Links to Exploration step
Le document en format XML
<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Crystal structure of a new benzoic acid inhibitor of influenza neuraminidase bound with a new tilt induced by overpacking subsite C6</title><author><name sortKey="Venkatramani, Lalitha" sort="Venkatramani, Lalitha" uniqKey="Venkatramani L" first="Lalitha" last="Venkatramani">Lalitha Venkatramani</name><affiliation><nlm:aff id="I1">Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, 941 Stanton L. Young Blvd, Oklahoma City, OK, 73104, USA</nlm:aff></affiliation></author><author><name sortKey="Johnson, Eric S" sort="Johnson, Eric S" uniqKey="Johnson E" first="Eric S" last="Johnson">Eric S. Johnson</name><affiliation><nlm:aff id="I2">Department of Chemistry and Center for Biophysical Sciences and Engineering, University of Alabama at Birmingham, 901 14th Street South, Birmingham, AL, 35294, USA</nlm:aff></affiliation></author><author><name sortKey="Kolavi, Gundurao" sort="Kolavi, Gundurao" uniqKey="Kolavi G" first="Gundurao" last="Kolavi">Gundurao Kolavi</name><affiliation><nlm:aff id="I2">Department of Chemistry and Center for Biophysical Sciences and Engineering, University of Alabama at Birmingham, 901 14th Street South, Birmingham, AL, 35294, USA</nlm:aff></affiliation></author><author><name sortKey="Air, Gillian M" sort="Air, Gillian M" uniqKey="Air G" first="Gillian M" last="Air">Gillian M. Air</name><affiliation><nlm:aff id="I1">Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, 941 Stanton L. Young Blvd, Oklahoma City, OK, 73104, USA</nlm:aff></affiliation></author><author><name sortKey="Brouillette, Wayne J" sort="Brouillette, Wayne J" uniqKey="Brouillette W" first="Wayne J" last="Brouillette">Wayne J. Brouillette</name><affiliation><nlm:aff id="I2">Department of Chemistry and Center for Biophysical Sciences and Engineering, University of Alabama at Birmingham, 901 14th Street South, Birmingham, AL, 35294, USA</nlm:aff></affiliation></author><author><name sortKey="Mooers, Blaine Hm" sort="Mooers, Blaine Hm" uniqKey="Mooers B" first="Blaine Hm" last="Mooers">Blaine Hm Mooers</name><affiliation><nlm:aff id="I1">Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, 941 Stanton L. Young Blvd, Oklahoma City, OK, 73104, USA</nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">22559154</idno><idno type="pmc">3416664</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3416664</idno><idno type="RBID">PMC:3416664</idno><idno type="doi">10.1186/1472-6807-12-7</idno><date when="2012">2012</date><idno type="wicri:Area/Pmc/Corpus">000592</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000592</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Crystal structure of a new benzoic acid inhibitor of influenza neuraminidase bound with a new tilt induced by overpacking subsite C6</title><author><name sortKey="Venkatramani, Lalitha" sort="Venkatramani, Lalitha" uniqKey="Venkatramani L" first="Lalitha" last="Venkatramani">Lalitha Venkatramani</name><affiliation><nlm:aff id="I1">Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, 941 Stanton L. Young Blvd, Oklahoma City, OK, 73104, USA</nlm:aff></affiliation></author><author><name sortKey="Johnson, Eric S" sort="Johnson, Eric S" uniqKey="Johnson E" first="Eric S" last="Johnson">Eric S. Johnson</name><affiliation><nlm:aff id="I2">Department of Chemistry and Center for Biophysical Sciences and Engineering, University of Alabama at Birmingham, 901 14th Street South, Birmingham, AL, 35294, USA</nlm:aff></affiliation></author><author><name sortKey="Kolavi, Gundurao" sort="Kolavi, Gundurao" uniqKey="Kolavi G" first="Gundurao" last="Kolavi">Gundurao Kolavi</name><affiliation><nlm:aff id="I2">Department of Chemistry and Center for Biophysical Sciences and Engineering, University of Alabama at Birmingham, 901 14th Street South, Birmingham, AL, 35294, USA</nlm:aff></affiliation></author><author><name sortKey="Air, Gillian M" sort="Air, Gillian M" uniqKey="Air G" first="Gillian M" last="Air">Gillian M. Air</name><affiliation><nlm:aff id="I1">Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, 941 Stanton L. Young Blvd, Oklahoma City, OK, 73104, USA</nlm:aff></affiliation></author><author><name sortKey="Brouillette, Wayne J" sort="Brouillette, Wayne J" uniqKey="Brouillette W" first="Wayne J" last="Brouillette">Wayne J. Brouillette</name><affiliation><nlm:aff id="I2">Department of Chemistry and Center for Biophysical Sciences and Engineering, University of Alabama at Birmingham, 901 14th Street South, Birmingham, AL, 35294, USA</nlm:aff></affiliation></author><author><name sortKey="Mooers, Blaine Hm" sort="Mooers, Blaine Hm" uniqKey="Mooers B" first="Blaine Hm" last="Mooers">Blaine Hm Mooers</name><affiliation><nlm:aff id="I1">Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, 941 Stanton L. Young