang</name>
</author>
<author><name sortKey="Shi, W" uniqKey="Shi W">W. Shi</name>
</author>
<author><name sortKey="Cai, L" uniqKey="Cai L">L. Cai</name>
</author>
<author><name sortKey="Lu, L" uniqKey="Lu L">L. Lu</name>
</author>
<author><name sortKey="Yu, F" uniqKey="Yu F">F. Yu</name>
</author>
<author><name sortKey="Wang, Q" uniqKey="Wang Q">Q. Wang</name>
</author>
<author><name sortKey="Jiang, X" uniqKey="Jiang X">X. Jiang</name>
</author>
<author><name sortKey="Xu, X" uniqKey="Xu X">X. Xu</name>
</author>
<author><name sortKey="Wang, K" uniqKey="Wang K">K. Wang</name>
</author>
<author><name sortKey="Xu, L" uniqKey="Xu L">L. Xu</name>
</author>
<author><name sortKey="Jiang, S" uniqKey="Jiang S">S. Jiang</name>
</author>
<author><name sortKey="Liu, K" uniqKey="Liu K">K. Liu</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Wuo, M" uniqKey="Wuo M">M. Wuo</name>
</author>
<author><name sortKey="Mahon, A B" uniqKey="Mahon A">A. B. Mahon</name>
</author>
<author><name sortKey="Arora, P S" uniqKey="Arora P">P. S. Arora</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Apostolovic, B" uniqKey="Apostolovic B">B. Apostolovic</name>
</author>
<author><name sortKey="Danial, M" uniqKey="Danial M">M. Danial</name>
</author>
<author><name sortKey="Klok, H A" uniqKey="Klok H">H. A. Klok</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Zheng, B" uniqKey="Zheng B">B. Zheng</name>
</author>
<author><name sortKey="Wang, K" uniqKey="Wang K">K. Wang</name>
</author>
<author><name sortKey="Lu, L" uniqKey="Lu L">L. Lu</name>
</author>
<author><name sortKey="Yu, F" uniqKey="Yu F">F. Yu</name>
</author>
<author><name sortKey="Cheng, M" uniqKey="Cheng M">M. Cheng</name>
</author>
<author><name sortKey="Jiang, S" uniqKey="Jiang S">S. Jiang</name>
</author>
<author><name sortKey="Liu, K" uniqKey="Liu K">K. Liu</name>
</author>
<author><name sortKey="Cai, L" uniqKey="Cai L">L. Cai</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Qi, Z" uniqKey="Qi Z">Z. Qi</name>
</author>
<author><name sortKey="Shi, W" uniqKey="Shi W">W. Shi</name>
</author>
<author><name sortKey="Xue, N" uniqKey="Xue N">N. Xue</name>
</author>
<author><name sortKey="Pan, C" uniqKey="Pan C">C. Pan</name>
</author>
<author><name sortKey="Jing, W" uniqKey="Jing W">W. Jing</name>
</author>
<author><name sortKey="Liu, K" uniqKey="Liu K">K. Liu</name>
</author>
<author><name sortKey="Jiang, S" uniqKey="Jiang S">S. Jiang</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Anderhuber, N" uniqKey="Anderhuber N">N. Anderhuber</name>
</author>
<author><name sortKey="Fladischer, P" uniqKey="Fladischer P">P. Fladischer</name>
</author>
<author><name sortKey="Gruber Khadjawi, M" uniqKey="Gruber Khadjawi M">M. Gruber-Khadjawi</name>
</author>
<author><name sortKey="Mairhofer, J" uniqKey="Mairhofer J">J. Mairhofer</name>
</author>
<author><name sortKey="Striedner, G" uniqKey="Striedner G">G. Striedner</name>
</author>
<author><name sortKey="Wiltschi, B" uniqKey="Wiltschi B">B. Wiltschi</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Fletcher, J M" uniqKey="Fletcher J">J. M. Fletcher</name>
</author>
<author><name sortKey="Boyle, A L" uniqKey="Boyle A">A. L. Boyle</name>
</author>
<author><name sortKey="Bruning, M" uniqKey="Bruning M">M. Bruning</name>
</author>
<author><name sortKey="Bartlett, G J" uniqKey="Bartlett G">G. J. Bartlett</name>
</author>
<author><name sortKey="Vincent, T L" uniqKey="Vincent T">T. L. Vincent</name>
</author>
<author><name sortKey="Zaccai, N R" uniqKey="Zaccai N">N. R. Zaccai</name>
</author>
<author><name sortKey="Armstrong, C T" uniqKey="Armstrong C">C. T. Armstrong</name>
</author>
<author><name sortKey="Bromley, E H C" uniqKey="Bromley E">E. H. C. Bromley</name>
</author>
<author><name sortKey="Booth, P J" uniqKey="Booth P">P. J. Booth</name>
</author>
<author><name sortKey="Brady, R L" uniqKey="Brady R">R. L. Brady</name>
</author>
<author><name sortKey="Thomson, A R" uniqKey="Thomson A">A. R. Thomson</name>
</author>
<author><name sortKey="Woolfson, D N" uniqKey="Woolfson D">D. N. Woolfson</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Cotten, M" uniqKey="Cotten M">M. Cotten</name>
</author>
<author><name sortKey="Watson, S J" uniqKey="Watson S">S. J. Watson</name>
</author>
<author><name sortKey="Zumla, A I" uniqKey="Zumla A">A. I. Zumla</name>
</author>
<author><name sortKey="Makhdoom, H Q" uniqKey="Makhdoom H">H. Q. Makhdoom</name>
</author>
<author><name sortKey="Palser, A L" uniqKey="Palser A">A. L. Palser</name>
</author>
<author><name sortKey="Ong, S H" uniqKey="Ong S">S. H. Ong</name>
</author>
<author><name sortKey="Al Rabeeah, A A" uniqKey="Al Rabeeah A">A. A. Al Rabeeah</name>
</author>
<author><name sortKey="Alhakeem, R F" uniqKey="Alhakeem R">R. F. Alhakeem</name>
</author>
<author><name sortKey="Assiri, A" uniqKey="Assiri A">A. Assiri</name>
</author>
<author><name sortKey="Al Tawfiq, J A" uniqKey="Al Tawfiq J">J. A. Al-Tawfiq</name>
</author>
<author><name sortKey="Albarrak, A" uniqKey="Albarrak A">A. Albarrak</name>
</author>
<author><name sortKey="Barry, M" uniqKey="Barry M">M. Barry</name>
</author>
<author><name sortKey="Shibl, A" uniqKey="Shibl A">A. Shibl</name>
</author>
<author><name sortKey="Alrabiah, F A" uniqKey="Alrabiah F">F. A. Alrabiah</name>
</author>
<author><name sortKey="Hajjar, S" uniqKey="Hajjar S">S. Hajjar</name>
</author>
<author><name sortKey="Balkhy, H H" uniqKey="Balkhy H">H. H. Balkhy</name>
</author>
<author><name sortKey="Flemban, H" uniqKey="Flemban H">H. Flemban</name>
</author>
<author><name sortKey="Rambaut, A" uniqKey="Rambaut A">A. Rambaut</name>
</author>
<author><name sortKey="Kellam, P" uniqKey="Kellam P">P. Kellam</name>
</author>
<author><name sortKey="Memish, Z A" uniqKey="Memish Z">Z. A. Memish</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Lawless, M K" uniqKey="Lawless M">M. K. Lawless</name>
</author>
<author><name sortKey="Barney, S" uniqKey="Barney S">S. Barney</name>
</author>
<author><name sortKey="Guthrie, K I" uniqKey="Guthrie K">K. I. Guthrie</name>
</author>
<author><name sortKey="Bucy, T B" uniqKey="Bucy T">T. B. Bucy</name>
</author>
<author><name sortKey="Petteway, S R" uniqKey="Petteway S">S. R. Petteway</name>
</author>
<author><name sortKey="Merutka, G" uniqKey="Merutka G">G. Merutka</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Cai, L F" uniqKey="Cai L">L. F. Cai</name>
</author>
<author><name sortKey="Balogh, E" uniqKey="Balogh E">E. Balogh</name>
</author>
<author><name sortKey="Gochin, M" uniqKey="Gochin M">M. Gochin</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Dwyer, J J" uniqKey="Dwyer J">J. J. Dwyer</name>
</author>
<author><name sortKey="Wilson, K L" uniqKey="Wilson K">K. L. Wilson</name>
</author>
<author><name sortKey="Davison, D K" uniqKey="Davison D">D. K. Davison</name>
</author>
<author><name sortKey="Freel, S A" uniqKey="Freel S">S. A. Freel</name>
</author>
<author><name sortKey="Seedorff, J E" uniqKey="Seedorff J">J. E. Seedorff</name>
</author>
<author><name sortKey="Wring, S A" uniqKey="Wring S">S. A. Wring</name>
</author>
<author><name sortKey="Tvermoes, N A" uniqKey="Tvermoes N">N. A. Tvermoes</name>
</author>
<author><name sortKey="Matthews, T J" uniqKey="Matthews T">T. J. Matthews</name>
</author>
<author><name sortKey="Greenberg, M L" uniqKey="Greenberg M">M. L. Greenberg</name>
</author>
<author><name sortKey="Delmedico, M K" uniqKey="Delmedico M">M. K. Delmedico</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Shi, W G" uniqKey="Shi W">W. G. Shi</name>
</author>
<author><name sortKey="Cai, L F" uniqKey="Cai L">L. F. Cai</name>
</author>
<author><name sortKey="Lu, L" uniqKey="Lu L">L. Lu</name>
</author>
<author><name sortKey="Wang, C" uniqKey="Wang C">C. Wang</name>
</author>
<author><name sortKey="Wang, K" uniqKey="Wang K">K. Wang</name>
</author>
<author><name sortKey="Xu, L" uniqKey="Xu L">L. Xu</name>
</author>
<author><name sortKey="Zhang, S" uniqKey="Zhang S">S. Zhang</name>
</author>
<author><name sortKey="Han, H" uniqKey="Han H">H. Han</name>
</author>
<author><name sortKey="Jiang, X F" uniqKey="Jiang X">X. F. Jiang</name>
</author>
<author><name sortKey="Zheng, B H" uniqKey="Zheng B">B. H. Zheng</name>
</author>
<author><name sortKey="Jiang, S B" uniqKey="Jiang S">S. B. Jiang</name>
</author>
<author><name sortKey="Liu, K L" uniqKey="Liu K">K. L. Liu</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Zhu, X J" uniqKey="Zhu X">X. J. Zhu</name>
</author>
<author><name sortKey="Zhu, Y" uniqKey="Zhu Y">Y. Zhu</name>
</author>
<author><name sortKey="Ye, S" uniqKey="Ye S">S. Ye</name>
</author>
<author><name sortKey="Wang, Q" uniqKey="Wang Q">Q. Wang</name>
</author>
<author><name sortKey="Xu, W" uniqKey="Xu W">W. Xu</name>
</author>
<author><name sortKey="Su, S" uniqKey="Su S">S. Su</name>
</author>
<author><name sortKey="Sun, Z W" uniqKey="Sun Z">Z. W. Sun</name>
</author>
<author><name sortKey="Yu, F" uniqKey="Yu F">F. Yu</name>
</author>
<author><name sortKey="Liu, Q" uniqKey="Liu Q">Q. Liu</name>
</author>
<author><name sortKey="Wang, C" uniqKey="Wang C">C. Wang</name>
</author>
<author><name sortKey="Zhang, T H" uniqKey="Zhang T">T. H. Zhang</name>
</author>
<author><name sortKey="Zhang, Z Q" uniqKey="Zhang Z">Z. Q. Zhang</name>
</author>
<author><name sortKey="Zhang, X Y" uniqKey="Zhang X">X. Y. Zhang</name>
</author>
<author><name sortKey="Xu, J Q" uniqKey="Xu J">J. Q. Xu</name>
</author>
<author><name sortKey="Du, L Y" uniqKey="Du L">L. Y. Du</name>
</author>
<author><name sortKey="Liu, K L" uniqKey="Liu K">K. L. Liu</name>
</author>
<author><name sortKey="Lu, L" uniqKey="Lu L">L. Lu</name>
</author>
<author><name sortKey="Zhang, R G" uniqKey="Zhang R">R. G. Zhang</name>
</author>
<author><name sortKey="Jiang, S B" uniqKey="Jiang S">S. B. Jiang</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Cui, H" uniqKey="Cui H">H. Cui</name>
</author>
<author><name sortKey="Qing, J" uniqKey="Qing J">J. Qing</name>
</author>
<author><name sortKey="Guo, Y" uniqKey="Guo Y">Y. Guo</name>
</author>
<author><name sortKey="Wang, Y" uniqKey="Wang Y">Y. Wang</name>
</author>
<author><name sortKey="Cui, L" uniqKey="Cui L">L. Cui</name>
</author>
<author><name sortKey="He, T" uniqKey="He T">T. He</name>
</author>
<author><name sortKey="Zhang, L" uniqKey="Zhang L">L. Zhang</name>
</author>
<author><name sortKey="Liu, L" uniqKey="Liu L">L. Liu</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Liu, Q" uniqKey="Liu Q">Q. Liu</name>
</author>
<author><name sortKey="Xia, S" uniqKey="Xia S">S. Xia</name>
</author>
<author><name sortKey="Sun, Z" uniqKey="Sun Z">Z. Sun</name>
</author>
<author><name sortKey="Wang, Q" uniqKey="Wang Q">Q. Wang</name>
</author>
<author><name sortKey="Du, L" uniqKey="Du L">L. Du</name>
</author>
<author><name sortKey="Lu, L" uniqKey="Lu L">L. Lu</name>
</author>
<author><name sortKey="Jiang, S" uniqKey="Jiang S">S. Jiang</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Xia, S" uniqKey="Xia S">S. Xia</name>
</author>
<author><name sortKey="Liu, Q" uniqKey="Liu Q">Q. Liu</name>
</author>
<author><name sortKey="Wang, Q" uniqKey="Wang Q">Q. Wang</name>
</author>
<author><name sortKey="Sun, Z" uniqKey="Sun Z">Z. Sun</name>
</author>
<author><name sortKey="Su, S" uniqKey="Su S">S. Su</name>
</author>
<author><name sortKey="Du, L" uniqKey="Du L">L. Du</name>
</author>
<author><name sortKey="Ying, T" uniqKey="Ying T">T. Ying</name>
</author>
<author><name sortKey="Lu, L" uniqKey="Lu L">L. Lu</name>
</author>
<author><name sortKey="Jiang, S" uniqKey="Jiang S">S. Jiang</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Zhao, G" uniqKey="Zhao G">G. Zhao</name>
</author>
<author><name sortKey="Du, L" uniqKey="Du L">L. Du</name>
</author>
<author><name sortKey="Ma, C" uniqKey="Ma C">C. Ma</name>
</author>
<author><name sortKey="Li, Y" uniqKey="Li Y">Y. Li</name>
</author>
<author><name sortKey="Li, L" uniqKey="Li L">L. Li</name>
</author>
<author><name sortKey="Poon, V K" uniqKey="Poon V">V. K. Poon</name>
</author>
<author><name sortKey="Wang, L" uniqKey="Wang L">L. Wang</name>
</author>
<author><name sortKey="Yu, F" uniqKey="Yu F">F. Yu</name>
</author>
<author><name sortKey="Zheng, B J" uniqKey="Zheng B">B. J. Zheng</name>
</author>
<author><name sortKey="Jiang, S" uniqKey="Jiang S">S. Jiang</name>
</author>
<author><name sortKey="Zhou, Y" uniqKey="Zhou Y">Y. Zhou</name>
</author>
</analytic>
</biblStruct>
</listBibl>
</div1>
</back>
</TEI>
<pmc article-type="research-article" xml:lang="EN"><pmc-dir>properties open_access</pmc-dir>
<front><journal-meta><journal-id journal-id-type="nlm-ta">J Med Chem</journal-id>
<journal-id journal-id-type="iso-abbrev">J. Med. Chem</journal-id>
<journal-id journal-id-type="publisher-id">jm</journal-id>
<journal-id journal-id-type="coden">jmcmar</journal-id>
<journal-title-group><journal-title>Journal of Medicinal Chemistry</journal-title>
</journal-title-group>
<issn pub-type="ppub">0022-2623</issn>
<issn pub-type="epub">1520-4804</issn>
<publisher><publisher-name>American Chemical
Society</publisher-name>
</publisher>
</journal-meta>
<article-meta><article-id pub-id-type="pmid">29442512</article-id>
<article-id pub-id-type="pmc">7075646</article-id>
<article-id pub-id-type="doi">10.1021/acs.jmedchem.7b01732</article-id>
<article-categories><subj-group><subject>Article</subject>
</subj-group>
</article-categories>
<title-group><article-title>Discovery of Hydrocarbon-Stapled
Short α-Helical
Peptides as Promising Middle East Respiratory Syndrome Coronavirus
(MERS-CoV) Fusion Inhibitors</article-title>
</title-group>
<contrib-group><contrib contrib-type="author" id="ath1"><name><surname>Wang</surname>
<given-names>Chao</given-names>
</name>
<xref rid="aff1" ref-type="aff">†</xref>
<xref rid="notes3" ref-type="notes">¶</xref>
</contrib>
<contrib contrib-type="author" id="ath2"><name><surname>Xia</surname>
<given-names>Shuai</given-names>
</name>
<xref rid="aff2" ref-type="aff">‡</xref>
<xref rid="notes3" ref-type="notes">¶</xref>
</contrib>
<contrib contrib-type="author" id="ath3"><name><surname>Zhang</surname>
<given-names>Peiyu</given-names>
</name>
<xref rid="aff3" ref-type="aff">§</xref>