Blvd, Oklahoma City, OK, 73104, USA</nlm:aff></affiliation></author></analytic><series><title level="j">BMC Structural Biology</title><idno type="eISSN">1472-6807</idno><imprint><date when="2012">2012</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><sec><title>Background</title><p>Influenza neuraminidase (NA) is an important target for antiviral inhibitors since its active site is highly conserved such that inhibitors can be cross-reactive against multiple types and subtypes of influenza. Here, we discuss the crystal structure of neuraminidase subtype N9 complexed with a new benzoic acid based inhibitor (<bold>2</bold>) that was designed to add contacts by overpacking one side of the active site pocket. Inhibitor <bold>2</bold> uses benzoic acid to mimic the pyranose ring, a bis-(hydroxymethyl)-substituted 2-pyrrolidinone ring in place of the <italic>N</italic>-acetyl group of the sialic acid, and a branched aliphatic structure to fill the sialic acid C6 subsite.</p></sec><sec><title>Results</title><p>Inhibitor <bold>2</bold> {4-[2,2-bis(hydroxymethyl)-5-oxo-pyrrolidin-1-yl]-3-[(dipropylamino)methyl)]benzoic acid} was soaked into crystals of neuraminidase of A/tern/Australia/G70c/75 (N9), and the structure refined with 1.55 Å X-ray data. The benzene ring of the inhibitor tilted 8.9° compared to the previous compound (<bold>1)</bold>, and the number of contacts, including hydrogen bonds, increased. However, the IC<sub>50</sub> for compound <bold>2</bold> remained in the low micromolar range, likely because one propyl group was disordered. In this high-resolution structure of NA isolated from virus grown in chicken eggs, we found electron density for additional sugar units on the N-linked glycans compared to previous neuraminidase structures. In particular, seven mannoses and two N-acetylglucosamines are visible in the glycan attached to Asn200. This long, branched high-mannose glycan makes significant contacts with the neighboring subunit.</p></sec><sec><title>Conclusions</title><p>We designed inhibitor <bold>2</bold> with an extended substituent at C4-corresponding to C6 of sialic acid-to increase the contact surface in the C6-subsite and to force the benzene ring to tilt to maximize these interactions while retaining the interactions of the carboxylate and the pyrolidinone substituents. The crystal structure at 1.55 Å showed that we partially succeeded in that the ring in <bold>2</bold> is tilted relative to <bold>1</bold> and the number of contacts increased, but one hydrophobic branch makes no contacts, perhaps explaining why the IC<sub>50</sub> did not decrease. Future design efforts will include branches of unequal length so that both branches may be accommodated in the C6-subsite without conformational disorder. The high-mannose glycan attached to Asn200 makes several inter-subunit contacts and appears to stabilize the tetramer.</p></sec></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Palese, P" uniqKey="Palese P">P Palese</name></author><author><name sortKey="Tobita, K" uniqKey="Tobita K">K Tobita</name></author><author><name sortKey="Ueda, M" uniqKey="Ueda M">M Ueda</name></author><author><name sortKey="Compans, Rw" uniqKey="Compans R">RW Compans</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Liu, C" uniqKey="Liu C">C Liu</name></author><author><name sortKey="Eichelberger, Mc" uniqKey="Eichelberger M">MC Eichelberger</name></author><author><name sortKey="Compans, Rw" uniqKey="Compans R">RW Compans</name></author><author><name sortKey="Air, Gm" uniqKey="Air G">GM Air</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Webster, Rg" uniqKey="Webster R">RG Webster</name></author><author><name sortKey="Govorkova, Ea" uniqKey="Govorkova E">EA Govorkova</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Rota, P" uniqKey="Rota P">P Rota</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Air, Gm" uniqKey="Air G">GM Air</name></author><author><name sortKey="Brouillette, Wj" uniqKey="Brouillette W">WJ Brouillette</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Colman, Pm" uniqKey="Colman P">PM Colman</name></author><author><name sortKey="Varghese, Jn" uniqKey="Varghese J">JN Varghese</name></author><author><name sortKey="Laver, Wg" uniqKey="Laver W">WG Laver</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Burmeister, Wp" uniqKey="Burmeister W">WP Burmeister</name></author><author><name sortKey="Ruigrok, Rw" uniqKey="Ruigrok R">RW Ruigrok</name></author><author><name sortKey="Cusack, S" uniqKey="Cusack S">S Cusack</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Bossart Whitaker, P" uniqKey="Bossart Whitaker P">P Bossart-Whitaker</name></author><author><name sortKey="Carson, M" uniqKey="Carson M">M Carson</name></author><author><name sortKey="Babu, Ys" uniqKey="Babu Y">YS Babu</name></author><author><name