<xref rid="notes3" ref-type="notes">¶</xref>
</contrib>
<contrib contrib-type="author" id="ath4"><name><surname>Zhang</surname>
<given-names>Tianhong</given-names>
</name>
<xref rid="aff1" ref-type="aff">†</xref>
</contrib>
<contrib contrib-type="author" id="ath5"><name><surname>Wang</surname>
<given-names>Weicong</given-names>
</name>
<xref rid="aff5" ref-type="aff">⊥</xref>
</contrib>
<contrib contrib-type="author" id="ath6"><name><surname>Tian</surname>
<given-names>Yangli</given-names>
</name>
<xref rid="aff1" ref-type="aff">†</xref>
</contrib>
<contrib contrib-type="author" id="ath7"><name><surname>Meng</surname>
<given-names>Guangpeng</given-names>
</name>
<xref rid="aff3" ref-type="aff">§</xref>
</contrib>
<contrib contrib-type="author" corresp="yes" id="ath8"><name><surname>Jiang</surname>
<given-names>Shibo</given-names>
</name>
<xref rid="cor2" ref-type="other">*</xref>
<xref rid="aff2" ref-type="aff">‡</xref>
<xref rid="aff4" ref-type="aff">∥</xref>
</contrib>
<contrib contrib-type="author" corresp="yes" id="ath9"><name><surname>Liu</surname>
<given-names>Keliang</given-names>
</name>
<xref rid="cor1" ref-type="other">*</xref>
<xref rid="aff1" ref-type="aff">†</xref>
<xref rid="aff3" ref-type="aff">§</xref>
</contrib>
<aff id="aff1"><label>†</label>
State Key Laboratory of Toxicology and Medical Countermeasures,<institution>Beijing Institute of Pharmacology and Toxicology</institution>
, 27 Tai-Ping Road, Beijing 100850,<country>China</country>
</aff>
<aff id="aff2"><label>‡</label>
Key Laboratory of Medical Molecular Virology of MOE/MOH, School of Basic Medical Sciences and Shanghai Public Health Clinical Center,<institution>Fudan University</institution>
, 130 Dong An Road, Shanghai 200032,<country>China</country>
</aff>
<aff id="aff3"><label>§</label>
Key Laboratory of Structure-Based Drug Design and Discovery of the Ministry of Education,<institution>Shenyang Pharmaceutical University</institution>
, Shenyang 110016,<country>China</country>
</aff>
<aff id="aff4"><label>∥</label>
Lindsley F. Kimball Research Institute,<institution>New York Blood Center</institution>
, New York, New York 10065,<country>United States</country>
</aff>
<aff id="aff5"><label>⊥</label>
Pharmaceutical Preparation Section, Plastic Surgery Hospital,<institution>Chinese Academy of Medical Sciences and Peking Union Medical College</institution>
, Beijing 100144,<country>China</country>
</aff>
</contrib-group>
<author-notes><corresp id="cor1"><label>*</label>
For K.L.: phone, <phone>86-10-6816-9363</phone>
; fax, <fax>86-10-6821-1656</fax>
; E-mail, <email>keliangliu55@126.com</email>
.</corresp>
<corresp id="cor2"><label>*</label>
For S.J.: phone, <phone>86-21-54237673</phone>
; fax, <fax>86-21-54237465</fax>
; E-mail, <email>shibojiang@fudan.edu.cn</email>
.</corresp>
</author-notes>
<pub-date pub-type="epub"><day>14</day>
<month>02</month>
<year>2018</year>
</pub-date>
<pub-date pub-type="ppub"><day>08</day>
<month>03</month>
<year>2018</year>
</pub-date>
<volume>61</volume>
<issue>5</issue>
<fpage>2018</fpage>
<lpage>2026</lpage>
<history><date date-type="received"><day>22</day>
<month>11</month>
<year>2017</year>
</date>
</history>
<permissions><copyright-statement>Copyright © 2018 American Chemical
Society</copyright-statement>
<copyright-year>2018</copyright-year>
<copyright-holder>American Chemical
Society</copyright-holder>
<license license-type="open-access"><license-p>This article is made available via the PMC Open Access Subset for unrestricted RESEARCH re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic.</license-p>
</license>
</permissions>
<abstract><p content-type="toc-graphic"><graphic xlink:href="jm7b01732_0006" id="ab-tgr1"></graphic>
</p>
<p>The hexameric α-helical coiled-coil
formed between the C-terminal
and N-terminal heptad repeat (CHR and NHR) regions of class I viral
fusion proteins plays an important role in mediating the fusion of
the viral and cellular membranes and provides a clear starting point
for molecular mimicry that drives viral fusion inhibitor design. Unfortunately,
such peptide mimicry of the short α-helical region in the CHR
of Middle East respiratory syndrome coronavirus (MERS-CoV) spike protein
has been thwarted by the loss of the peptide’s native α-helical
conformation when taken out of the parent protein structure. Here,
we describe that appropriate all-hydrocarbon stapling of the short
helical portion-based peptide to reinforce its bioactive secondary
structure remarkably improves antiviral potency. The resultant stapled
peptide P21S10 could effectively inhibit infection by MERS-CoV pseudovirus
and its spike protein-mediated cell–cell fusion; additionally,
P21S10 exhibits improved pharmacokinetic properties than HR2P-M2,
suggesting strong potential for development as an anti-MERS-CoV therapeutic.</p>
</abstract>
<custom-meta-group><custom-meta><meta-name>document-id-old-9</meta-name>
<meta-value>jm7b01732</meta-value>
</custom-meta>
<custom-meta><meta-name>document-id-new-14</meta-name>
<meta-value>jm7b01732</meta-value>
</custom-meta>
<custom-meta><meta-name>ccc-price</meta-name>
<meta-value></meta-value>
</custom-meta>
</custom-meta-group>
</article-meta>
<notes id="notes-d1e21-autogenerated"><fn-group><fn fn-type="" id="d30e234"><p>This article is made available for a limited time sponsored by ACS under the <ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/page/policy/freetoread/index.html">ACS Free to Read License</ext-link>
, which permits copying and redistribution of the article for non-commercial scholarly purposes.</p>
</fn>
</fn-group>
</notes>
</front>
<body><sec id="sec1"><title>Introduction</title>
<p>Class I viral fusion
proteins, such as those of the corona-, retro-,
filo-, orthomyxo-, and paramyxoviruses, share similarities in their
apparent use of a trimer of α-helical hairpins, or six-helix
bundle (6-HB), to mediate membrane fusion processes of virus to target
cell.<sup><xref ref-type="bibr" rid="ref1">1</xref>
,<xref ref-type="bibr" rid="ref2">2</xref>
</sup>
In the 6-HB viral fusion apparatus, the
C-terminal heptad repeat (CHR) segment of the trimeric virus glycoproteins
zips up into three α-helices along the conserved hydrophobic
grooves on the periphery of an internal three-stranded coiled coil
formed by the N-terminal heptad repeat (NHR) region of those fusion
proteins.<sup><xref ref-type="bibr" rid="ref3">3</xref>
,<xref ref-type="bibr" rid="ref4">4</xref>
</sup>
Despite the topologically similar conformation
of the fusion-promoting hexameric helical scaffold, their α-helical
portion of the CHR domain displays dramatic differences in length.
For example, the fusion core structure of HIV, a <italic>Retroviridae</italic>
family member, contains regular α-helical CHR domains involving
approximately 10 helical turns that bind to the central trimeric NHR
helices.<sup><xref ref-type="bibr" rid="ref5">5</xref>
,<xref ref-type="bibr" rid="ref6">6</xref>
</sup>
In contrast, some other class I enveloped
viruses, including Middle East respiratory syndrome coronavirus (MERS-CoV)
of the <italic>Coronaviridae</italic>
family, encode a notable dimorphism
in the length of its NHR and CHR helices.<sup><xref ref-type="bibr" rid="ref7">7</xref>
−<xref ref-type="bibr" rid="ref10">10</xref>
</sup>
In the MERS-CoV 6-HB assembly,
the NHR trimers have ∼21 helical turns, whereas the central
CHR helices are limited to ∼4.5 turns within the longer heptad-repeat
sequence (<xref rid="fig1" ref-type="fig">Figure <xref rid="fig1" ref-type="fig">1</xref>
</xref>
A).<sup><xref ref-type="bibr" rid="ref11">11</xref>
</sup>
</p>
<fig id="fig1" position="float"><label>Figure 1</label>
<caption><p>Crystal structure of the MERS-CoV six-helix bundle (6HB)
fusion
core and the design of peptides based on the interaction between the
NHR and CHR domains. (A) Cartoon representations of the MERS-CoV and
HIV 6HBs, in which the NHR trimers and CHR segments are colored in
gray and green, respectively. In the HIV 6HB structure (PDB 1AIK), the CHR segments
form regular helices that pack against the central NHR core. In contrast,
only a short helical domain is found within the MERS-CoV CHR (PDB 4NJL). (B) Helical wheel
representation of the 6HB. In the CHR, the residues at the <italic>a</italic>
–<italic>d</italic>
positions (yellow) in direct
contact with the NHR domains are buried in the 6HB. (C) Interaction
between the NHR and CHR peptides, as well as the designed P21 peptide.
The dashed lines between the NHR and CHR domains indicate the interaction
between the residues located at the <italic>a</italic>
–<italic>d</italic>
positions in the CHR and the <italic>e</italic>
–<italic>g</italic>
positions in the NHR to form the 6HB. The helical domain
sequence in MERS-CoV CHR is highlighted in red.</p>
</caption>
<graphic xlink:href="jm7b01732_0001" id="gr1" position="float"></graphic>
</fig>
<p>Blocking α-helix-mediated NHR/CHR interactions using
CHR-derived
peptides that target the transiently exposed NHR coiled coil is a
promising approach to inhibit membrane fusion and viral infection.<sup><xref ref-type="bibr" rid="ref12">12</xref>
,<xref ref-type="bibr" rid="ref13">13</xref>
</sup>
With this strategy, a family of decoy α-helices structurally
mimicking the lengthy C-terminal helices of HIV was identified as
low-nanomolar inhibitors of viral entry.<sup><xref ref-type="bibr" rid="ref14">14</xref>
,<xref ref-type="bibr" rid="ref15">15</xref>
</sup>
It was disappointing that a synthetic peptide spanning the α-helical
region of the MERS-CoV CHR domain sequence showed no anti-MERS-CoV
activity.<sup><xref ref-type="bibr" rid="ref11">11</xref>
,<xref ref-type="bibr" rid="ref16">16</xref>
</sup>
Although the typical α-helical region
is an important recognition motif for MERS-CoV fusion core formation,
this short CHR peptide alone does not retain its native conformation
owing to the lack of structural reinforcement provided by the parent
protein, which, in turn, retards its binding to partner NHR helices.<sup><xref ref-type="bibr" rid="ref11">11</xref>
</sup>
</p>
<p>Peptide stapling based on different macrocyclization
chemistry
is a key technique for constraining short peptides in α-helical
structures and thereby preorganizing them into their bound conformations
to modulate helix-mediated protein–protein interactions with
reduced entropic penalty.<sup><xref ref-type="bibr" rid="ref17">17</xref>
,<xref ref-type="bibr" rid="ref18">18</xref>
</sup>
Among numerous stapling
chemistries, ruthenium-catalyzed RCM is one of the most noteworthy
methods.<sup><xref ref-type="bibr" rid="ref19">19</xref>
,<xref ref-type="bibr" rid="ref20">20</xref>
</sup>
This method of producing all-hydrocarbon-stapled
α-helical peptides affords high levels of α-helix induction
and nucleation, thus directly resulting in peptide target-binding
proclivity, cell permeability, and serum stability.<sup><xref ref-type="bibr" rid="ref21">21</xref>
,<xref ref-type="bibr" rid="ref22">22</xref>
</sup>
Furthermore, the therapeutic potential of stapled peptides has been
showcased in a growing diversity of biological settings. Two hydrocarbon-stapled
peptides developed by Aileron Therapeutics have reached clinical trials,
epitomizing the early indications regarding the clinical translational
potential of these stabilized α-helical peptidomimetics.<sup><xref ref-type="bibr" rid="ref23">23</xref>
,<xref ref-type="bibr" rid="ref24">24</xref>
</sup>
</p>
<p>In the present study, we employed hydrocarbon stapling to
recapitulate
the topography of α-helical motifs found in the MERS-CoV CHR
region to inhibit MERS-CoV infection and its spike (S) protein-mediated
cell–cell fusion. In the process of developing these stapled
α-helical peptides, we performed a detailed study to identify
the optimal stapling sites and the effect of hydrophobic amino acid
incorporation to generate promising therapeutics.</p>
</sec>
<sec id="sec2"><title>Design</title>
<p>The primary structure of the MERS-CoV S protein hexameric coiled-coil
fusion complex exhibits a heptad repeat pattern usually denoted as <italic>a</italic>
–<italic>b</italic>
–<italic>c</italic>
–<italic>d</italic>
–<italic>e</italic>
–<italic>f</italic>
–<italic>g</italic>
.<sup><xref ref-type="bibr" rid="ref11">11</xref>
,<xref ref-type="bibr" rid="ref16">16</xref>
</sup>
In structural terms, three NHR
helices form a trimeric coiled-coil inner core using their <italic>a</italic>
–<italic>d</italic>
residues for self-association,
while those at the <italic>e</italic>
–<italic>g</italic>
positions
participate in the interhelical knob-in-hole packing interaction with
residues at the <italic>a</italic>
–<italic>d</italic>
positions
of CHR helices, which are considered critical for stabilizing the
6-HB structure (<xref rid="fig1" ref-type="fig">Figure <xref rid="fig1" ref-type="fig">1</xref>
</xref>
B).<sup><xref ref-type="bibr" rid="ref25">25</xref>
,<xref ref-type="bibr" rid="ref26">26</xref>
</sup>
According to the crystal structure of the
MERS-CoV fusion core, residues L1262 at position <italic>d</italic>
to Y1280 at position <italic>a</italic>
in the CHR domain adopt
a canonical α-helical region, binding to the deep grooves of
the NHR trimers. Seminal work investigating the stability of the coiled
coil suggests that helices consisting of fewer than three heptads
generally do not create enough opportunities for hydrophobic interactions
in the core to favor superhelical assembly.<sup><xref ref-type="bibr" rid="ref27">27</xref>
,<xref ref-type="bibr" rid="ref28">28</xref>
</sup>
On the basis of the X-ray crystallography and coiled-coil sequence-to-structure
relationship study, we engineered a peptide containing the three integral
heptads, designated as P21, spanning residues 1260–1280 of
the MERS-CoV S protein CHR as a template for inhibitor design (<xref rid="fig1" ref-type="fig">Figure <xref rid="fig1" ref-type="fig">1</xref>
</xref>
C). Analysis of the
crystal structure of CHR in complex with NHR helices shows that six
residues at the <italic>a</italic>
and <italic>d</italic>
positions
(L1262, M1266, L1269, V1273, L1276, and Y1280) are buried in the 6-HB.