sortKey="Smith, Cd" uniqKey="Smith C">CD Smith</name></author><author><name sortKey="Laver, Wg" uniqKey="Laver W">WG Laver</name></author><author><name sortKey="Air, Gm" uniqKey="Air G">GM Air</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Air, Gm" uniqKey="Air G">GM Air</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Von Itzstein, M" uniqKey="Von Itzstein M">M von Itzstein</name></author><author><name sortKey="Wu, Wy" uniqKey="Wu W">WY Wu</name></author><author><name sortKey="Kok, Gb" uniqKey="Kok G">GB Kok</name></author><author><name sortKey="Pegg, Ms" uniqKey="Pegg M">MS Pegg</name></author><author><name sortKey="Dyason, Jc" uniqKey="Dyason J">JC Dyason</name></author><author><name sortKey="Jin, B" uniqKey="Jin B">B Jin</name></author><author><name sortKey="Van Phan, T" uniqKey="Van Phan T">T Van Phan</name></author><author><name sortKey="Smythe, Ml" uniqKey="Smythe M">ML Smythe</name></author><author><name sortKey="White, Hf" uniqKey="White H">HF White</name></author><author><name sortKey="Oliver, Sw" uniqKey="Oliver S">SW Oliver</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Kim, Cu" uniqKey="Kim C">CU Kim</name></author><author><name sortKey="Lew, W" uniqKey="Lew W">W Lew</name></author><author><name sortKey="Williams, Ma" uniqKey="Williams M">MA Williams</name></author><author><name sortKey="Liu, H" uniqKey="Liu H">H Liu</name></author><author><name sortKey="Zhang, L" uniqKey="Zhang L">L Zhang</name></author><author><name sortKey="Swaminathan, S" uniqKey="Swaminathan S">S Swaminathan</name></author><author><name sortKey="Bischofberger, N" uniqKey="Bischofberger N">N Bischofberger</name></author><author><name sortKey="Chen, Ms" uniqKey="Chen M">MS Chen</name></author><author><name sortKey="Mendel, Db" uniqKey="Mendel D">DB Mendel</name></author><author><name sortKey="Tai, Cy" uniqKey="Tai C">CY Tai</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Brouillette, Wj" uniqKey="Brouillette W">WJ Brouillette</name></author><author><name sortKey="Atigadda, Vr" uniqKey="Atigadda V">VR Atigadda</name></author><author><name sortKey="Luo, M" uniqKey="Luo M">M Luo</name></author><author><name sortKey="Air, Gm" uniqKey="Air G">GM Air</name></author><author><name sortKey="Babu, Ys" uniqKey="Babu Y">YS Babu</name></author><author><name sortKey="Bantia, S" uniqKey="Bantia S">S Bantia</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Kim, Cu" uniqKey="Kim C">CU Kim</name></author><author><name sortKey="Lew, W" uniqKey="Lew W">W Lew</name></author><author><name sortKey="Williams, Ma" uniqKey="Williams M">MA Williams</name></author><author><name sortKey="Wu, H" uniqKey="Wu H">H Wu</name></author><author><name sortKey="Zhang, L" uniqKey="Zhang L">L Zhang</name></author><author><name sortKey="Chen, X" uniqKey="Chen X">X Chen</name></author><author><name sortKey="Escarpe, Pa" uniqKey="Escarpe P">PA Escarpe</name></author><author><name sortKey="Mendel, Db" uniqKey="Mendel D">DB Mendel</name></author><author><name sortKey="Laver, Wg" uniqKey="Laver W">WG Laver</name></author><author><name sortKey="Stevens, Rc" uniqKey="Stevens R">RC Stevens</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Babu, Ys" uniqKey="Babu Y">YS Babu</name></author><author><name sortKey="Chand, P" uniqKey="Chand P">P Chand</name></author><author><name sortKey="Bantia, S" uniqKey="Bantia S">S Bantia</name></author><author><name sortKey="Kotian, P" uniqKey="Kotian P">P Kotian</name></author><author><name sortKey="Dehghani, A" uniqKey="Dehghani A">A Dehghani</name></author><author><name sortKey="El Kattan, Y" uniqKey="El Kattan Y">Y El-Kattan</name></author><author><name sortKey="Lin, Th" uniqKey="Lin T">TH Lin</name></author><author><name sortKey="Hutchison, Tl" uniqKey="Hutchison T">TL Hutchison</name></author><author><name sortKey="Elliott, Aj" uniqKey="Elliott A">AJ Elliott</name></author><author><name sortKey="Parker, Cd" uniqKey="Parker C">CD Parker</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Jedrzejas, Mj" uniqKey="Jedrzejas M">MJ Jedrzejas</name></author><author><name sortKey="Singh, S" uniqKey="Singh S">S Singh</name></author><author><name sortKey="Brouillette, Wj" uniqKey="Brouillette W">WJ Brouillette</name></author><author><name sortKey="Air, Gm" uniqKey="Air G">GM Air</name></author><author><name sortKey="Luo, M" uniqKey="Luo M">M Luo</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Singh, S" uniqKey="Singh S">S Singh</name></author><author><name sortKey="Jedrzejas, Mj" uniqKey="Jedrzejas M">MJ Jedrzejas</name></author><author><name sortKey="Air, Gm" uniqKey="Air G">GM Air</name></author><author><name sortKey="Luo, M" uniqKey="Luo M">M Luo</name></author><author><name sortKey="Laver, Wg" uniqKey="Laver W">WG Laver</name></author><author><name sortKey="Brouillette, Wj" uniqKey="Brouillette W">WJ Brouillette</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Brouillette, Wj" uniqKey="Brouillette