In addition to the hydrophobic interactions contributed by their side
chains, residues L1262, M1266, V1273, and Y1280 form four hydrogen
bonds with NHR-helical trimer residues, including Q1023, N1016, Q1009,
and K1000, via their main-chain amino or oxygen groups. Meanwhile,
the hydroxyl group of the Y1280 side chain accepts a hydrogen bond
from the NH group of Q1009 in NHR helices.<sup><xref ref-type="bibr" rid="ref11">11</xref>
,<xref ref-type="bibr" rid="ref16">16</xref>
</sup>
Together with critical hydrophobic stacking forces, these hydrogen
bonds constitute a polar contact network that further tightly ties
CHR to NHR helix. We initially utilized the “staple scanning”
approach along the length of the peptide helix, but away from the
critical <italic>a</italic>
–<italic>d</italic>
residues, to
identify the optimal stapling sites. We substituted pairs of (<italic>S</italic>
)-2-(4-pentenyl)alanine (abbreviated as S5 residues) at
the <italic>i</italic>
and <italic>i</italic>
+4 positions because
it is the most widely used unnatural amino acid for such type of staple.<sup><xref ref-type="bibr" rid="ref21">21</xref>
</sup>
Accumulated evidence that underline the structure
and stability of coiled-coils has shown that the superhelix stability
can generally be improved by inserting residues with more hydrophobic
side chains at the <italic>a</italic>
–<italic>d</italic>
positions.<sup><xref ref-type="bibr" rid="ref28">28</xref>
,<xref ref-type="bibr" rid="ref29">29</xref>
</sup>
Among the six buried <italic>a</italic>
–<italic>d</italic>
residues, Leu and Val, who possess high hydropathy indices,<sup><xref ref-type="bibr" rid="ref30">30</xref>
</sup>
can strongly bind to the grooves on the surface
of the N-helix trimer through their fully hydrophobic side chain interactions
but Met and Tyr are polar residues. On the basis of the scanning study
results, we elected to create stapled structures with hydrophobic
mutations in the buried <italic>a</italic>
–<italic>d</italic>
residues through individual substitution of M1266 or Y1280 with <sc>l</sc>
-norleucine, an isosteric Met analogue with less polar and
more hydrophobic properties,<sup><xref ref-type="bibr" rid="ref31">31</xref>
</sup>
or F, respectively,
or a combination of both replacements, thus expecting to form a potential
hydrophobic binding core.</p>
</sec>
<sec id="sec3"><title>Results and Discussion</title>
<sec id="sec3.1"><title>Effect of Stapling on Inhibition
of MERS-CoV S Protein-Mediated
Cell–Cell Fusion</title>
<p>First, peptides P21S1–P21S10
were tested for their ability to inhibit MERS-CoV fusion with its
target cell using our previously developed MERS-CoV S protein-mediated
cell–cell fusion assay.<sup><xref ref-type="bibr" rid="ref11">11</xref>
</sup>
As shown
in <xref rid="tbl1" ref-type="other">Table <xref rid="tbl1" ref-type="other">1</xref>
</xref>
, the linear
wild-type P21 exhibited no inhibition at a concentration of up to
50 μM but when the peptides were cyclized markedly improved
inhibitory activity was demonstrated. Among these α-helical
mimetics, P21S8 and P21S10 strongly inhibited S protein-mediated cell–cell
fusion, with EC<sub>50</sub>
values of 0.26 and 0.33 μM, respectively,
which are even more potent than the most active MERS-CoV fusion inhibitor
so far, namely the 36-mer peptide HR2P-M2.<sup><xref ref-type="bibr" rid="ref11">11</xref>
</sup>
Importantly, their unmetathesized analogues, i.e., peptides containing
un-cross-linked S5 residues had a dramatically decreased membrane
fusion inhibitory potency (<xref rid="tbl1" ref-type="other">Table <xref rid="tbl1" ref-type="other">1</xref>
</xref>
). These results reveal the critical contribution of
hydrocarbon stapling to replicate the local topography of MERS-CoV
short C-terminal helices for therapeutic benefit. In addition, shifting
the position of the all-hydrocarbon staple had a significant effect
on anti-MERS-CoV activity. Peptides P21S2, P21S4, P21S5, and P21S9,
with a 2/6, 5/9, 8/12, and 15/19 staple, respectively, exhibited only
similar activity compared to their relevant unstapled peptides, whereas
peptides P21S1, P21S3, P21S6, and P21S7 had no activity. Compared
to P21S8 whose cross-link had the <italic>S</italic>
,<italic>S</italic>
-configuration, its <italic>R</italic>
,<italic>R</italic>
-configured
counterpart, P21R8, provided 62-fold weaker anti-MERS-CoV activity,
consistent with previous publications revealing the requirement of <italic>S</italic>
-form building blocks for an <italic>i</italic>
, <italic>i</italic>
+4 staple type.<sup><xref ref-type="bibr" rid="ref21">21</xref>
</sup>
Finally, we
explored whether the incorporation of hydrophobic mutations at the
binding interface could lead to an improvement in biological activity.
In our assay, replacing M1266 with <sc>l</sc>
-norleucine (i.e., Z),
which led to P21S8Z, retained the high activity of P21S8 whereas Y1280F
alone and the combination of M1266Z and Y1280F showed decreased potencies
compared to that of P21S8, indicating the critical role of hydrogen
bond formation between Y1280 and Q1009 of the S protein NHR trimer.
Although buried hydrophobic residues may contribute more energy toward
stabilization of a coiled-coil structure than polar interactions,<sup><xref ref-type="bibr" rid="ref28">28</xref>
,<xref ref-type="bibr" rid="ref32">32</xref>
</sup>
subtle specific interacting networks are required for hydrocarbon-stapled
peptides to stop the MERS-CoV–cell fusion process efficiently.</p>
<table-wrap id="tbl1" position="float"><label>Table 1</label>
<caption><title>Inhibitory Activities of Stapled Peptides
on MERS-CoV S Protein-Mediated Cell–Cell Fusion</title>
</caption>
<table frame="hsides" rules="groups" border="0"><colgroup><col align="left"></col>
<col align="left"></col>
<col align="center"></col>
</colgroup>
<thead><tr><th style="border:none;" align="center">compd</th>
<th style="border:none;" align="center">sequence<xref rid="t1fn1" ref-type="table-fn">a</xref>
</th>
<th style="border:none;" align="center">EC<sub>50</sub>
(μM)<xref rid="t1fn5" ref-type="table-fn">e</xref>
</th>
</tr>
</thead>
<tbody><tr><td colspan="3" style="border:none;" align="center">Hydrocarbon Stapled Peptides<xref rid="t1fn2" ref-type="table-fn">b</xref>
</td>
</tr>
<tr><td style="border:none;" align="left">P21S1</td>
<td style="border:none;" align="left"><bold>*</bold>
DLT<bold>*</bold>
EM LSLQQVV KALNESY</td>
<td style="border:none;" align="center">>50</td>
</tr>
<tr><td style="border:none;" align="left">P21S2</td>
<td style="border:none;" align="left">L<bold>*</bold>
LTY<bold>*</bold>
M LSLQQVV KALNESY</td>
<td style="border:none;" align="center">3.90 ± 1.1</td>
</tr>
<tr><td style="border:none;" align="left">P21S3</td>
<td style="border:none;" align="left">LDL<bold>*</bold>
YEM <bold>*</bold>
SLQQVV KALNESY</td>
<td style="border:none;" align="center">>50</td>
</tr>
<tr><td style="border:none;" align="left">P21S4</td>
<td style="border:none;" align="left">LDLT<bold>*</bold>
EM L<bold>*</bold>
LQQVV KALNESY</td>
<td style="border:none;" align="center">7.14 ± 0.7</td>
</tr>
<tr><td style="border:none;" align="left">P21S5</td>
<td style="border:none;" align="left">LDLTYEM <bold>*</bold>
SLQ<bold>*</bold>
VV KALNESY</td>
<td style="border:none;" align="center">10.7 ± 2.6</td>
</tr>
<tr><td style="border:none;" align="left">P21S6</td>
<td style="border:none;" align="left">LDLTYEM L<bold>*</bold>
LQQ<bold>*</bold>
V KALNESY</td>
<td style="border:none;" align="center">>50</td>
</tr>
<tr><td style="border:none;" align="left">P21S7</td>
<td style="border:none;" align="left">LDLTYEM LSL<bold>*</bold>
QVV <bold>*</bold>
ALNESY</td>
<td style="border:none;" align="center">>50</td>
</tr>
<tr><td style="border:none;" align="left">P21S8</td>
<td style="border:none;" align="left">LDLTYEM LSLQ<bold>*</bold>
VV K<bold>*</bold>
LNESY</td>
<td style="border:none;" align="center">0.26 ± 0.05</td>
</tr>
<tr><td style="border:none;" align="left">P21S9</td>
<td style="border:none;" align="left">LDLTYEM LSLQQVV <bold>*</bold>
ALN<bold>*</bold>
SY</td>
<td style="border:none;" align="center">14.1 ± 2.3</td>
</tr>
<tr><td style="border:none;" align="left">P21S10</td>
<td style="border:none;" align="left">LDLTYEM LSLQQVV K<bold>*</bold>
LNE<bold>*</bold>
Y</td>
<td style="border:none;" align="center">0.33 ± 0.04</td>
</tr>
<tr><td colspan="3" style="border:none;" align="center">Unstapled Peptides<xref rid="t1fn3" ref-type="table-fn">c</xref>
</td>
</tr>
<tr><td style="border:none;" align="left">P21L2</td>
<td style="border:none;" align="left">L<bold>X</bold>
LTY<bold>X</bold>
M LSLQQVV KALNESY</td>
<td style="border:none;" align="center">10.9 ± 1.1</td>
</tr>
<tr><td style="border:none;" align="left">P21L4</td>
<td style="border:none;" align="left">LDLT<bold>X</bold>
EM L<bold>X</bold>
LQQVV KALNESY</td>
<td style="border:none;" align="center">8.21 ± 0.9</td>
</tr>
<tr><td style="border:none;" align="left">P21L5</td>
<td style="border:none;" align="left">LDLTYEM <bold>X</bold>
SLQ<bold>X</bold>
VV KALNESY</td>
<td style="border:none;" align="center">4.49 ± 0.6</td>
</tr>
<tr><td style="border:none;" align="left">P21L8</td>
<td style="border:none;" align="left">LDLTYEM LSLQ<bold>X</bold>
VV K<bold>X</bold>
LNESY</td>
<td style="border:none;" align="center">20.6 ± 3.3</td>
</tr>
<tr><td style="border:none;" align="left">P21L9</td>
<td style="border:none;" align="left">LDLTYEM LSLQQVV <bold>X</bold>
ALN<bold>X</bold>
SY</td>
<td style="border:none;" align="center">10.9 ± 1.0</td>
</tr>
<tr><td style="border:none;" align="left">P21L10</td>
<td style="border:none;" align="left">LDLTYEM LSLQQVV K<bold>X</bold>
LNE<bold>X</bold>
Y</td>
<td style="border:none;" align="center">3.55 ± 0.2</td>
</tr>
<tr><td colspan="3" style="border:none;" align="center">P21S8 Mutated Peptides<xref rid="t1fn4" ref-type="table-fn">d</xref>
</td>
</tr>
<tr><td style="border:none;" align="left">P21R8</td>
<td style="border:none;" align="left">LDLTYEM LSLQ<bold><sup>∧</sup>
</bold>
VV K<bold><sup>∧</sup>
</bold>
LNESY</td>
<td style="border:none;" align="center">16.3 ± 1.1</td>
</tr>
<tr><td style="border:none;" align="left">P21S8Z</td>
<td style="border:none;" align="left">LDLTYE<bold>Z</bold>
LSLQ<bold>*</bold>
VV K<bold>*</bold>
LNESY</td>
<td style="border:none;" align="center">0.63 ± 0.05</td>
</tr>
<tr><td style="border:none;" align="left">P21S8F</td>
<td style="border:none;" align="left">LDLTYEM LSLQ<bold>*</bold>
VV K<bold>*</bold>
LNES<bold>F</bold>
</td>
<td style="border:none;" align="center">2.16 ± 1.1</td>
</tr>
<tr><td style="border:none;" align="left">P21S8ZF</td>
<td style="border:none;" align="left">LDLTYE<bold>Z</bold>
LSLQ<bold>*</bold>
VV K<bold>*</bold>
LNES<bold>F</bold>
</td>
<td style="border:none;" align="center">3.89 ± 0.8</td>
</tr>
<tr><td colspan="3" style="border:none;" align="center">Control Peptides</td>
</tr>
<tr><td style="border:none;" align="left">P21</td>
<td style="border:none;" align="left">LDLTYEM LSLQQVV KALNESY</td>
<td style="border:none;" align="center">>50</td>
</tr>
<tr><td style="border:none;" align="left">HR2P-M2</td>
<td style="border:none;" align="left">SLTQINTTLLDLEYEMKKLEEVVKKLEESYIDLKEL</td>
<td style="border:none;" align="center">0.75 ± 0.09</td>
</tr>
</tbody>
</table>
<table-wrap-foot><fn id="t1fn1"><label>a</label>
<p>These peptides
have an acetyl group
at the N-terminus and carboxyamide at the C-terminus.</p>
</fn>
<fn id="t1fn2"><label>b</label>
<p>Asterisks indicate the positions
of the S5 residues, which react to form the all-hydrocarbon staple.</p>
</fn>
<fn id="t1fn3"><label>c</label>
<p><bold>X</bold>
indicates the
positions
of the S5 amino acids left uncyclized.</p>
</fn>
<fn id="t1fn4"><label>d</label>
<p><bold><sup>∧</sup>
</bold>
denotes the positions
of the R5 amino acids, which react to form
staples.</p>
</fn>
<fn id="t1fn5"><label>e</label>
<p>EC<sub>50</sub>
data were derived
from the results of three independent experiments and are expressed
as the mean ± standard deviation.</p>
</fn>
</table-wrap-foot>
</table-wrap>
</sec>
<sec id="sec3.2"><title>Stapled Peptides As Inhibitors of Pseudotyped MERS-CoV Infection</title>
<p>Subsequently, we tested the potential inhibitory activities of
the P21S8, P21S10, and P21S8Z peptides, which exhibited promising
inhibitory potency in the cell–cell fusion assay, on MERS-CoV
pseudovirus infection in Huh-7 cells. As shown in <xref rid="tbl2" ref-type="other">Table <xref rid="tbl2" ref-type="other">2</xref>
</xref>
and Supporting Information, <ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acs.jmedchem.7b01732/suppl_file/jm7b01732_si_001.pdf">Figure S1</ext-link>
, P21S8, P21S10, and P21S8Z could significantly
inhibit the MERS-CoV pseudovirus carrying the wild-type S protein,
with EC<sub>50</sub>
values of 3.03, 0.97, and 2.80 μM, respectively.
Although P21S8 exhibited 2.8-fold greater potency than the positive
control HR2P-M2 in inhibiting cell–cell fusion, it was 2.8-fold
less active than HR2P-M2 against wild-type MERS-CoV pseudovirus. Notably,
P21S10 displayed potency similar to that of HR2P-M2 in both cell–cell
and virus–cell fusion inhibition. These results suggest that,
unlike P21S10, P21S8 does not have good correlation between its blockage
of S protein-mediated membrane fusion and inhibition of wild-type
MERS-CoV infection. One of the possible explanations for this discrepancy
is that P21S8 may have lower target binding capability than P21S10
during the inhibition of virus infection. In addition, none of these
viral fusion blockers displayed significant cytotoxicity to Huh-7
cells up to 100 μM (<xref rid="tbl2" ref-type="other">Table <xref rid="tbl2" ref-type="other">2</xref>
</xref>
and Supporting Information, <ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acs.jmedchem.7b01732/suppl_file/jm7b01732_si_001.pdf">Figure S2</ext-link>
). The selectivity index (SI = CC<sub>50</sub>
/EC<sub>50</sub>
) of P21S10 was greater than 103. Among clinical MERS-CoV strains,
S protein amino acid changes of Q1020H and Q1020R located in the NHR
domain were under positive selection and present in nearly all variants.<sup><xref ref-type="bibr" rid="ref33">33</xref>
</sup>
Therefore, we next investigated whether P21S8,
P21S10, and P21S8Z would be effective against MERS-CoV strains with
the Q1020H and Q1020R mutations. Strikingly, these peptides were effective
for inhibiting infection by MERS-CoV pseudoviruses with mutated S
protein, with EC<sub>50</sub>
values similar to those for inhibiting
pseudovirus carrying the wild-type S protein (<xref rid="tbl2" ref-type="other">Table <xref rid="tbl2" ref-type="other">2</xref>
</xref>
and Supporting Information, <ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acs.jmedchem.7b01732/suppl_file/jm7b01732_si_001.pdf">Figure S1</ext-link>
). P21S8 and P21S8Z displayed about
3-fold less potency than HR2P-M2 for inhibiting infection by pseudoviruses
carrying S protein with Q1020H or Q1020R mutation; however, P21S10
possessed potency similar to that of HR2P-M2. Because MERS-CoV is
primarily infecting the human respiratory tract, we further tested
the inhibitory activities of these stapled peptides on MERS-CoV pseudovirus
infection in a human lung-derived cell line Calu-3. Strikingly, P21S8,
P21S10, and P21S8Z could inhibit MERS-CoV pseudovirus infection in
Calu-3 cells with EC<sub>50</sub>
values of 2.21, 1.58, and 2.57 μM,
respectively, in an agreement with result observed in experiment using
Huh-7 cells (Supporting Information, <ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acs.jmedchem.7b01732/suppl_file/jm7b01732_si_001.pdf">Table S1</ext-link>
and <ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acs.jmedchem.7b01732/suppl_file/jm7b01732_si_001.pdf">Figure S1</ext-link>
). They also had no significant
cytotoxicity to Calu-3 cells at the concentrations as high as 100
μM (Supporting Information, <ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acs.jmedchem.7b01732/suppl_file/jm7b01732_si_001.pdf">Figure S3</ext-link>
). Moreover, we evaluated the aqueous solubility of these stapled
peptides. P21S8 and P21S10 had aqueous solubilities of 6.47 and 10.5
mg/mL at pH 7.4, respectively, which were 14-fold and 22-fold higher
than that of P21S8Z (Supporting Information, <ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acs.jmedchem.7b01732/suppl_file/jm7b01732_si_001.pdf">Table S2</ext-link>
), suggesting their good potential for further development.</p>
<table-wrap id="tbl2" position="float"><label>Table 2</label>
<caption><title>Inhibitory Activity of P21S8, P21S10,
and P21S8Z on Infection by Pseudotyped MERS-CoV<xref rid="t2fn1" ref-type="table-fn">a</xref>
</title>
</caption>
<table frame="hsides" rules="groups" border="0"><colgroup><col align="left"></col>
<col align="char" char="±"></col>
<col align="char" char="±"></col>
<col align="char" char="±"></col>
<col align="char" char="."></col>
<col align="char" char="."></col>
</colgroup>
<thead><tr><th style="border:none;" align="center"> </th>
<th colspan="3" align="center" char="±">EC<sub>50</sub>
(μM) for inhibiting<hr></hr>
</th>
<th style="border:none;" align="center" char="."> </th>
<th style="border:none;" align="center" char="."> </th>
</tr>
<tr><th style="border:none;" align="center">compd</th>
<th style="border:none;" align="center" char="±">WT MERS-CoV pseudovirus</th>
<th style="border:none;" align="center" char="±">Q1020H-MERS-CoV pseudovirus</th>
<th style="border:none;" align="center" char="±">Q1020R-MERS-CoV pseudovirus</th>
<th style="border:none;" align="center" char=".">CC<sub>50</sub>
(μM)</th>
<th style="border:none;" align="center" char=".">SI<xref rid="t2fn2" ref-type="table-fn">b</xref>
</th>
</tr>
</thead>
<tbody><tr><td style="border:none;" align="left">P21S8</td>
<td style="border:none;" align="char" char="±">3.03 ± 0.29</td>
<td style="border:none;" align="char" char="±">4.06 ± 0.34</td>
<td style="border:none;" align="char" char="±">1.98 ± 0.28</td>
<td style="border:none;" align="char" char=".">>100</td>
<td style="border:none;" align="char" char=".">>33</td>
</tr>
<tr><td style="border:none;" align="left">P21S10</td>
<td style="border:none;" align="char" char="±">0.97 ± 0.08</td>
<td style="border:none;" align="char" char="±">1.82 ± 0.28</td>
<td style="border:none;" align="char" char="±">0.89 ± 0.07</td>
<td style="border:none;" align="char" char=".">>100</td>
<td style="border:none;" align="char" char=".">>103</td>
</tr>
<tr><td style="border:none;" align="left">P21S8Z</td>
<td style="border:none;" align="char" char="±">2.80 ± 0.74</td>
<td style="border:none;" align="char" char="±">4.15 ± 0.25</td>
<td style="border:none;" align="char" char="±">2.49 ± 0.18</td>
<td style="border:none;" align="char" char=".">>100</td>
<td style="border:none;" align="char" char=".">>36</td>
</tr>
<tr><td style="border:none;" align="left">HR2P-M2</td>
<td style="border:none;" align="char" char="±">1.07 ± 0.21</td>
<td style="border:none;" align="char" char="±">1.25 ± 0.18</td>
<td style="border:none;" align="char" char="±">0.64 ± 0.16</td>
<td style="border:none;" align="char" char=".">>100</td>
<td style="border:none;" align="char" char=".">>93</td>
</tr>
</tbody>
</table>
<table-wrap-foot><fn id="t2fn1"><label>a</label>
<p>Data were derived from the results
of three independent experiments and are presented as the mean ±
standard deviation.</p>
</fn>
<fn id="t2fn2"><label>b</label>
<p>SI (selectivity
index) = CC<sub>50</sub>
/EC<sub>50</sub>
for inhibiting WT MERS-CoV
pseudovirus infection.</p>
</fn>
</table-wrap-foot>
</table-wrap>
</sec>
<sec id="sec3.3"><title>Effect
of Stapling on Helical Propensity</title>
<p>Next, we sought
to determine whether hydrocarbon stapling could effectively recapitulate
the native conformation of the α-helical region in the MERS-CoV
CHR domain. CD spectroscopy provides a typical signature for α-helices
with a maximum near 190 nm and double minima at 208 and 222 nm. CD
spectra showed that P21 displayed a random coil conformation in phosphate
buffer. Strikingly, with the exception of P21S2 that was too insoluble
to make accurate measurements, the other hydrocarbon-stapled peptides
exhibited α-helical structure with 15.6–54.5% helicity.
Among them, the highly effective stapled peptides P21S8 and P21S10
were found to have greatly improved helical propensity, compared to
the linear wild-type peptide, with helical contents of 53.7% and 46.5%,
respectively (<xref rid="fig2" ref-type="fig">Figure <xref rid="fig2" ref-type="fig">2</xref>
</xref>
). Furthermore, these stapled peptides were indeed more helical than
their correponding uncyclized linear precursors (Supporting Information, <ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acs.jmedchem.7b01732/suppl_file/jm7b01732_si_001.pdf">Table S3–S4</ext-link>
). These data further confirmed
the successful mimicry of the CHR helix via cyclization using the
stapling strategy. Interestingly, although the CD spectra for peptides
P21S8 and P21S10 were similar to those of P21S6 and P21S7, a difference
in inhibitory potency of more than 193-fold was observed between them,
suggesting that the much higher biological activity cannot be merely
attributed to the helical propensity. Hence, the following target-binding
studies were performed to further elucidate their mechanisim of action.</p>
<fig id="fig2" position="float"><label>Figure 2</label>
<caption><p>CD spectra
of the isolated peptides P21S8 and P21S10. The corresponding
unstapled counterparts and linear peptide P21 are included for structural
comparison. The final concentration of each peptide in PBS (pH 7.2)
was 50 μM.</p>
</caption>
<graphic xlink:href="jm7b01732_0002" id="gr2" position="float"></graphic>
</fig>
</sec>
<sec id="sec3.4"><title>Binding of Stapled Peptides
to Their Complementary S Protein
NHR Domain</title>
<p>Previous studies have shown that peptides derived
from the MERS-CoV S protein CHR domain can bind to their NHR target
to form a stable α-helix hexamer.<sup><xref ref-type="bibr" rid="ref11">11</xref>
</sup>
Here, we first sought to determine the interaction of the stapled
helical peptides with their native NHR ligand, designated HR1P, to
form stable α-helical complexes using CD spectroscopy. The significant
increase over the sum of the individual spectra of two peptides mixed
together in equimolar concentrations indicates that the interaction
between them led to enhanced helical structures.<sup><xref ref-type="bibr" rid="ref34">34</xref>
,<xref ref-type="bibr" rid="ref35">35</xref>
</sup>
The isolated HR1P displays a random coil structure in phosphate
buffer (pH 7.2) as judged by CD spectroscopy, which was consistent
with previous reports.<sup><xref ref-type="bibr" rid="ref11">11</xref>
</sup>
Both P21S8 and
P21S10 interacted with HR1P, resulting in 35.5% and 45.6% α-helical
content in the complex, respectively. The CD signals of the P21S8/HR1P
mixture and the P21S10/HR1P mixture at 222 nm were dramatically greater
than the mathematical sum of those of the isolated peptides, indicating
the induction of a large α-helical structure upon their interaction
(<xref rid="fig3" ref-type="fig">Figure <xref rid="fig3" ref-type="fig">3</xref>
</xref>
A). Strikingly,
the thermal unfolding transition (<italic>T</italic>
<sub>m</sub>
)
values of P21S8/HR1P and P21S10/HR1P reached 71.1 and 76.4 °C,
respectively (<xref rid="fig3" ref-type="fig">Figure <xref rid="fig3" ref-type="fig">3</xref>
</xref>
B). Together, these CD data are consistent with P21S8/HR1P and P21S10/HR1P
folding to form thermally stable and cooperatively folded helical
bundles. Stapled peptides with moderate activity, including P21S4,
P21S5, and P21S9, resulted in a less stable interaction with <italic>T</italic>
<sub>m</sub>
values ranging from 56.6 to 66.6 °C while
peptides with very poor antiviral activity showed no, or weak, coiled-coil
interactions (Supporting Information, <ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acs.jmedchem.7b01732/suppl_file/jm7b01732_si_001.pdf">Table S4</ext-link>
). These results demonstrated a strong correlation between the biological
activities and the thermal stabilities of the stapled peptides/HR1P
complexes.</p>
<fig id="fig3" position="float"><label>Figure 3</label>
<caption><p>Binding of P21S8 and P21S10 to the NHR peptide HR1P. (A) CD spectrum
of peptide mixtures (Spec(C+N), solid symbols) and the sum of the
spectra of the related isolated peptides (Spec(C) + Spec(N), open
symbols) are shown for comparison. The hydrocarbon-stapled peptide/HR1P
interaction induces more α-helix content than the sum of the
single peptides. The final concentration of each peptide in PBS was
50 μM. (B) The thermostability of the complexes formed by P21S8
or P21S10 and the target mimic NHR peptide HR1P was determined by
CD spectroscopy. The final concentration of each peptide in PBS was
50 μM. (C) N-PAGE analysis of the stapled peptides, HR1P, and
their complexes. Lane 1, HR1P; lane 2, P21S8 + HR1P; lane 3, P21S8;
lane 4, P21S10 + HR1P; lane 5, P21S10; lane 6, P21S9 + HR1P; lane
7, P21S9; lane 8, P21S3 + HR1P; lane 9, P21S3. (D) Sedimentation velocity
analysis of the P21S8/HR1P mixture and the P21S10/HR1P mixture. The
sedimentation coefficient (s) and the observed molecular mass (kDa)
of each peak are indicated in parentheses.</p>
</caption>
<graphic xlink:href="jm7b01732_0003" id="gr3" position="float"></graphic>
</fig>
<p>The binding of the stapled peptides to the NHR-derived peptide
HR1P was further confirmed using native (N)-PAGE. In N-PAGE, owing
to its net positive charge, HR1P did not appear on the gel as it migrated
into the solvent under the native electrophoresis conditions; these
results are consistent with previous observations.<sup><xref ref-type="bibr" rid="ref11">11</xref>
</sup>
The respective stapled helical peptides with various antiviral
potencies were mixed with HR1P and subjected to N-PAGE analysis. As
shown in <xref rid="fig3" ref-type="fig">Figure <xref rid="fig3" ref-type="fig">3</xref>
</xref>
C,
both P21S8 and P21S10 formed stable complexes with HR1P, as evidenced
by new bands at the upper position in the gel concomitant with the
fading or disappearance of the stapled peptide bands, suggesting that
oligomeric complexes formed via coiled-coil interactions. The N-PAGE
results also indicated a stronger binding of either P21S8 or P21S10
to HR1P than the bindings of P21S4, P21S5, and P21S9 to HR1P based
on the densities of the residue-stapled peptide bands observed in
the gel (<xref rid="fig3" ref-type="fig">Figure <xref rid="fig3" ref-type="fig">3</xref>
</xref>
C
and Supporting Information, <ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acs.jmedchem.7b01732/suppl_file/jm7b01732_si_001.pdf">Figure S4</ext-link>
).
Consistent with the cell–cell fusion assay and CD spectroscopy
results, the inactive peptide P21S1, P21S3, and P21S6 exhibited no
NHR binding. Interestingly, the mixture of P21S7/HR1P did not show
new band at the upper position in the gel, while the P21S7 band in
the mixture had a much lower intensity than the corresponding P21S7
band alone (Supporting Information, <ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acs.jmedchem.7b01732/suppl_file/jm7b01732_si_001.pdf">Figure S4</ext-link>
). This is in agreement with the CD data, where the signal of the
P21S7/HR1P mixture was dramatically smaller than that of the mathematical
sum of the isolated peptides, suggesting that addition of P21S7 to
HR1P distorts the α-helical conformation of P21S7 due to their
interaction. The combined CD and N-PAGE data indicate that P21S7 may
associate with the viral N-helices to form nonhelical complexes or
aggregates that cannot migrate in the gel, thus resulting in no inhibitory
activity against MERS-CoV infection.</p>
<p>The sizes of the complexes
formed between HR1P and P21S8 or P21S10
were further confirmed by sedimentation velocity analysis (SVA). As
analyzed by SVA, the sedimentation coefficients of the P21S8/HR1P
complex and the P21S10/HR1P complex were 2.27 and 1.94 s, corresponding
to 22.6 and 22.3 kDa, respectively. Compared to the expected molecular
masses of 7.07 kDa for the P21S8/HR1P heterodimer and 7.10 kDa for
the P21S10/HR1P heterodimer, we concluded that P21S8 and P21S10 could
both associate with HR1P to form a heterogeneous 6HB complex, thus
blocking MERS-CoV entry into the host cells (<xref rid="fig3" ref-type="fig">Figure <xref rid="fig3" ref-type="fig">3</xref>
</xref>
D and Supporting Information, <ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acs.jmedchem.7b01732/suppl_file/jm7b01732_si_001.pdf">Table S5</ext-link>
).</p>
</sec>
<sec id="sec3.5"><title>Pharmacokinetic Studies</title>
<p>The pharmacokinetic behavior
of P21S10, P21S8, and HR2P-M2 was comparatively evaluated in rats.
Sprague–Dawley rats (200 ± 10 g) were injected intravenously
with the three peptides (4 mg/kg). Seven blood samples were collected
sequentially over a 240 min period (three animals at each time point),
and the concentrations of the intact peptides in plasma were determined
by LC/MS/MS analysis. As shown in <xref rid="fig4" ref-type="fig">Figure <xref rid="fig4" ref-type="fig">4</xref>
</xref>
and <xref rid="tbl3" ref-type="other">Table <xref rid="tbl3" ref-type="other">3</xref>
</xref>
, although these three peptides exhibited similar in
vivo half-lives, the mean value of the maximum blood concentration
(<italic>C</italic>
<sub>max</sub>
) for P21S10 was 3.6–5.8-fold
greater than those for P21S8 and HR2P-M2. Meanwhile, we observed dramatic
differences among the areas under the plasma concentration–time
curve extrapolated to the last time point (AUC<sub>0–<italic>t</italic>
</sub>
), a measure of the total systemic exposure to a
drug, for the three compounds. The AUC<sub>0–<italic>t</italic>
</sub>
was 91.3 μg/(mL·h) for P21S10 as compared to 44.1
μg/(mL·h) for P21S8 and 3.36 μg/(mL·h) for HR2P-M2.
Consequently, P21S10 showed the lowest systemic clearance (0.041 (L/h)/kg),
which was approximately 25-fold less than that of HR2P-M2 (1.04 (L/h)/kg).
Thus, the current results indicate that P21S10 should be further developed
as a potential drug candidate on the basis of its high potency in
inhibiting infection of divergent MERS-CoV strains and its improved
pharmacokinetics compared with HR2P-M2.</p>
<fig id="fig4" position="float"><label>Figure 4</label>
<caption><p>Pharmacokinetic profiles
of P21S10, P21S8, and HR2P-M2 in plasma
following the administration of a single intravenous dose (4 mg/kg)
to Sprague–Dawley rats (<italic>n</italic>
= 3).</p>
</caption>
<graphic xlink:href="jm7b01732_0004" id="gr4" position="float"></graphic>
</fig>
<table-wrap id="tbl3" position="float"><label>Table 3</label>
<caption><title>Pharmacokinetic Parameters of P21S10,
P21S8, and HR2P-M2 in Rats Following a Single Dose iv Administration
Calculated by Noncompartmental Analysis by Using DAS, version 3.2.8<xref rid="t3fn1" ref-type="table-fn">a</xref>
</title>
</caption>
<table frame="hsides" rules="groups" border="0"><colgroup><col align="left"></col>
<col align="char" char="±"></col>
<col align="char" char="±"></col>
<col align="char" char="±"></col>
<col align="char" char="±"></col>
<col align="char" char="±"></col>
<col align="char" char="±"></col>
</colgroup>
<thead><tr><th style="border:none;" align="center">compd</th>
<th style="border:none;" align="center" char="±">AUC (0–<italic>t</italic>
) (μg/(mL·h))</th>
<th style="border:none;" align="center" char="±">MRT (0–<italic>t</italic>
) (h)</th>
<th style="border:none;" align="center" char="±"><italic>t</italic>
<sub>1/2</sub>
(h)</th>
<th style="border:none;" align="center" char="±">CL ((L/h)/kg)</th>
<th style="border:none;" align="center" char="±"><italic>C</italic>
<sub>max</sub>
(μg/mL)</th>
<th style="border:none;" align="center" char="±"><italic>V</italic>
<sub>d</sub>
(L/kg)</th>
</tr>
</thead>
<tbody><tr><td style="border:none;" align="left">P21S10</td>
<td style="border:none;" align="char" char="±">91.3 ± 9.8</td>
<td style="border:none;" align="char" char="±">1.08 ± 0.04</td>
<td style="border:none;" align="char" char="±">1.15 ± 0.15</td>
<td style="border:none;" align="char" char="±">0.041 ± 0.006</td>
<td style="border:none;" align="char" char="±">98.7 ± 6.5</td>
<td style="border:none;" align="char" char="±">0.066 ± 0.004</td>
</tr>
<tr><td style="border:none;" align="left">P21S8</td>
<td style="border:none;" align="char" char="±">44.1 ± 1.0</td>
<td style="border:none;" align="char" char="±">1.03 ± 0.04</td>
<td style="border:none;" align="char" char="±">1.32 ± 0.49</td>
<td style="border:none;" align="char" char="±">0.082 ± 0.004</td>
<td style="border:none;" align="char" char="±">61.8 ± 13.6</td>
<td style="border:none;" align="char" char="±">0.156 ± 0.052</td>
</tr>
<tr><td style="border:none;" align="left">HR2P-M2</td>
<td style="border:none;" align="char" char="±">3.35 ± 0.5</td>
<td style="border:none;" align="char" char="±">1.34 ± 0.14</td>
<td style="border:none;" align="char" char="±">1.54 ± 0.73</td>
<td style="border:none;" align="char" char="±">1.04 ± 0.26</td>
<td style="border:none;" align="char" char="±">17.0 ± 1.3</td>
<td style="border:none;" align="char" char="±">2.188 ± 0.603</td>
</tr>
</tbody>
</table>
<table-wrap-foot><fn id="t3fn1"><label>a</label>
<p>MRT, mean residence
time; CL, clearance; <italic>C</italic>
<sub>max</sub>
, maximum drug
concentration; <italic>V</italic>
<sub>d</sub>
, volume of distribution.</p>
</fn>
</table-wrap-foot>
</table-wrap>
</sec>
</sec>
<sec id="sec4"><title>Conclusions</title>
<p>We
provide the first example of all-hydrocarbon-stapled peptides
mimicking the native conformation of the C-terminal short α-helical
region within the MERS-CoV S protein. These stapled peptides were
able to block the formation of the hexameric coiled-coil fusion complex
and thus inhibit viral–host cell membrane fusion. Our data
showed that one of these stapled peptides, P21S10, not only preserves
the biologically active α-helical conformation, as found in
the MERS-CoV fusion machinery, but also has good potential to be developed
into anti-MERS therapeutics based on its promising antiviral activity,
effective native ligand-binding capability, and favorable pharmacokinetics.