W">WJ Brouillette</name></author><author><name sortKey="Bajpai, Sn" uniqKey="Bajpai S">SN Bajpai</name></author><author><name sortKey="Ali, Sm" uniqKey="Ali S">SM Ali</name></author><author><name sortKey="Velu, Se" uniqKey="Velu S">SE Velu</name></author><author><name sortKey="Atigadda, Vr" uniqKey="Atigadda V">VR Atigadda</name></author><author><name sortKey="Lommer, Bs" uniqKey="Lommer B">BS Lommer</name></author><author><name sortKey="Finley, Jb" uniqKey="Finley J">JB Finley</name></author><author><name sortKey="Luo, M" uniqKey="Luo M">M Luo</name></author><author><name sortKey="Air, Gm" uniqKey="Air G">GM Air</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Atigadda, Vr" uniqKey="Atigadda V">VR Atigadda</name></author><author><name sortKey="Brouillette, Wj" uniqKey="Brouillette W">WJ Brouillette</name></author><author><name sortKey="Duarte, F" uniqKey="Duarte F">F Duarte</name></author><author><name sortKey="Ali, Sm" uniqKey="Ali S">SM Ali</name></author><author><name sortKey="Babu, Ys" uniqKey="Babu Y">YS Babu</name></author><author><name sortKey="Bantia, S" uniqKey="Bantia S">S Bantia</name></author><author><name sortKey="Chand, P" uniqKey="Chand P">P Chand</name></author><author><name sortKey="Chu, N" uniqKey="Chu N">N Chu</name></author><author><name sortKey="Montgomery, Ja" uniqKey="Montgomery J">JA Montgomery</name></author><author><name sortKey="Walsh, Da" uniqKey="Walsh D">DA Walsh</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Smith, Pw" uniqKey="Smith P">PW Smith</name></author><author><name sortKey="Robinson, Je" uniqKey="Robinson J">JE Robinson</name></author><author><name sortKey="Evans, Dn" uniqKey="Evans D">DN Evans</name></author><author><name sortKey="Sollis, Sl" uniqKey="Sollis S">SL Sollis</name></author><author><name sortKey="Howes, Pd" uniqKey="Howes P">PD Howes</name></author><author><name sortKey="Trivedi, N" uniqKey="Trivedi N">N Trivedi</name></author><author><name sortKey="Bethell, Rc" uniqKey="Bethell R">RC Bethell</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Russell, Rj" uniqKey="Russell R">RJ Russell</name></author><author><name sortKey="Haire, Lf" uniqKey="Haire L">LF Haire</name></author><author><name sortKey="Stevens, Dj" uniqKey="Stevens D">DJ Stevens</name></author><author><name sortKey="Collins, Pj" uniqKey="Collins P">PJ Collins</name></author><author><name sortKey="Lin, Yp" uniqKey="Lin Y">YP Lin</name></author><author><name sortKey="Blackburn, Gm" uniqKey="Blackburn G">GM Blackburn</name></author><author><name sortKey="Hay, Aj" uniqKey="Hay A">AJ Hay</name></author><author><name sortKey="Gamblin, Sj" uniqKey="Gamblin S">SJ Gamblin</name></author><author><name sortKey="Skehel, Jj" uniqKey="Skehel J">JJ Skehel</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Finley, Jb" uniqKey="Finley J">JB Finley</name></author><author><name sortKey="Atigadda, Vr" uniqKey="Atigadda V">VR Atigadda</name></author><author><name sortKey="Duarte, F" uniqKey="Duarte F">F Duarte</name></author><author><name sortKey="Zhao, Jj" uniqKey="Zhao J">JJ Zhao</name></author><author><name sortKey="Brouillette, Wj" uniqKey="Brouillette W">WJ Brouillette</name></author><author><name sortKey="Air, Gm" uniqKey="Air G">GM Air</name></author><author><name sortKey="Luo, M" uniqKey="Luo M">M Luo</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Rarey, M" uniqKey="Rarey M">M Rarey</name></author><author><name sortKey="Kramer, B" uniqKey="Kramer B">B Kramer</name></author><author><name sortKey="Lengauer, T" uniqKey="Lengauer T">T Lengauer</name></author><author><name sortKey="Klebe, G" uniqKey="Klebe G">G Klebe</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Kolavi, G" uniqKey="Kolavi G">G Kolavi</name></author><author><name sortKey="Li, Y" uniqKey="Li Y">Y Li</name></author><author><name sortKey="Johnson, Es" uniqKey="Johnson E">ES Johnson</name></author><author><name sortKey="Gulati, S" uniqKey="Gulati S">S Gulati</name></author><author><name sortKey="Air, Gm" uniqKey="Air G">GM Air</name></author><author><name sortKey="Brouillette, Wj" uniqKey="Brouillette W">WJ Brouillette</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Varghese, Jn" uniqKey="Varghese J">JN Varghese</name></author><author><name sortKey="Smith, Pw" uniqKey="Smith P">PW Smith</name></author><author><name sortKey="Sollis, Sl" uniqKey="Sollis S">SL Sollis</name></author><author><name sortKey="Blick, Tj" uniqKey="Blick T">TJ Blick</name></author><author><name sortKey="Sahasrabudhe, A" uniqKey="Sahasrabudhe A">A Sahasrabudhe</name></author><author><name sortKey="Mckimm Breschkin, Jl" uniqKey="Mckimm Breschkin J">JL McKimm-Breschkin</name></author><author><name sortKey="Colman, Pm" uniqKey="Colman P">PM Colman</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Laver, Wg" uniqKey="Laver W">WG Laver</name></author><author><name