Formation of MERS-CoV 6-HB fusion core between CHR and NHR is basically
the result of α-helix-mediated protein–protein interactions
wherein the CHR helix can be considered to have two distinct faces,
i.e., a buried hydrophobic surface and a solvent-exposed surface.<sup><xref ref-type="bibr" rid="ref29">29</xref>
</sup>
The established rules of thumb that underline
the sequence and structure in coiled coils reveal that stabilization
of individual helices will enhance the overall stability of the superhelical
protein structural motif.<sup><xref ref-type="bibr" rid="ref28">28</xref>
</sup>
Therefore,
strategies used for reinforcing the secondary structure of constituent
α-helices in a coiled coil such as introducing intrahelical
salt bridges at solvent accessible site and substitution of noncore
residues with helix-favoring amino acids have been successfully applied
to HIV fusion inhibitor design.<sup><xref ref-type="bibr" rid="ref36">36</xref>
</sup>
We envisioned
that these strategies would also be applicable to the further optimization
of our lead compounds found in this study. In addition, rather than
generating hydrophobic mutations in buried residues, for future design
one should also consider the place of polar amino acids at the interactive
site of stapled peptides, which could establish attractive interhelical
electrostatic interactions or hydrogen bonds, thus increasing their
aqueous solubility and target-binding affinity.<sup><xref ref-type="bibr" rid="ref37">37</xref>
,<xref ref-type="bibr" rid="ref38">38</xref>
</sup>
Overall, considering the universal 6HB fusion mechanism employed
by the class I enveloped viruses, our study establishes the paradigm
that hydrocarbon stapling may be an attractive strategy for developing
viral entry inhibitors designed to control viral epidemics through
recapitulating certain α-helical peptides of the viral fusion
core structures so that NHR/CHR coiled coil-mediated interactions
are inhibited.</p>
</sec>
<sec id="sec5"><title>Experimental Section</title>
<sec id="sec5.1"><title>General
Peptide Synthesis</title>
<p>Peptides were prepared using
a CEM Liberty automated microwave peptide synthesizer together with
the Fmoc solid-phase peptide synthesis protocol on Rink-Amide resin
with a loading capacity of 0.44 mmol/g. Briefly, the Fmoc protecting
group of the resin was removed with 20% piperidine/DMF (2 × 30
min). Natural amino acids were coupled using <italic>O</italic>
-benzotriazol-1-yl-<italic>N</italic>
,<italic>N</italic>
,<italic>N</italic>
′,<italic>N</italic>
′-tetramethyl-uronium hexafluorophosphate (3 equiv)
in DMF and diisopropylethylamine (DIEA) (6 equiv) in NMP as coupling
reagents, 3 equiv of <italic>N</italic>
<sup>α</sup>
-protected
amino acid, and 3 equiv of 1-hydroxy-benzotriazole. After each coupling,
or Fmoc removal, the resin was washed with DMF (5 × 1 min) and
DCM (3 × 1 min). For the assembly of olefin-containing amino
acids, a double coupling method was performed to achieve a complete
reaction, as described previously.<sup><xref ref-type="bibr" rid="ref39">39</xref>
</sup>
After
final deprotection, the peptide-bound resin was treated with acetic
acid anhydride–DIEA (1:1, v/v) (2 × 30 min). For synthesis
of the all-hydrocarbon-stapled peptides, the RCM reactions of the
protected peptides containing S5 ((<italic>S</italic>
)-2-(4-pentenyl)alanine)
or R5 ((<italic>R</italic>
)-2-(4-pentenyl)alanine) were performed
on-resin using the first-generation Grubbs catalyst. First, the reaction
bottle containing peptide-bound resin was kept under lucifugal conditions
by silver paper and then protected under nitrogen. Second, Grubbs
I catalyst (0.3 equiv) in dichloroethane was added to the resin, the
mixture was stirred for 6 h at room temperature, and then repeated
treatments with fresh catalyst were carried out for 6 h to obtain
more complete conversion. After the reaction solution was drained,
the resin was washed with DMF (2 × 1 min) and DCM (2 × 1
min). The resin cleavage was performed by treatment with a cleavage
cocktail containing 85% TFA, 5% thioanisole, 5% <italic>m</italic>
-cresol, and 5% water. After cleavage from resin, crude peptides
were precipitated using cold diethyl ether. The crude products were
purified by preparative reversed-phase (RP) HPLC, and the purity of
each compound was confirmed to be ≥95% by analytical RP-HPLC.
Such information is provided in the Supporting Information (<ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acs.jmedchem.7b01732/suppl_file/jm7b01732_si_001.pdf">Tables S6–S7</ext-link>
). The molecular weight of
the peptides was confirmed by Q-FT-ICR-MS (Apex Qe, Bruker, Germany).</p>
</sec>
<sec id="sec5.2"><title>Inhibition of MERS-CoV S-Protein-Mediated Cell–Cell Fusion
Assay</title>
<p>MERS-CoV S protein-mediated cell–cell fusion
was assessed, as described previously.<sup><xref ref-type="bibr" rid="ref40">40</xref>
,<xref ref-type="bibr" rid="ref41">41</xref>
</sup>
In brief,
293T cells expressing MERS-CoV S protein and enhanced GFP (EGFP) were
used as the effector cells (293T/MERS/EGFP), and Huh-7 cells expressing
the MERS-CoV receptor DPP4 were used as the target cells. 293T/EGFP
cells, which express only EGFP, were used as negative control cells.
Huh-7 cells were plated in a 96-well plate (5 × 10<sup>4</sup>
per well) and cultured at 37 °C for 5 h. Inhibitors at the
indicated concentrations were then added, followed by the addition
of 293T/MERS/EGFP cells or 293T/EGFP cells (1 × 10<sup>4</sup>
per well). After coculture at 37 °C for 4 h, the 293T/MERS/EGFP
cells and 293T/EGFP cells fused or unfused with Huh-7 cells were counted
under an inverted fluorescence microscope (Nikon Eclipse Ti–S).</p>
</sec>
<sec id="sec5.3"><title>Inhibition of Pseudotyped MERS-CoV Infection</title>
<p>MERS-CoV
pseudovirus was generated via cotransfection of 293T cells with the
plasmid pNL4-3.luc.RE as well as the pcDNA3.1-MERS-CoV-S plasmid,
as previously described.<sup><xref ref-type="bibr" rid="ref42">42</xref>
</sup>
Peptides at
graded concentrations were mixed with the pseudovirus and then incubated
for 1 h at room temperature. The mixture was added to Huh-7 or Calu-3
cells, fresh medium was added 12 h later, and the cells were incubated
for an additional 48 h at 37 °C. Fluorescence was determined
immediately using a luciferase kit (Promega) and an Ultra 384 luminometer
(Tecan) after the addition of luciferase substrate (Promega).</p>
</sec>
<sec id="sec5.4"><title>Cytotoxicity
Assessment</title>
<p>The cytotoxicity of compounds
on Huh-7 cells used for testing pseudotyped MERS-CoV infection was
measured using a cell counting kit (CCK-8, Dojindo, Kumamoto, Japan).
Briefly, 5 μL of test peptide at graded concentrations were
added to 100 μL of cells (1 × 10<sup>5</sup>
per well)
in wells of a 96-well plate incubated at 37 °C for 12 h before
the addition. After incubation at 37 °C for 48 h, 100 μL
of CCK-8 were added. The absorbance at 450 nm was measured 2 h later.</p>
</sec>
<sec id="sec5.5"><title>Aqueous Solubility Determination</title>
<p>Solubility was measured
by using an HPLC-UV method. Stapled peptides (∼2 mg) were added
to 1.5 mL Eppendorf tubes and either pH 7.4 phosphate buffer (100
μL) or double-distilled H<sub>2</sub>
O (ddH<sub>2</sub>
O) (100
μL) was added for dissolution with shaking for 24 h at 25 °C,
followed by centrifugation of the mixture at 10000 rpm for 15 min.
The saturated supernatant solution was filtered through a 0.45 μm
filter membrane and then transferred to other vials for analysis by
HPLC with UV detection. Each sample was assayed in triplicate. For
quantification, analytical RP-HPLC was used with a Zorbax Eclipse
XDB-C8 column (4.6 mm × 150 mm, 5 μm). Solvent A, 0.1%
TFA in H<sub>2</sub>
O; Solvent B, 0.1% TFA in 70% CH<sub>3</sub>
CN/H<sub>2</sub>
O; flow rate, 1 mL/min; gradient, 5–100% solvent B
in solvent A over 25 min. The aqueous concentration was determined
by comparison of the peak area of the saturated solution with a standard
curve plotted for the peak area versus known concentrations, which
was prepared by solutions of test compound in PBS or ddH<sub>2</sub>
O at 20, 10, 5, 2.5, 0.5, and 0.05 mg/mL.</p>
</sec>
<sec id="sec5.6"><title>CD Spectroscopy</title>
<p>Peptides were dissolved in PBS (50
mM, pH = 7.2) to a final concentration of 50 μM. Equimolar mixtures
of HR1P, and test peptides were incubated at 25 °C for 30 min
at a concentration of 50 μM. The CD spectra were obtained on
a Chirascan Plus spectropolarimeter (Applied Photophysics Ltd., Leatherhead,
Surrey, UK) at 4 °C. The measurement parameters were set up as
follows: wavelength, 190–260 nm; step resolution, 0.5 nm; speed,
20 nm/min. The CD data are shown as the mean residue ellipticity,
and the [θ]<sub>222</sub>
value of −33000° cm<sup>2</sup>
/dmol was taken to correspond to 100% α-helicity. Thermal
denaturation was monitored at 222 nm by implementation of a thermal
gradient of 2 °C/min from 10 to 90 °C.</p>
</sec>
<sec id="sec5.7"><title>N-PAGE</title>
<p>Stapled peptides and HR1P were dissolved in
50 mM PBS and ddH<sub>2</sub>
O at a concentration of 200 μM,
respectively. Each stapled peptide was incubated with HR1P at 25 °C
for 30 min, respectively. After the addition of Tris-glycine native
sample buffer (BioRad, Hercules, CA), the samples were loaded onto
an 18% Tris-glycine gel with a Tris-glycine sample buffer (pH 8.3).
Gel electrophoresis was carried out at a constant voltage of 120 V
at room temperature for 3 h, and then the gel was stained with Coomassie
Blue R250. The images were obtained by the ChampGel 6000 Imaging System
(SageCreation Ltd., Beijing, China).</p>
</sec>
<sec id="sec5.8"><title>SVA</title>
<p>SVA was performed
using a ProteomelabTMXL-A/XL-I
analytical ultracentrifuge (Beckman Coulter, Fullerton, CA) equipped
with a three-channel cell in an An-60 Ti rotor. All samples were prepared
at a final concentration of 250 μM in 50 mM PBS. The HR1P/P21S8
and HR1P/P21S10 mixtures were incubated at 25 °C for 30 min and
were initially scanned at 3000 rpm for 10 min. Data were obtained
at 60000 rpm at a wavelength of 280 nm and 20 °C for 7 h. Weight-averaged
molecular weights were calculated and fitted by processing with the
SEDFIT program.</p>
</sec>
<sec id="sec5.9"><title>Pharmacokinetic Assessments</title>
<p>Sprague–Dawley
rats
weighing 200 ± 10 g each were obtained from the Animal Center
of Beijing Institute of Pharmacology and Toxicology and were used
for pharmacokinetic assessments. Animals were treated in accordance
with the Animal Welfare Act and the “Guide for the Care and
Use of Laboratory Animals” (NIH Publication 86-23, revised
1985). Complete pharmacokinetic experimental procedures are provided
in the <ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acs.jmedchem.7b01732/suppl_file/jm7b01732_si_001.pdf">Supporting Information</ext-link>
.</p>
</sec>
</sec>
</body>
<back><notes id="notes1" notes-type="si"><title>Supporting Information Available</title>
<p>The Supporting
Information is
available free of charge on the <ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org">ACS Publications website</ext-link>
at DOI: <ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/abs/10.1021/acs.jmedchem.7b01732">10.1021/acs.jmedchem.7b01732</ext-link>
.<list id="silist" list-type="simple"><list-item><p>Inhibitory activity
of peptides on MERS-CoV infection
in Calu-3 cells; solubility of P21S8, P21S10, and P21S8Z; biophysical
properties and biological activity of uncyclized peptides; biophysical
properties of the stapled peptides; summary of the SVA results of
P21S8/HR1P and P21S10/HR1P complexes; HPLC methods used for the purification
and analysis of peptide compounds; stapled peptides as inhibitors
of MERS-CoV infection, pharmacokinetic experiments, as well as HPLC
purity and characterization data of compounds (<ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acs.jmedchem.7b01732/suppl_file/jm7b01732_si_001.pdf">PDF</ext-link>
)</p>
</list-item>
</list>
</p>
</notes>
<sec sec-type="supplementary-material"><title>Supplementary Material</title>
<supplementary-material content-type="local-data" id="sifile1"><media xlink:href="jm7b01732_si_001.pdf"><caption><p>jm7b01732_si_001.pdf</p>
</caption>
</media>
</supplementary-material>
</sec>
<notes notes-type="" id="notes3"><title>Author Contributions</title>
<p><sup>¶</sup>
Chao Wang,
Shuai Xia, and Peiyu Zhang contributed equally to this
work. Prof. Keliang Liu, Prof. Shibo Jiang, and Dr. Chao Wang conceived
and designed the study. Peiyu Zhang, Guangpeng Meng, and Yangli Tian
performed synthesis. Shuai Xia performed biological evaluation. Peiyu
Zhang performed biophysical analysis. Dr. Tianhong Zhang and Weicong
Wang analyzed the pharmacokinetics data. The manuscript was written
by Dr. Chao Wang.</p>
</notes>
<notes notes-type="COI-statement" id="NOTES-d155e1759-autogenerated"><p>The authors declare no
competing financial interest.</p>
</notes>
<ack><title>Acknowledgments</title>
<p>This research
was supported, in part, by grants
from the National Science Foundation of China (81573266 and 81590766)
and the National Key Research and Development Program of China (2016YFC1201000)</p>
</ack>
<glossary id="dl1"><def-list><title>Abbreviations Used</title>
<def-item><term>CHR</term>
<def><p>C-terminal heptad repeat</p>
</def>
</def-item>
<def-item><term>NHR</term>
<def><p>N-terminal heptad repeat</p>
</def>
</def-item>
<def-item><term>6HB</term>
<def><p>six-helix bundle</p>
</def>
</def-item>
<def-item><term>Da</term>
<def><p>Dalton</p>
</def>
</def-item>
<def-item><term>SVA</term>
<def><p>sedimentation velocity
analysis</p>
</def>
</def-item>
</def-list>
</glossary>
<ref-list><title>References</title>
<ref id="ref1"><mixed-citation publication-type="journal" id="cit1"><name><surname>Harrison</surname>
<given-names>S. C.</given-names>
</name>
<article-title>Viral membrane
fusion</article-title>
. <source>Nat. Struct. Mol. Biol.</source>
<year>2008</year>
, <volume>15</volume>
, <fpage>690</fpage>
–<lpage>698</lpage>
. <pub-id pub-id-type="doi">10.1038/nsmb.1456</pub-id>
.<pub-id pub-id-type="pmid">18596815</pub-id>
</mixed-citation>
</ref>
<ref id="ref2"><mixed-citation publication-type="journal" id="cit2"><name><surname>Dimitrov</surname>
<given-names>D. S.</given-names>
</name>
<article-title>Virus entry:
Molecular mechanisms and biomedical applications</article-title>
. <source>Nat. Rev. Microbiol.</source>
<year>2004</year>
, <volume>2</volume>
, <fpage>109</fpage>
–<lpage>122</lpage>
. <pub-id pub-id-type="doi">10.1038/nrmicro817</pub-id>
.<pub-id pub-id-type="pmid">15043007</pub-id>
</mixed-citation>
</ref>
<ref id="ref3"><mixed-citation publication-type="journal" id="cit3"><name><surname>Eckert</surname>
<given-names>D. M.</given-names>
</name>
; <name><surname>Kim</surname>
<given-names>P. S.</given-names>
</name>
<article-title>Mechanisms of viral membrane fusion and its inhibition</article-title>
. <source>Annu. Rev. Biochem.</source>
<year>2001</year>
, <volume>70</volume>
, <fpage>777</fpage>
–<lpage>810</lpage>
. <pub-id pub-id-type="doi">10.1146/annurev.biochem.70.1.777</pub-id>
.<pub-id pub-id-type="pmid">11395423</pub-id>
</mixed-citation>
</ref>
<ref id="ref4"><mixed-citation publication-type="journal" id="cit4"><name><surname>Vigant</surname>
<given-names>F.</given-names>
</name>
; <name><surname>Santos</surname>
<given-names>N. C.</given-names>
</name>
; <name><surname>Lee</surname>
<given-names>B.</given-names>
</name>
<article-title>Broad-spectrum
antivirals against
viral fusion</article-title>
. <source>Nat. Rev. Microbiol.</source>
<year>2015</year>
, <volume>13</volume>
, <fpage>426</fpage>
–<lpage>437</lpage>
. <pub-id pub-id-type="doi">10.1038/nrmicro3475</pub-id>
.<pub-id pub-id-type="pmid">26075364</pub-id>
</mixed-citation>
</ref>
<ref id="ref5"><mixed-citation publication-type="journal" id="cit5"><name><surname>Lu</surname>
<given-names>M.</given-names>
</name>
; <name><surname>Blacklow</surname>
<given-names>S. C.</given-names>
</name>
; <name><surname>Kim</surname>
<given-names>P. S. A.</given-names>
</name>
<article-title>Trimeric
structural domain of the
HIV-1 transmembrane glycoprotein</article-title>
. <source>Nat. Struct.
Mol. Biol.</source>
<year>1995</year>
, <volume>2</volume>
, <fpage>1075</fpage>
–<lpage>1082</lpage>
. <pub-id pub-id-type="doi">10.1038/nsb1295-1075</pub-id>
.</mixed-citation>
</ref>
<ref id="ref6"><mixed-citation publication-type="journal" id="cit6"><name><surname>Lai</surname>
<given-names>W.</given-names>
</name>
; <name><surname>Wang</surname>
<given-names>C.</given-names>
</name>
; <name><surname>Yu</surname>
<given-names>F.</given-names>
</name>
; <name><surname>Lu</surname>
<given-names>L.</given-names>
</name>
; <name><surname>Wang</surname>
<given-names>Q.</given-names>
</name>
; <name><surname>Jiang</surname>
<given-names>X.</given-names>
</name>
; <name><surname>Xu</surname>
<given-names>X.</given-names>
</name>
; <name><surname>Zhang</surname>
<given-names>T.</given-names>
</name>
; <name><surname>Wu</surname>
<given-names>S.</given-names>
</name>
; <name><surname>Zheng</surname>
<given-names>X.</given-names>
</name>
; <name><surname>Zhang</surname>
<given-names>Z.</given-names>
</name>
; <name><surname>Dong</surname>
<given-names>F.</given-names>
</name>
; <name><surname>Jiang</surname>
<given-names>S.</given-names>
</name>
; <name><surname>Liu</surname>
<given-names>K.</given-names>
</name>
<article-title>An effective strategy
for recapitulating N-terminal heptad repeat
trimers in enveloped virus surface glycoproteins for therapeutic applications</article-title>
. <source>Chem. Sci.</source>
<year>2016</year>
, <volume>7</volume>
, <fpage>2145</fpage>
–<lpage>2150</lpage>
. <pub-id pub-id-type="doi">10.1039/C5SC04046A</pub-id>
.<pub-id pub-id-type="pmid">29899942</pub-id>
</mixed-citation>
</ref>
<ref id="ref7"><mixed-citation publication-type="journal" id="cit7"><name><surname>Follis</surname>
<given-names>K. E.</given-names>
</name>
; <name><surname>York</surname>
<given-names>J.</given-names>
</name>
; <name><surname>Nunberg</surname>
<given-names>J. H.</given-names>
</name>
<article-title>Serine-scanning mutagenesis studies
of the C-terminal heptad repeats in the SARS coronavirus S glycoprotein
highlight the important role of the short helical region</article-title>
. <source>Virology</source>
<year>2005</year>
, <volume>341</volume>
, <fpage>122</fpage>
–<lpage>129</lpage>
. <pub-id pub-id-type="doi">10.1016/j.virol.2005.07.005</pub-id>
.<pub-id pub-id-type="pmid">16081124</pub-id>
</mixed-citation>
</ref>
<ref id="ref8"><mixed-citation publication-type="journal" id="cit8"><name><surname>Bullough</surname>
<given-names>P. A.</given-names>
</name>
; <name><surname>Hughson</surname>
<given-names>F. M.</given-names>
</name>
; <name><surname>Skehel</surname>
<given-names>J. J.</given-names>
</name>
; <name><surname>Wiley</surname>
<given-names>D. C.</given-names>
</name>
<article-title>Structure
of influenza
haemagglutinin at the pH of membrane fusion</article-title>
. <source>Nature</source>
<year>1994</year>
, <volume>371</volume>
, <fpage>37</fpage>
–<lpage>43</lpage>
. <pub-id pub-id-type="doi">10.1038/371037a0</pub-id>
.<pub-id pub-id-type="pmid">8072525</pub-id>
</mixed-citation>
</ref>
<ref id="ref9"><mixed-citation publication-type="journal" id="cit9"><name><surname>Liu</surname>
<given-names>S.</given-names>
</name>
; <name><surname>Xiao</surname>
<given-names>G.</given-names>
</name>
; <name><surname>Chen</surname>
<given-names>Y.</given-names>
</name>
; <name><surname>He</surname>
<given-names>Y. X.</given-names>
</name>
; <name><surname>Niu</surname>
<given-names>J. K.</given-names>
</name>
; <name><surname>Escalante</surname>
<given-names>C. R.</given-names>
</name>
; <name><surname>Xiong</surname>
<given-names>H.</given-names>
</name>
; <name><surname>Farmar</surname>
<given-names>J.</given-names>
</name>
; <name><surname>Debnath</surname>
<given-names>A. K.</given-names>
</name>
; <name><surname>Tien</surname>
<given-names>P.</given-names>
</name>
; <name><surname>Jiang</surname>
<given-names>S.</given-names>
</name>
<article-title>Interaction between heptad repeat
1 and 2 regions in spike protein of SARS-associated coronavirus: implications
for virus fusogenic mechanism and identification of fusion inhibitors</article-title>
. <source>Lancet</source>
<year>2004</year>
, <volume>363</volume>
, <fpage>938</fpage>
–<lpage>947</lpage>
. <pub-id pub-id-type="doi">10.1016/S0140-6736(04)15788-7</pub-id>
.<pub-id pub-id-type="pmid">15043961</pub-id>
</mixed-citation>
</ref>
<ref id="ref10"><mixed-citation publication-type="journal" id="cit10"><name><surname>Miller</surname>
<given-names>E. H.</given-names>
</name>
; <name><surname>Harrison</surname>
<given-names>J. S.</given-names>
</name>
; <name><surname>Radoshitzky</surname>
<given-names>S. R.</given-names>
</name>
; <name><surname>Higgins</surname>
<given-names>C. D.</given-names>
</name>
; <name><surname>Chi</surname>
<given-names>X.</given-names>
</name>
; <name><surname>Dong</surname>
<given-names>L.</given-names>
</name>
; <name><surname>Kuhn</surname>
<given-names>J. H.</given-names>
</name>
; <name><surname>Bavari</surname>
<given-names>S.</given-names>
</name>
; <name><surname>Lai</surname>
<given-names>J.</given-names>
</name>
; <name><surname>Chandran</surname>
<given-names>K.</given-names>
</name>
<article-title>Inhibition of Ebola
virus entry by a C-peptide targeted
to endosomes</article-title>
. <source>J. Biol. Chem.</source>
<year>2011</year>
, <volume>286</volume>
, <fpage>15854</fpage>
–<lpage>15861</lpage>
. <pub-id pub-id-type="doi">10.1074/jbc.M110.207084</pub-id>
.<pub-id pub-id-type="pmid">21454542</pub-id>
</mixed-citation>
</ref>
<ref id="ref11"><mixed-citation publication-type="journal" id="cit11"><name><surname>Lu</surname>
<given-names>L.</given-names>
</name>
; <name><surname>Liu</surname>
<given-names>Q.</given-names>
</name>
; <name><surname>Zhu</surname>
<given-names>Y.</given-names>
</name>
; <name><surname>Chan</surname>
<given-names>K.</given-names>
</name>
; <name><surname>Qin</surname>
<given-names>L.</given-names>
</name>
; <name><surname>Li</surname>
<given-names>Y.</given-names>
</name>
; <name><surname>Wang</surname>
<given-names>Q.</given-names>
</name>
; <name><surname>Chan</surname>
<given-names>J. F.</given-names>
</name>
; <name><surname>Du</surname>
<given-names>L.</given-names>
</name>
; <name><surname>Yu</surname>
<given-names>F.</given-names>
</name>
; <name><surname>Ma</surname>
<given-names>C.</given-names>
</name>
; <name><surname>Ye</surname>
<given-names>S.</given-names>
</name>
; <name><surname>Yuen</surname>
<given-names>K.</given-names>
</name>
; <name><surname>Zhang</surname>
<given-names>R.</given-names>
</name>
; <name><surname>Jiang</surname>
<given-names>S.</given-names>
</name>
<article-title>Structure-based
discovery of Middle East respiratory syndrome coronavirus fusion inhibitor</article-title>
. <source>Nat. Commun.</source>
<year>2014</year>
, <volume>5</volume>
, <fpage>3067</fpage>
<pub-id pub-id-type="doi">10.1038/ncomms4067</pub-id>
.<pub-id pub-id-type="pmid">24473083</pub-id>
</mixed-citation>
</ref>
<ref id="ref12"><mixed-citation publication-type="journal" id="cit12"><name><surname>Berkhout</surname>
<given-names>B.</given-names>
</name>
; <name><surname>Eggink</surname>
<given-names>D.</given-names>
</name>
; <name><surname>Sanders</surname>
<given-names>R. W.</given-names>
</name>
<article-title>Is there
a future for antiviral fusion
inhibitors?</article-title>
. <source>Curr. Opin. Virol.</source>
<year>2012</year>
, <volume>2</volume>
, <fpage>50</fpage>
–<lpage>59</lpage>
. <pub-id pub-id-type="doi">10.1016/j.coviro.2012.01.002</pub-id>
.<pub-id pub-id-type="pmid">22440966</pub-id>
</mixed-citation>
</ref>
<ref id="ref13"><mixed-citation publication-type="journal" id="cit13"><name><surname>Azzarito</surname>
<given-names>V.</given-names>
</name>
; <name><surname>Long</surname>
<given-names>K.</given-names>
</name>
; <name><surname>Murphy</surname>
<given-names>N. S.</given-names>
</name>
; <name><surname>Wilson</surname>
<given-names>A. J.</given-names>
</name>
<article-title>Inhibition of alpha-helix-mediated
protein-protein interactions using designed molecules</article-title>
. <source>Nat. Chem.</source>
<year>2013</year>
, <volume>5</volume>
, <fpage>161</fpage>
–<lpage>173</lpage>
. <pub-id pub-id-type="doi">10.1038/nchem.1568</pub-id>
.<pub-id pub-id-type="pmid">23422557</pub-id>
</mixed-citation>
</ref>
<ref id="ref14"><mixed-citation publication-type="journal" id="cit14"><name><surname>Cai</surname>
<given-names>L.</given-names>
</name>
; <name><surname>Jiang</surname>
<given-names>S.</given-names>
</name>
<article-title>Development of peptide and small-molecule HIV-1 fusion inhibitors
that target gp41</article-title>
. <source>ChemMedChem</source>
<year>2010</year>
, <volume>5</volume>
, <fpage>1813</fpage>
–<lpage>1824</lpage>
. <pub-id pub-id-type="doi">10.1002/cmdc.201000289</pub-id>
.<pub-id pub-id-type="pmid">20845360</pub-id>
</mixed-citation>
</ref>
<ref id="ref15"><mixed-citation publication-type="journal" id="cit15"><name><surname>Pan</surname>
<given-names>C.</given-names>
</name>
; <name><surname>Liu</surname>
<given-names>S.</given-names>
</name>
; <name><surname>Jiang</surname>
<given-names>S.</given-names>
</name>
<article-title>HIV-1 gp41
fusion intermediate: a target for HIV therapeutics</article-title>
. <source>J. Formosan Med. Assoc.</source>
<year>2010</year>
, <volume>109</volume>
, <fpage>94</fpage>
–<lpage>105</lpage>
. <pub-id pub-id-type="doi">10.1016/S0929-6646(10)60029-0</pub-id>
.<pub-id pub-id-type="pmid">20206833</pub-id>
</mixed-citation>
</ref>
<ref id="ref16"><mixed-citation publication-type="journal" id="cit16"><name><surname>Gao</surname>
<given-names>J.</given-names>
</name>
; <name><surname>Lu</surname>
<given-names>G.</given-names>
</name>
; <name><surname>Qi</surname>
<given-names>J.</given-names>
</name>
; <name><surname>Li</surname>
<given-names>Y.</given-names>
</name>
; <name><surname>Wu</surname>
<given-names>Y.</given-names>
</name>
; <name><surname>Deng</surname>
<given-names>Y.</given-names>
</name>
; <name><surname>Geng</surname>
<given-names>H.</given-names>
</name>
; <name><surname>Li</surname>
<given-names>H.</given-names>
</name>
; <name><surname>Wang</surname>
<given-names>Q.</given-names>
</name>
; <name><surname>Xiao</surname>
<given-names>H.</given-names>
</name>
; <name><surname>Tan</surname>
<given-names>W.</given-names>
</name>
; <name><surname>Yan</surname>
<given-names>J.</given-names>
</name>
; <name><surname>Gao</surname>
<given-names>G.</given-names>
</name>
<article-title>Structure
of the fusion core and
inhibition of fusion by a heptad repeat peptide derived from the S
protein of Middle East respiratory syndrome coronavirus</article-title>
. <source>J. Virol.</source>
<year>2013</year>
, <volume>87</volume>
, <fpage>13134</fpage>
–<lpage>13140</lpage>
. <pub-id pub-id-type="doi">10.1128/JVI.02433-13</pub-id>
.<pub-id pub-id-type="pmid">24067982</pub-id>
</mixed-citation>
</ref>
<ref id="ref17"><mixed-citation publication-type="journal" id="cit17"><name><surname>Lau</surname>
<given-names>Y.</given-names>
</name>
; <name><surname>De Andrade</surname>
<given-names>P.</given-names>
</name>
; <name><surname>Wu</surname>
<given-names>Y.</given-names>
</name>
; <name><surname>Spring</surname>
<given-names>D. R.</given-names>
</name>
<article-title>Peptide stapling
techniques based on different macrocyclisation chemistries</article-title>
. <source>Chem. Soc. Rev.</source>
<year>2015</year>
, <volume>44</volume>
, <fpage>91</fpage>
–<lpage>102</lpage>
. <pub-id pub-id-type="doi">10.1039/C4CS00246F</pub-id>
.<pub-id pub-id-type="pmid">25199043</pub-id>
</mixed-citation>
</ref>
<ref id="ref18"><mixed-citation publication-type="journal" id="cit18"><name><surname>Walensky</surname>
<given-names>L. D.</given-names>
</name>
; <name><surname>Bird</surname>
<given-names>G. H.</given-names>
</name>
<article-title>Hydrocarbon-stapled peptides: principles, practice,
and progress</article-title>
. <source>J. Med. Chem.</source>
<year>2014</year>
, <volume>57</volume>
, <fpage>6275</fpage>
–<lpage>6288</lpage>
. <pub-id pub-id-type="doi">10.1021/jm4011675</pub-id>
.<pub-id pub-id-type="pmid">24601557</pub-id>
</mixed-citation>
</ref>
<ref id="ref19"><mixed-citation publication-type="journal" id="cit19"><name><surname>Tan</surname>
<given-names>Y.</given-names>
</name>
; <name><surname>Lane</surname>
<given-names>D. P.</given-names>
</name>
; <name><surname>Verma</surname>
<given-names>C. S.</given-names>
</name>
<article-title>Stapled
peptide design: principles
and roles of computation</article-title>
. <source>Drug Discovery Today</source>
<year>2016</year>
, <volume>21</volume>
, <fpage>1642</fpage>
–<lpage>1653</lpage>
. <pub-id pub-id-type="doi">10.1016/j.drudis.2016.06.012</pub-id>
.<pub-id pub-id-type="pmid">27326912</pub-id>
</mixed-citation>
</ref>
<ref id="ref20"><mixed-citation publication-type="journal" id="cit20"><name><surname>Kawamoto</surname>
<given-names>S. A.</given-names>
</name>
; <name><surname>Coleska</surname>
<given-names>A.</given-names>
</name>
; <name><surname>Ran</surname>
<given-names>X.</given-names>
</name>
; <name><surname>Yi</surname>
<given-names>H.</given-names>
</name>
; <name><surname>Yang</surname>
<given-names>C.</given-names>
</name>
; <name><surname>Wang</surname>
<given-names>S.</given-names>
</name>
<article-title>Design of triazole-stapled BCL9 alpha-helical
peptides to target
the beta-Catenin/B-Cell CLL/lymphoma 9 (BCL9) protein-protein interaction</article-title>
. <source>J. Med. Chem.</source>
<year>2012</year>
, <volume>55</volume>
, <fpage>1137</fpage>
–<lpage>1146</lpage>
. <pub-id pub-id-type="doi">10.1021/jm201125d</pub-id>