sortKey="Colman, Pm" uniqKey="Colman P">PM Colman</name></author><author><name sortKey="Webster, Rg" uniqKey="Webster R">RG Webster</name></author><author><name sortKey="Hinshaw, Vs" uniqKey="Hinshaw V">VS Hinshaw</name></author><author><name sortKey="Air, Gm" uniqKey="Air G">GM Air</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Varghese, Jn" uniqKey="Varghese J">JN Varghese</name></author><author><name sortKey="Colman, Pm" uniqKey="Colman P">PM Colman</name></author><author><name sortKey="Van Donkelaar, A" uniqKey="Van Donkelaar A">A van Donkelaar</name></author><author><name sortKey="Blick, Tj" uniqKey="Blick T">TJ Blick</name></author><author><name sortKey="Sahasrabudhe, A" uniqKey="Sahasrabudhe A">A Sahasrabudhe</name></author><author><name sortKey="Mckimm Breschkin, Jl" uniqKey="Mckimm Breschkin J">JL McKimm-Breschkin</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Weiss, Ms" uniqKey="Weiss M">MS Weiss</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Pettersen, Ef" uniqKey="Pettersen E">EF Pettersen</name></author><author><name sortKey="Goddard, Td" uniqKey="Goddard T">TD Goddard</name></author><author><name sortKey="Huang, Cc" uniqKey="Huang C">CC Huang</name></author><author><name sortKey="Couch, Gs" uniqKey="Couch G">GS Couch</name></author><author><name sortKey="Greenblatt, Dm" uniqKey="Greenblatt D">DM Greenblatt</name></author><author><name sortKey="Meng, Ec" uniqKey="Meng E">EC Meng</name></author><author><name sortKey="Ferrin, Te" uniqKey="Ferrin T">TE Ferrin</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Mcdonald, I" uniqKey="Mcdonald I">I McDonald</name></author><author><name sortKey="Thornton, J" uniqKey="Thornton J">J Thornton</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Copeland, Ra" uniqKey="Copeland R">RA Copeland</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Baker, En" uniqKey="Baker E">EN Baker</name></author><author><name sortKey="Hubbard, Re" uniqKey="Hubbard R">RE Hubbard</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Lommer, Bs" uniqKey="Lommer B">BS Lommer</name></author><author><name sortKey="Ali, Sm" uniqKey="Ali S">SM Ali</name></author><author><name sortKey="Bajpai, Sn" uniqKey="Bajpai S">SN Bajpai</name></author><author><name sortKey="Brouillette, Wj" uniqKey="Brouillette W">WJ Brouillette</name></author><author><name sortKey="Air, Gm" uniqKey="Air G">GM Air</name></author><author><name sortKey="Luo, M" uniqKey="Luo M">M Luo</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Wang, T" uniqKey="Wang T">T Wang</name></author><author><name sortKey="Wade, Rc" uniqKey="Wade R">RC Wade</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Vagin, Aa" uniqKey="Vagin A">AA Vagin</name></author><author><name sortKey="Steiner, Ra" uniqKey="Steiner R">RA Steiner</name></author><author><name sortKey="Lebedev, Aa" uniqKey="Lebedev A">AA Lebedev</name></author><author><name sortKey="Potterton, L" uniqKey="Potterton L">L Potterton</name></author><author><name sortKey="Mcnicholas, S" uniqKey="Mcnicholas S">S McNicholas</name></author><author><name sortKey="Long, F" uniqKey="Long F">F Long</name></author><author><name sortKey="Murshudov, Gn" uniqKey="Murshudov G">GN Murshudov</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Varghese, Jn" uniqKey="Varghese J">JN Varghese</name></author><author><name sortKey="Mckimm Breschkin, Jl" uniqKey="Mckimm Breschkin J">JL McKimm-Breschkin</name></author><author><name sortKey="Caldwell, Jb" uniqKey="Caldwell J">JB Caldwell</name></author><author><name sortKey="Kortt, Aa" uniqKey="Kortt A">AA Kortt</name></author><author><name sortKey="Colman, Pm" uniqKey="Colman P">PM Colman</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Krissinel, E" uniqKey="Krissinel E">E Krissinel</name></author><author><name sortKey="Henrick, K" uniqKey="Henrick K">K Henrick</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Emsley, P" uniqKey="Emsley P">P Emsley</name></author><author><name sortKey="Lohkamp, B" uniqKey="Lohkamp B">B Lohkamp</name></author><author><name sortKey="Scott, Wg" uniqKey="Scott W">WG Scott</name></author><author><name sortKey="Cowtan, K" uniqKey="Cowtan K">K Cowtan</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Collins, Pj" uniqKey="Collins P">PJ Collins</name></author><author><name sortKey="Haire, Lf" uniqKey="Haire L">LF Haire</name></author><author><name sortKey="Lin, Yp" uniqKey="Lin Y">YP Lin</name></author><author><name sortKey="Liu, J" uniqKey="Liu J">J Liu</name></author><author><name sortKey="Russell, Rj" uniqKey="Russell R">RJ Russell</name></author><author><name sortKey="Walker, Pa" uniqKey="Walker P">PA Walker</name></author><author><name sortKey="Skehel, Jj" uniqKey="Skehel J">JJ Skehel</name></author><author><name sortKey="Martin, Sr" uniqKey="Martin S">SR Martin</name></author><author><name sortKey="Hay, Aj" uniqKey="Hay A">AJ Hay</name></author><author><name sortKey="Gamblin, Sj" uniqKey="Gamblin S">SJ Gamblin</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Jedrzejas, Mj" uniqKey="Jedrzejas M">MJ Jedrzejas</name></author><author><name sortKey="Singh, S" uniqKey="Singh S">S Singh</name></author><author><name sortKey="Brouillette, Wj" uniqKey="Brouillette W">WJ Brouillette</name></author><author><name sortKey="Laver, Wg" uniqKey="Laver W">WG Laver</name></author><author><name sortKey="Air, Gm" uniqKey="Air G">GM Air</name></author><author><name sortKey="Luo, M" uniqKey="Luo M">M Luo</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Holm, L" uniqKey="Holm L">L Holm</name></author><author><name sortKey="Park, J" uniqKey="Park J">J Park</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Venkatramani, L" uniqKey="Venkatramani L">L Venkatramani</name></author><author><name sortKey="Bochkareva, E" uniqKey="Bochkareva E">E Bochkareva</name></author><author><name sortKey="Lee, Jt" uniqKey="Lee J">JT Lee</name></author><author><name sortKey="Gulati, U" uniqKey="Gulati U">U Gulati</name></author><author><name sortKey="Graeme Laver, W" uniqKey="Graeme Laver W">W Graeme Laver</name></author><author><name sortKey="Bochkarev, A" uniqKey="Bochkarev A">A Bochkarev</name></author><author><name sortKey="Air, Gm" uniqKey="Air G">GM Air</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Lee, Jt" uniqKey="Lee J">JT Lee</name></author><author><name sortKey="Air, Gm" uniqKey="Air G">GM Air</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Ward, Cw" uniqKey="Ward C">CW Ward</name></author><author><name sortKey="Murray, Jm" uniqKey="Murray J">JM Murray</name></author><author><name sortKey="Roxburgh, Cm" uniqKey="Roxburgh C">CM Roxburgh</name></author><author><name sortKey="Jackson, Dc" uniqKey="Jackson D">DC Jackson</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Wu, Z" uniqKey="Wu Z">Z Wu</name></author><author><name sortKey="Ethen, C" uniqKey="Ethen C">C Ethen</name></author><author><name sortKey="Hickey, G" uniqKey="Hickey G">G Hickey</name></author><author><name sortKey="Jiang, W" uniqKey="Jiang W">W Jiang</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Ceroni, A" uniqKey="Ceroni A">A Ceroni</name></author><author><name sortKey="Dell, A" uniqKey="Dell A">A Dell</name></author><author><name sortKey="Haslam, S" uniqKey="Haslam S">S Haslam</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Garman, E" uniqKey="Garman E">E Garman</name></author></analytic></biblStruct><biblStruct></biblStruct><biblStruct><analytic><author><name sortKey="Kabsch, W" uniqKey="Kabsch W">W Kabsch</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Winn, Md" uniqKey="Winn M">MD Winn</name></author><author><name sortKey="Ballard, Cc" uniqKey="Ballard C">CC Ballard</name></author><author><name sortKey="Cowtan, Kd" uniqKey="Cowtan K">KD Cowtan</name></author><author><name sortKey="Dodson, Ej" uniqKey="Dodson E">EJ Dodson</name></author><author><name sortKey="Emsley, P" uniqKey="Emsley P">P Emsley</name></author><author><name sortKey="Evans, Pr" uniqKey="Evans P">PR Evans</name></author><author><name sortKey="Keegan, Rm" uniqKey="Keegan R">RM Keegan</name></author><author><name sortKey="Krissinel, Eb" uniqKey="Krissinel E">EB Krissinel</name></author><author><name sortKey="Leslie, Ag" uniqKey="Leslie A">AG Leslie</name></author><author><name sortKey="Mccoy, A" uniqKey="Mccoy A">A McCoy</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Evans, P" uniqKey="Evans P">P Evans</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="French, S" uniqKey="French S">S French</name></author><author><name sortKey="Wilson, K" uniqKey="Wilson K">K Wilson</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Murshudov, Gn" uniqKey="Murshudov G">GN Murshudov</name></author><author><name sortKey="Vagin, Aa" uniqKey="Vagin A">AA Vagin</name></author><author><name sortKey="Dodson, Ej" uniqKey="Dodson E">EJ Dodson</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Adams, Pd" uniqKey="Adams P">PD Adams</name></author><author><name sortKey="Afonine, Pv" uniqKey="Afonine P">PV Afonine</name></author><author><name sortKey="Bunkoczi, G" uniqKey="Bunkoczi G">G Bunkoczi</name></author><author><name sortKey="Chen, Vb" uniqKey="Chen V">VB Chen</name></author><author><name sortKey="Davis, Iw" uniqKey="Davis I">IW Davis</name></author><author><name sortKey="Echols, N" uniqKey="Echols N">N Echols</name></author><author><name sortKey="Headd, Jj" uniqKey="Headd J">JJ Headd</name></author><author><name sortKey="Hung, Lw" uniqKey="Hung L">LW Hung</name></author><author><name sortKey="Kapral, Gj" uniqKey="Kapral G">GJ Kapral</name></author><author><name sortKey="Grosse Kunstleve, Rw" uniqKey="Grosse Kunstleve R">RW Grosse-Kunstleve</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Schuttelkopf, Aw" uniqKey="Schuttelkopf