.<pub-id pub-id-type="pmid">22196480</pub-id>
</mixed-citation>
</ref>
<ref id="ref21"><mixed-citation publication-type="journal" id="cit21"><name><surname>Hojo</surname>
<given-names>K.</given-names>
</name>
; <name><surname>Hossain</surname>
<given-names>M. A.</given-names>
</name>
; <name><surname>Tailhades</surname>
<given-names>J.</given-names>
</name>
; <name><surname>Shabanpoor</surname>
<given-names>F.</given-names>
</name>
; <name><surname>Wong</surname>
<given-names>L. L.L.</given-names>
</name>
; <name><surname>Ong-Palsson</surname>
<given-names>E. E. K.</given-names>
</name>
; <name><surname>Kastman</surname>
<given-names>H. E.</given-names>
</name>
; <name><surname>Ma</surname>
<given-names>S.</given-names>
</name>
; <name><surname>Gundlach</surname>
<given-names>A. L.</given-names>
</name>
; <name><surname>Rosengren</surname>
<given-names>K. J.</given-names>
</name>
; <name><surname>Wade</surname>
<given-names>J. D.</given-names>
</name>
; <name><surname>Bathgate</surname>
<given-names>R. A. D.</given-names>
</name>
<article-title>Development
of a single-chain peptide agonist of the relaxin-3 receptor using
hydrocarbon stapling</article-title>
. <source>J. Med. Chem.</source>
<year>2016</year>
, <volume>59</volume>
, <fpage>7445</fpage>
–<lpage>7456</lpage>
. <pub-id pub-id-type="doi">10.1021/acs.jmedchem.6b00265</pub-id>
.<pub-id pub-id-type="pmid">27464307</pub-id>
</mixed-citation>
</ref>
<ref id="ref22"><mixed-citation publication-type="journal" id="cit22"><name><surname>Frank</surname>
<given-names>A. O.</given-names>
</name>
; <name><surname>Vangamudi</surname>
<given-names>B.</given-names>
</name>
; <name><surname>Feldkamp</surname>
<given-names>M. D.</given-names>
</name>
; <name><surname>Souza-Fagundes</surname>
<given-names>E. M.</given-names>
</name>
; <name><surname>Luzwick</surname>
<given-names>J. W.</given-names>
</name>
; <name><surname>Cortez</surname>
<given-names>D.</given-names>
</name>
; <name><surname>Olejniczak</surname>
<given-names>E. T.</given-names>
</name>
; <name><surname>Waterson</surname>
<given-names>A. G.</given-names>
</name>
; <name><surname>Rossanese</surname>
<given-names>O. W.</given-names>
</name>
; <name><surname>Chazin</surname>
<given-names>W. J.</given-names>
</name>
; <name><surname>Fesik</surname>
<given-names>S. W.</given-names>
</name>
<article-title>Discovery
of a potent stapled helix peptide that binds to the 70N domain of
replication protein A</article-title>
. <source>J. Med. Chem.</source>
<year>2014</year>
, <volume>57</volume>
, <fpage>2455</fpage>
–<lpage>2461</lpage>
. <pub-id pub-id-type="doi">10.1021/jm401730y</pub-id>
.<pub-id pub-id-type="pmid">24491171</pub-id>
</mixed-citation>
</ref>
<ref id="ref23"><mixed-citation publication-type="journal" id="cit23"><name><surname>Hilinski</surname>
<given-names>G. J.</given-names>
</name>
; <name><surname>Kim</surname>
<given-names>Y. W.</given-names>
</name>
; <name><surname>Hong</surname>
<given-names>J.</given-names>
</name>
; <name><surname>Kutchukian</surname>
<given-names>P. S.</given-names>
</name>
; <name><surname>Crenshaw</surname>
<given-names>C. M.</given-names>
</name>
; <name><surname>Berkovitch</surname>
<given-names>S. S.</given-names>
</name>
; <name><surname>Chang</surname>
<given-names>A.</given-names>
</name>
; <name><surname>Ham</surname>
<given-names>S.</given-names>
</name>
; <name><surname>Verdine</surname>
<given-names>G. L.</given-names>
</name>
<article-title>Stitched alpha-helical
peptides via bis ring-closing
metathesis</article-title>
. <source>J. Am. Chem. Soc.</source>
<year>2014</year>
, <volume>136</volume>
, <fpage>12314</fpage>
–<lpage>12322</lpage>
. <pub-id pub-id-type="doi">10.1021/ja505141j</pub-id>
.<pub-id pub-id-type="pmid">25105213</pub-id>
</mixed-citation>
</ref>
<ref id="ref24"><mixed-citation publication-type="journal" id="cit24"><name><surname>Chang</surname>
<given-names>Y.</given-names>
</name>
; <name><surname>Graves</surname>
<given-names>B.</given-names>
</name>
; <name><surname>Guerlavais</surname>
<given-names>V.</given-names>
</name>
; <name><surname>Tovar</surname>
<given-names>C.</given-names>
</name>
; <name><surname>Packman</surname>
<given-names>K.</given-names>
</name>
; <name><surname>To</surname>
<given-names>K. H.</given-names>
</name>
; <name><surname>Olson</surname>
<given-names>K. A.</given-names>
</name>
; <name><surname>Kesavan</surname>
<given-names>K.</given-names>
</name>
; <name><surname>Gangurde</surname>
<given-names>P.</given-names>
</name>
; <name><surname>Mukherjee</surname>
<given-names>A.</given-names>
</name>
; <name><surname>Baker</surname>
<given-names>T.</given-names>
</name>
; <name><surname>Darlak</surname>
<given-names>K.</given-names>
</name>
; <name><surname>Elkin</surname>
<given-names>C.</given-names>
</name>
; <name><surname>Filipovic</surname>
<given-names>Z.</given-names>
</name>
; <name><surname>Qureshi</surname>
<given-names>F. Z.</given-names>
</name>
; <name><surname>Cai</surname>
<given-names>H.</given-names>
</name>
; <name><surname>Berry</surname>
<given-names>P.</given-names>
</name>
; <name><surname>Feyfant</surname>
<given-names>E.</given-names>
</name>
; <name><surname>Shi</surname>
<given-names>X. E.</given-names>
</name>
; <name><surname>Horstick</surname>
<given-names>J.</given-names>
</name>
; <name><surname>Annis</surname>
<given-names>D. A.</given-names>
</name>
; <name><surname>Manning</surname>
<given-names>A. M.</given-names>
</name>
; <name><surname>Fotouhi</surname>
<given-names>N.</given-names>
</name>
; <name><surname>Nash</surname>
<given-names>H.</given-names>
</name>
; <name><surname>Vassilev</surname>
<given-names>L. T.</given-names>
</name>
; <name><surname>Sawyer</surname>
<given-names>T. K.</given-names>
</name>
<article-title>Stapled
alpha-helical peptide drug development: a potent
dual inhibitor of MDM2 and MDMX for p53-dependent cancer therapy</article-title>
. <source>Proc. Natl. Acad. Sci. U. S. A.</source>
<year>2013</year>
, <volume>110</volume>
, <fpage>E3445</fpage>
–<lpage>54</lpage>
. <pub-id pub-id-type="doi">10.1073/pnas.1303002110</pub-id>
.<pub-id pub-id-type="pmid">23946421</pub-id>
</mixed-citation>
</ref>
<ref id="ref25"><mixed-citation publication-type="journal" id="cit25"><name><surname>Wang</surname>
<given-names>C.</given-names>
</name>
; <name><surname>Shi</surname>
<given-names>W.</given-names>
</name>
; <name><surname>Cai</surname>
<given-names>L.</given-names>
</name>
; <name><surname>Lu</surname>
<given-names>L.</given-names>
</name>
; <name><surname>Wang</surname>
<given-names>Q.</given-names>
</name>
; <name><surname>Zhang</surname>
<given-names>T.</given-names>
</name>
; <name><surname>Li</surname>
<given-names>J.</given-names>
</name>
; <name><surname>Zhang</surname>
<given-names>Z.</given-names>
</name>
; <name><surname>Wang</surname>
<given-names>K.</given-names>
</name>
; <name><surname>Xu</surname>
<given-names>L.</given-names>
</name>
; <name><surname>Jiang</surname>
<given-names>X.</given-names>
</name>
; <name><surname>Jiang</surname>
<given-names>S.</given-names>
</name>
; <name><surname>Liu</surname>
<given-names>K.</given-names>
</name>
<article-title>Design, synthesis, and biological
evaluation of highly potent small molecule-peptide conjugates as new
HIV-1 fusion inhibitors</article-title>
. <source>J. Med. Chem.</source>
<year>2013</year>
, <volume>56</volume>
, <fpage>2527</fpage>
–<lpage>2539</lpage>
. <pub-id pub-id-type="doi">10.1021/jm3018964</pub-id>
.<pub-id pub-id-type="pmid">23458727</pub-id>
</mixed-citation>
</ref>
<ref id="ref26"><mixed-citation publication-type="journal" id="cit26"><name><surname>Wang</surname>
<given-names>C.</given-names>
</name>
; <name><surname>Shi</surname>
<given-names>W.</given-names>
</name>
; <name><surname>Cai</surname>
<given-names>L.</given-names>
</name>
; <name><surname>Lu</surname>
<given-names>L.</given-names>
</name>
; <name><surname>Yu</surname>
<given-names>F.</given-names>
</name>
; <name><surname>Wang</surname>
<given-names>Q.</given-names>
</name>
; <name><surname>Jiang</surname>
<given-names>X.</given-names>
</name>
; <name><surname>Xu</surname>
<given-names>X.</given-names>
</name>
; <name><surname>Wang</surname>
<given-names>K.</given-names>
</name>
; <name><surname>Xu</surname>
<given-names>L.</given-names>
</name>
; <name><surname>Jiang</surname>
<given-names>S.</given-names>
</name>
; <name><surname>Liu</surname>
<given-names>K.</given-names>
</name>
<article-title>Artificial
peptides conjugated with
cholesterol and pocket-specific small molecules potently inhibit infection
by laboratory-adapted and primary HIV-1 isolates and enfuvirtide-resistant
HIV-1 strains</article-title>
. <source>J. Antimicrob. Chemother.</source>
<year>2014</year>
, <volume>69</volume>
, <fpage>1537</fpage>
–<lpage>1545</lpage>
. <pub-id pub-id-type="doi">10.1093/jac/dku010</pub-id>
.<pub-id pub-id-type="pmid">24500189</pub-id>
</mixed-citation>
</ref>
<ref id="ref27"><mixed-citation publication-type="journal" id="cit27"><name><surname>Wuo</surname>
<given-names>M.</given-names>
</name>
; <name><surname>Mahon</surname>
<given-names>A. B.</given-names>
</name>
; <name><surname>Arora</surname>
<given-names>P. S.</given-names>
</name>
<article-title>An effective
strategy for stabilizing
minimal coiled coil mimetics</article-title>
. <source>J. Am. Chem. Soc.</source>
<year>2015</year>
, <volume>137</volume>
, <fpage>11618</fpage>
–<lpage>11621</lpage>
. <pub-id pub-id-type="doi">10.1021/jacs.5b05525</pub-id>
.<pub-id pub-id-type="pmid">26340721</pub-id>
</mixed-citation>
</ref>
<ref id="ref28"><mixed-citation publication-type="journal" id="cit28"><name><surname>Apostolovic</surname>
<given-names>B.</given-names>
</name>
; <name><surname>Danial</surname>
<given-names>M.</given-names>
</name>
; <name><surname>Klok</surname>
<given-names>H. A.</given-names>
</name>
<article-title>Coiled
coils: attractive protein
folding motifs for the fabrication of self-assembled, responsive and
bioactive materials</article-title>
. <source>Chem. Soc. Rev.</source>
<year>2010</year>
, <volume>39</volume>
, <fpage>3541</fpage>
–<lpage>3575</lpage>
. <pub-id pub-id-type="doi">10.1039/b914339b</pub-id>
.<pub-id pub-id-type="pmid">20676430</pub-id>
</mixed-citation>
</ref>
<ref id="ref29"><mixed-citation publication-type="journal" id="cit29"><name><surname>Zheng</surname>
<given-names>B.</given-names>
</name>
; <name><surname>Wang</surname>
<given-names>K.</given-names>
</name>
; <name><surname>Lu</surname>
<given-names>L.</given-names>
</name>
; <name><surname>Yu</surname>
<given-names>F.</given-names>
</name>
; <name><surname>Cheng</surname>
<given-names>M.</given-names>
</name>
; <name><surname>Jiang</surname>
<given-names>S.</given-names>
</name>
; <name><surname>Liu</surname>
<given-names>K.</given-names>
</name>
; <name><surname>Cai</surname>
<given-names>L.</given-names>
</name>
<article-title>Hydrophobic
mutations
in buried polar residues enhance HIV-1 gp41 N-terminal heptad repeat-C-terminal
heptad repeat interactions and C-peptides’ anti-HIV activity</article-title>
. <source>AIDS</source>
<year>2014</year>
, <volume>28</volume>
, <fpage>1251</fpage>
–<lpage>1260</lpage>
. <pub-id pub-id-type="doi">10.1097/QAD.0000000000000255</pub-id>
.<pub-id pub-id-type="pmid">24625369</pub-id>
</mixed-citation>
</ref>
<ref id="ref30"><mixed-citation publication-type="journal" id="cit30"><name><surname>Qi</surname>
<given-names>Z.</given-names>
</name>
; <name><surname>Shi</surname>
<given-names>W.</given-names>
</name>
; <name><surname>Xue</surname>
<given-names>N.</given-names>
</name>
; <name><surname>Pan</surname>
<given-names>C.</given-names>
</name>
; <name><surname>Jing</surname>
<given-names>W.</given-names>
</name>
; <name><surname>Liu</surname>
<given-names>K.</given-names>
</name>
; <name><surname>Jiang</surname>
<given-names>S.</given-names>
</name>
<article-title>Rationally
designed anti-HIV peptides containing multifunctional
domains as molecule probes for studying the mechanisms of action of
the first and second generation HIV fusion inhibitors</article-title>
. <source>J. Biol. Chem.</source>
<year>2008</year>
, <volume>283</volume>
, <fpage>30376</fpage>
–<lpage>30384</lpage>
. <pub-id pub-id-type="doi">10.1074/jbc.M804672200</pub-id>
.<pub-id pub-id-type="pmid">18662985</pub-id>
</mixed-citation>
</ref>
<ref id="ref31"><mixed-citation publication-type="journal" id="cit31"><name><surname>Anderhuber</surname>
<given-names>N.</given-names>
</name>
; <name><surname>Fladischer</surname>
<given-names>P.</given-names>
</name>
; <name><surname>Gruber-Khadjawi</surname>
<given-names>M.</given-names>
</name>
; <name><surname>Mairhofer</surname>
<given-names>J.</given-names>
</name>
; <name><surname>Striedner</surname>
<given-names>G.</given-names>
</name>
; <name><surname>Wiltschi</surname>
<given-names>B.</given-names>
</name>
<article-title>High-level biosynthesis of norleucine
in E.coli for the economic labeling of proteins</article-title>
. <source>J. Biotechnol.</source>
<year>2016</year>
, <volume>235</volume>
, <fpage>100</fpage>
–<lpage>111</lpage>
. <pub-id pub-id-type="doi">10.1016/j.jbiotec.2016.04.033</pub-id>
.<pub-id pub-id-type="pmid">27107466</pub-id>
</mixed-citation>
</ref>
<ref id="ref32"><mixed-citation publication-type="journal" id="cit32"><name><surname>Fletcher</surname>
<given-names>J. M.</given-names>
</name>
; <name><surname>Boyle</surname>
<given-names>A. L.</given-names>
</name>
; <name><surname>Bruning</surname>
<given-names>M.</given-names>
</name>
; <name><surname>Bartlett</surname>
<given-names>G. J.</given-names>