A">AW Schuttelkopf</name></author><author><name sortKey="Van Aalten, Dm" uniqKey="Van Aalten D">DM van Aalten</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Word, Jm" uniqKey="Word J">JM Word</name></author><author><name sortKey="Lovell, Sc" uniqKey="Lovell S">SC Lovell</name></author><author><name sortKey="Richardson, Js" uniqKey="Richardson J">JS Richardson</name></author><author><name sortKey="Richardson, Dc" uniqKey="Richardson D">DC Richardson</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Laskowski, Ra" uniqKey="Laskowski R">RA Laskowski</name></author><author><name sortKey="Swindells, Mb" uniqKey="Swindells M">MB Swindells</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">BMC Struct Biol</journal-id><journal-id journal-id-type="iso-abbrev">BMC Struct. Biol</journal-id><journal-title-group><journal-title>BMC Structural Biology</journal-title></journal-title-group><issn pub-type="epub">1472-6807</issn><publisher><publisher-name>BioMed Central</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">22559154</article-id><article-id pub-id-type="pmc">3416664</article-id><article-id pub-id-type="publisher-id">1472-6807-12-7</article-id><article-id pub-id-type="doi">10.1186/1472-6807-12-7</article-id><article-categories><subj-group subj-group-type="heading"><subject>Research Article</subject></subj-group></article-categories><title-group><article-title>Crystal structure of a new benzoic acid inhibitor of influenza neuraminidase bound with a new tilt induced by overpacking subsite C6</article-title></title-group><contrib-group><contrib contrib-type="author" id="A1"><name><surname>Venkatramani</surname><given-names>Lalitha</given-names></name><xref ref-type="aff" rid="I1">1</xref><email>lalitha-venkatramani@ouhsc.edu</email></contrib><contrib contrib-type="author" id="A2"><name><surname>Johnson</surname><given-names>Eric S</given-names></name><xref ref-type="aff" rid="I2">2</xref><email>esjohnson74@gmail.com</email></contrib><contrib contrib-type="author" id="A3"><name><surname>Kolavi</surname><given-names>Gundurao</given-names></name><xref ref-type="aff" rid="I2">2</xref><email>gkolavichem@gmail.com</email></contrib><contrib contrib-type="author" id="A4"><name><surname>Air</surname><given-names>Gillian M</given-names></name><xref ref-type="aff" rid="I1">1</xref><email>gillian-air@ouhsc.edu</email></contrib><contrib contrib-type="author" corresp="yes" id="A5"><name><surname>Brouillette</surname><given-names>Wayne J</given-names></name><xref ref-type="aff" rid="I2">2</xref><email>wbrou@uab.edu</email></contrib><contrib contrib-type="author" corresp="yes" id="A6"><name><surname>Mooers</surname><given-names>Blaine HM</given-names></name><xref ref-type="aff" rid="I1">1</xref><email>blaine-mooers@ouhsc.edu</email></contrib></contrib-group><aff id="I1"><label>1</label>Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, 941 Stanton L. Young Blvd, Oklahoma City, OK, 73104, USA</aff><aff id="I2"><label>2</label>Department of Chemistry and Center for Biophysical Sciences and Engineering, University of Alabama at Birmingham, 901 14th Street South, Birmingham, AL, 35294, USA</aff><pub-date pub-type="collection"><year>2012</year></pub-date><pub-date pub-type="epub"><day>6</day><month>5</month><year>2012</year></pub-date><volume>12</volume><fpage>7</fpage><lpage>7</lpage><history><date date-type="received"><day>24</day><month>2</month><year>2012</year></date><date date-type="accepted"><day>6</day><month>5</month><year>2012</year></date></history><permissions><copyright-statement>Copyright ©2012 Venkatramani et al.; licensee BioMed Central Ltd.</copyright-statement><copyright-year>2012</copyright-year><copyright-holder>Venkatramani et al.; licensee BioMed Central Ltd.</copyright-holder><license license-type="open-access" xlink:href="http://creativecommons.org/licenses/by/2.0"><license-p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/2.0">http://creativecommons.org/licenses/by/2.0</ext-link>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</license-p></license></permissions><self-uri xlink:href="http://www.biomedcentral.com/1472-6807/12/7"></self-uri><abstract><sec><title>Background</title><p>Influenza neuraminidase (NA) is an important target for antiviral inhibitors since its active site is highly conserved such that inhibitors can be cross-reactive against multiple types and subtypes of influenza. Here, we discuss the crystal structure of neuraminidase subtype N9 complexed with a new benzoic acid based inhibitor (<bold>2</bold>) that was designed to add contacts by overpacking one side of the active site pocket. Inhibitor <bold>2</bold> uses benzoic acid to mimic the pyranose ring, a bis-(hydroxymethyl)-substituted 2-pyrrolidinone ring in place of the <italic>N</italic>-acetyl group of the sialic acid, and a branched aliphatic structure to fill the sialic acid C6 subsite.