</name>
; <name><surname>Vincent</surname>
<given-names>T. L.</given-names>
</name>
; <name><surname>Zaccai</surname>
<given-names>N. R.</given-names>
</name>
; <name><surname>Armstrong</surname>
<given-names>C. T.</given-names>
</name>
; <name><surname>Bromley</surname>
<given-names>E. H. C.</given-names>
</name>
; <name><surname>Booth</surname>
<given-names>P. J.</given-names>
</name>
; <name><surname>Brady</surname>
<given-names>R. L.</given-names>
</name>
; <name><surname>Thomson</surname>
<given-names>A. R.</given-names>
</name>
; <name><surname>Woolfson</surname>
<given-names>D. N.</given-names>
</name>
<article-title>A basis set of de
novo coiled-coil peptide oligomers for rational protein design and
synthetic biology</article-title>
. <source>ACS Synth. Biol.</source>
<year>2012</year>
, <volume>1</volume>
, <fpage>240</fpage>
–<lpage>250</lpage>
. <pub-id pub-id-type="doi">10.1021/sb300028q</pub-id>
.<pub-id pub-id-type="pmid">23651206</pub-id>
</mixed-citation>
</ref>
<ref id="ref33"><mixed-citation publication-type="journal" id="cit33"><name><surname>Cotten</surname>
<given-names>M.</given-names>
</name>
; <name><surname>Watson</surname>
<given-names>S. J.</given-names>
</name>
; <name><surname>Zumla</surname>
<given-names>A. I.</given-names>
</name>
; <name><surname>Makhdoom</surname>
<given-names>H. Q.</given-names>
</name>
; <name><surname>Palser</surname>
<given-names>A. L.</given-names>
</name>
; <name><surname>Ong</surname>
<given-names>S. H.</given-names>
</name>
; <name><surname>Al Rabeeah</surname>
<given-names>A. A.</given-names>
</name>
; <name><surname>Alhakeem</surname>
<given-names>R. F.</given-names>
</name>
; <name><surname>Assiri</surname>
<given-names>A.</given-names>
</name>
; <name><surname>Al-Tawfiq</surname>
<given-names>J. A.</given-names>
</name>
; <name><surname>Albarrak</surname>
<given-names>A.</given-names>
</name>
; <name><surname>Barry</surname>
<given-names>M.</given-names>
</name>
; <name><surname>Shibl</surname>
<given-names>A.</given-names>
</name>
; <name><surname>Alrabiah</surname>
<given-names>F. A.</given-names>
</name>
; <name><surname>Hajjar</surname>
<given-names>S.</given-names>
</name>
; <name><surname>Balkhy</surname>
<given-names>H. H.</given-names>
</name>
; <name><surname>Flemban</surname>
<given-names>H.</given-names>
</name>
; <name><surname>Rambaut</surname>
<given-names>A.</given-names>
</name>
; <name><surname>Kellam</surname>
<given-names>P.</given-names>
</name>
; <name><surname>Memish</surname>
<given-names>Z. A.</given-names>
</name>
<article-title>Spread, circulation,
and evolution of the Middle East respiratory syndrome coronavirus</article-title>
. <source>mBio</source>
<year>2014</year>
, <volume>5</volume>
, <fpage>e01062-13</fpage>
–<lpage>13</lpage>
. <pub-id pub-id-type="doi">10.1128/mBio.01062-13</pub-id>
.<pub-id pub-id-type="pmid">24549846</pub-id>
</mixed-citation>
</ref>
<ref id="ref34"><mixed-citation publication-type="journal" id="cit34"><name><surname>Lawless</surname>
<given-names>M. K.</given-names>
</name>
; <name><surname>Barney</surname>
<given-names>S.</given-names>
</name>
; <name><surname>Guthrie</surname>
<given-names>K. I.</given-names>
</name>
; <name><surname>Bucy</surname>
<given-names>T. B.</given-names>
</name>
; <name><surname>Petteway</surname>
<given-names>S. R.</given-names>
<suffix>Jr.</suffix>
</name>
; <name><surname>Merutka</surname>
<given-names>G.</given-names>
</name>
<article-title>HIV-1 membrane fusion mechanism:
structural studies of the interactions between biologically-active
peptides from gp41</article-title>
. <source>Biochemistry</source>
<year>1996</year>
, <volume>35</volume>
, <fpage>13697</fpage>
–<lpage>13708</lpage>
. <pub-id pub-id-type="doi">10.1021/bi9606962</pub-id>
.<pub-id pub-id-type="pmid">8885850</pub-id>
</mixed-citation>
</ref>
<ref id="ref35"><mixed-citation publication-type="journal" id="cit35"><name><surname>Cai</surname>
<given-names>L. F.</given-names>
</name>
; <name><surname>Balogh</surname>
<given-names>E.</given-names>
</name>
; <name><surname>Gochin</surname>
<given-names>M.</given-names>
</name>
<article-title>Stable extended
human immunodeficiency
virus type 1 gp41 coiled coil as an effective target in an assay for
high-affinity fusion inhibitors</article-title>
. <source>Antimicrob.
Agents Chemother.</source>
<year>2009</year>
, <volume>53</volume>
, <fpage>2444</fpage>
–<lpage>2449</lpage>
. <pub-id pub-id-type="doi">10.1128/AAC.00150-09</pub-id>
.<pub-id pub-id-type="pmid">19364877</pub-id>
</mixed-citation>
</ref>
<ref id="ref36"><mixed-citation publication-type="journal" id="cit36"><name><surname>Dwyer</surname>
<given-names>J. J.</given-names>
</name>
; <name><surname>Wilson</surname>
<given-names>K. L.</given-names>
</name>
; <name><surname>Davison</surname>
<given-names>D. K.</given-names>
</name>
; <name><surname>Freel</surname>
<given-names>S. A.</given-names>
</name>
; <name><surname>Seedorff</surname>
<given-names>J. E.</given-names>
</name>
; <name><surname>Wring</surname>
<given-names>S. A.</given-names>
</name>
; <name><surname>Tvermoes</surname>
<given-names>N. A.</given-names>
</name>
; <name><surname>Matthews</surname>
<given-names>T. J.</given-names>
</name>
; <name><surname>Greenberg</surname>
<given-names>M. L.</given-names>
</name>
; <name><surname>Delmedico</surname>
<given-names>M. K.</given-names>
</name>
<article-title>Design of helical, oligomeric HIV-1 fusion inhibitor
peptides with potent activity against enfuvirtide-resistant virus</article-title>
. <source>Proc. Natl. Acad. Sci. U. S. A.</source>
<year>2007</year>
, <volume>104</volume>
, <fpage>12772</fpage>
–<lpage>12777</lpage>
. <pub-id pub-id-type="doi">10.1073/pnas.0701478104</pub-id>
.<pub-id pub-id-type="pmid">17640899</pub-id>
</mixed-citation>
</ref>
<ref id="ref37"><mixed-citation publication-type="journal" id="cit37"><name><surname>Shi</surname>
<given-names>W. G.</given-names>
</name>
; <name><surname>Cai</surname>
<given-names>L. F.</given-names>
</name>
; <name><surname>Lu</surname>
<given-names>L.</given-names>
</name>
; <name><surname>Wang</surname>
<given-names>C.</given-names>
</name>
; <name><surname>Wang</surname>
<given-names>K.</given-names>
</name>
; <name><surname>Xu</surname>
<given-names>L.</given-names>
</name>
; <name><surname>Zhang</surname>
<given-names>S.</given-names>
</name>
; <name><surname>Han</surname>
<given-names>H.</given-names>
</name>
; <name><surname>Jiang</surname>
<given-names>X. F.</given-names>
</name>
; <name><surname>Zheng</surname>
<given-names>B. H.</given-names>
</name>
; <name><surname>Jiang</surname>
<given-names>S. B.</given-names>
</name>
; <name><surname>Liu</surname>
<given-names>K. L.</given-names>
</name>
<article-title>Design
of highly potent HIV fusion
inhibitors based on artificial peptide sequences</article-title>
. <source>Chem. Commun.</source>
<year>2012</year>
, <volume>48</volume>
, <fpage>11579</fpage>
–<lpage>11581</lpage>
. <pub-id pub-id-type="doi">10.1039/c2cc35973a</pub-id>
.</mixed-citation>
</ref>
<ref id="ref38"><mixed-citation publication-type="journal" id="cit38"><name><surname>Zhu</surname>
<given-names>X. J.</given-names>
</name>
; <name><surname>Zhu</surname>
<given-names>Y.</given-names>
</name>
; <name><surname>Ye</surname>
<given-names>S.</given-names>
</name>
; <name><surname>Wang</surname>
<given-names>Q.</given-names>
</name>
; <name><surname>Xu</surname>
<given-names>W.</given-names>
</name>
; <name><surname>Su</surname>
<given-names>S.</given-names>
</name>
; <name><surname>Sun</surname>
<given-names>Z. W.</given-names>
</name>
; <name><surname>Yu</surname>
<given-names>F.</given-names>
</name>
; <name><surname>Liu</surname>
<given-names>Q.</given-names>
</name>
; <name><surname>Wang</surname>
<given-names>C.</given-names>
</name>
; <name><surname>Zhang</surname>
<given-names>T. H.</given-names>
</name>
; <name><surname>Zhang</surname>
<given-names>Z. Q.</given-names>
</name>
; <name><surname>Zhang</surname>
<given-names>X. Y.</given-names>
</name>
; <name><surname>Xu</surname>
<given-names>J. Q.</given-names>
</name>
; <name><surname>Du</surname>
<given-names>L. Y.</given-names>
</name>
; <name><surname>Liu</surname>
<given-names>K. L.</given-names>
</name>
; <name><surname>Lu</surname>
<given-names>L.</given-names>
</name>
; <name><surname>Zhang</surname>
<given-names>R. G.</given-names>
</name>
; <name><surname>Jiang</surname>
<given-names>S. B.</given-names>
</name>
<article-title>Improved pharmacological and structural properties
of HIV fusion inhibitor AP3 over enfuvirtide: highlighting advantages
of artificial peptide strategy</article-title>
. <source>Sci. Rep.</source>
<year>2015</year>
, <volume>5</volume>
, <fpage>13028</fpage>
<pub-id pub-id-type="doi">10.1038/srep13028</pub-id>
.<pub-id pub-id-type="pmid">26286358</pub-id>
</mixed-citation>
</ref>
<ref id="ref39"><mixed-citation publication-type="journal" id="cit39"><name><surname>Cui</surname>
<given-names>H.</given-names>
</name>
; <name><surname>Qing</surname>
<given-names>J.</given-names>
</name>
; <name><surname>Guo</surname>
<given-names>Y.</given-names>
</name>
; <name><surname>Wang</surname>
<given-names>Y.</given-names>
</name>
; <name><surname>Cui</surname>
<given-names>L.</given-names>
</name>
; <name><surname>He</surname>
<given-names>T.</given-names>
</name>
; <name><surname>Zhang</surname>
<given-names>L.</given-names>
</name>
; <name><surname>Liu</surname>
<given-names>L.</given-names>
</name>
<article-title>Stapled peptide-based membrane fusion
inhibitors of hepatitis C virus</article-title>
. <source>Bioorg. Med.
Chem.</source>
<year>2013</year>
, <volume>21</volume>
, <fpage>3547</fpage>
–<lpage>3554</lpage>
. <pub-id pub-id-type="doi">10.1016/j.bmc.2013.02.011</pub-id>
.<pub-id pub-id-type="pmid">23490158</pub-id>
</mixed-citation>
</ref>
<ref id="ref40"><mixed-citation publication-type="journal" id="cit40"><name><surname>Liu</surname>
<given-names>Q.</given-names>
</name>
; <name><surname>Xia</surname>
<given-names>S.</given-names>
</name>
; <name><surname>Sun</surname>
<given-names>Z.</given-names>
</name>
; <name><surname>Wang</surname>
<given-names>Q.</given-names>
</name>
; <name><surname>Du</surname>
<given-names>L.</given-names>
</name>
; <name><surname>Lu</surname>
<given-names>L.</given-names>
</name>
; <name><surname>Jiang</surname>
<given-names>S.</given-names>
</name>
<article-title>Testing of
Middle East respiratory syndrome coronavirus replication
inhibitors for the ability to block viral entry</article-title>
. <source>Antimicrob. Agents Chemother.</source>
<year>2015</year>
, <volume>59</volume>
, <fpage>742</fpage>
–<lpage>744</lpage>
. <pub-id pub-id-type="doi">10.1128/AAC.03977-14</pub-id>
.<pub-id pub-id-type="pmid">25331705</pub-id>
</mixed-citation>
</ref>
<ref id="ref41"><mixed-citation publication-type="journal" id="cit41"><name><surname>Xia</surname>
<given-names>S.</given-names>
</name>
; <name><surname>Liu</surname>
<given-names>Q.</given-names>
</name>
; <name><surname>Wang</surname>
<given-names>Q.</given-names>
</name>
; <name><surname>Sun</surname>
<given-names>Z.</given-names>
</name>
; <name><surname>Su</surname>
<given-names>S.</given-names>
</name>
; <name><surname>Du</surname>
<given-names>L.</given-names>
</name>
; <name><surname>Ying</surname>
<given-names>T.</given-names>
</name>
; <name><surname>Lu</surname>
<given-names>L.</given-names>
</name>
; <name><surname>Jiang</surname>
<given-names>S.</given-names>
</name>
<article-title>Middle East respiratory
syndrome coronavirus (MERS-CoV) entry inhibitors targeting spike protein</article-title>
. <source>Virus Res.</source>
<year>2014</year>
, <volume>194</volume>
, <fpage>200</fpage>
–<lpage>210</lpage>
. <pub-id pub-id-type="doi">10.1016/j.virusres.2014.10.007</pub-id>
.<pub-id pub-id-type="pmid">25451066</pub-id>
</mixed-citation>
</ref>
<ref id="ref42"><mixed-citation publication-type="journal" id="cit42"><name><surname>Zhao</surname>
<given-names>G.</given-names>
</name>
; <name><surname>Du</surname>
<given-names>L.</given-names>
</name>
; <name><surname>Ma</surname>
<given-names>C.</given-names>
</name>
; <name><surname>Li</surname>
<given-names>Y.</given-names>
</name>
; <name><surname>Li</surname>
<given-names>L.</given-names>
</name>
; <name><surname>Poon</surname>
<given-names>V. K.</given-names>
</name>
; <name><surname>Wang</surname>
<given-names>L.</given-names>
</name>
; <name><surname>Yu</surname>
<given-names>F.</given-names>
</name>
; <name><surname>Zheng</surname>
<given-names>B. J.</given-names>
</name>
; <name><surname>Jiang</surname>
<given-names>S.</given-names>
</name>
; <name><surname>Zhou</surname>
<given-names>Y.</given-names>
</name>
<article-title>A safe and convenient
pseudovirus-based inhibition assay to detect
neutralizing antibodies and screen for viral entry inhibitors against
the novel human coronavirus MERS-CoV</article-title>
. <source>Virol.
J.</source>
<year>2013</year>
, <volume>10</volume>
, <fpage>266</fpage>
<pub-id pub-id-type="doi">10.1186/1743-422X-10-266</pub-id>
.<pub-id pub-id-type="pmid">23978242</pub-id>
</mixed-citation>
</ref>
</ref-list>
</back>
</pmc>
</record>
Pour manipuler ce document sous Unix (Dilib)
EXPLOR_STEP=$WICRI_ROOT/Sante/explor/MersV1/Data/Pmc/Corpus
HfdSelect -h $EXPLOR_STEP/biblio.hfd -nk 000130 | SxmlIndent | more
Ou
HfdSelect -h $EXPLOR_AREA/Data/Pmc/Corpus/biblio.hfd -nk 000130 | SxmlIndent | more
Pour mettre un lien sur cette page dans le réseau Wicri
{{Explor lien |wiki= Sante |area= MersV1 |flux= Pmc |étape= Corpus |type= RBID |clé= |texte= }}
![]() | This area was generated with Dilib version V0.6.33. | ![]() |