</p></sec><sec><title>Results</title><p>Inhibitor <bold>2</bold> {4-[2,2-bis(hydroxymethyl)-5-oxo-pyrrolidin-1-yl]-3-[(dipropylamino)methyl)]benzoic acid} was soaked into crystals of neuraminidase of A/tern/Australia/G70c/75 (N9), and the structure refined with 1.55 Å X-ray data. The benzene ring of the inhibitor tilted 8.9° compared to the previous compound (<bold>1)</bold>, and the number of contacts, including hydrogen bonds, increased. However, the IC<sub>50</sub> for compound <bold>2</bold> remained in the low micromolar range, likely because one propyl group was disordered. In this high-resolution structure of NA isolated from virus grown in chicken eggs, we found electron density for additional sugar units on the N-linked glycans compared to previous neuraminidase structures. In particular, seven mannoses and two N-acetylglucosamines are visible in the glycan attached to Asn200. This long, branched high-mannose glycan makes significant contacts with the neighboring subunit.</p></sec><sec><title>Conclusions</title><p>We designed inhibitor <bold>2</bold> with an extended substituent at C4-corresponding to C6 of sialic acid-to increase the contact surface in the C6-subsite and to force the benzene ring to tilt to maximize these interactions while retaining the interactions of the carboxylate and the pyrolidinone substituents. The crystal structure at 1.55 Å showed that we partially succeeded in that the ring in <bold>2</bold> is tilted relative to <bold>1</bold> and the number of contacts increased, but one hydrophobic branch makes no contacts, perhaps explaining why the IC<sub>50</sub> did not decrease. Future design efforts will include branches of unequal length so that both branches may be accommodated in the C6-subsite without conformational disorder. The high-mannose glycan attached to Asn200 makes several inter-subunit contacts and appears to stabilize the tetramer.</p></sec></abstract><kwd-group><kwd>Influenza neuraminidase inhibitor</kwd><kwd>Enzyme-ligand complex</kwd><kwd>Antiviral</kwd><kwd>Structure-based drug design</kwd><kwd>Glycoprotein</kwd><kwd>Glycan structure</kwd><kwd>Influenza virus</kwd><kwd>Benzoic acid</kwd><kwd>Pyrrolidinone</kwd></kwd-group></article-meta></front><body><sec><title>Background</title><p>Influenza A viruses display two membrane-anchored glycoproteins, hemagglutinin (HA) and neuraminidase (NA). HA mediates attachment of the virus to sialic acid receptors on host cells to initiate virus infection. After virus replication, NA removes sialic acid residues from viral and cellular glycoproteins to facilitate virus release and allow spread of infection to new cells. In the absence of NA activity, the progeny virions aggregate and infection ends [<xref ref-type="bibr" rid="B1">1</xref>,<xref ref-type="bibr" rid="B2">2</xref>].</p><p>The distinct antigenic properties of HA and NA from different viruses are used to classify influenza type A into subtypes—16 for HA and 9 for NA. H1N1 (1918), H2N2 (1957) and H3N2 (1968) caused the major influenza pandemics of the 20<sup>th</sup> century. Evidence that the new HA and sometimes NA genes originated in wild birds before appearing in humans has raised concerns about the recent spread of highly pathogenic avian H5N1 viruses. These viruses have the potential to gain transmissibility among humans and thereby devastate immunologically naïve human populations [<xref ref-type="bibr" rid="B3">3</xref>]. Type B viruses are not carried by birds and are not divided into antigenic subtypes, but since the mid-1980s, two lineages, B/Victoria and B/Yamagata, have been evolving concurrently in humans [<xref ref-type="bibr" rid="B4">4</xref>].</p><p>NA is a good target for structure-based enzyme inhibitor design because its well-characterized active site is conserved across all influenza A and B viruses [<xref ref-type="bibr" rid="B5">5</xref>]. The 11 amino acids in the active site that interact with sialic acid remain unchanged amidst extensive genetic variation in the rest of the sequence [<xref ref-type="bibr" rid="B6">6</xref>-<xref ref-type="bibr" rid="B8">8</xref>]; for an alignment, see [<xref ref-type="bibr" rid="B9">9</xref>]. Structure-based drug design led to the successful antiviral drugs zanamivir (GG167) [<xref ref-type="bibr" rid="B10">10</xref>] and oseltamivir (GS4071) [<xref ref-type="bibr" rid="B11">11</xref>] that are effective against different types and subtypes of influenza. Zanamivir is based on a transition state analogue of sialic acid while oseltamivir replaces sialic acid's pyranose ring with a cyclohexene scaffold (Figure <xref ref-type="fig" rid="F1">1</xref>). The high potency and oral activity of oseltamivir encourages the use of alternative scaffolds to replace the sugar ring of sialic acid [<xref ref-type="bibr" rid="B12">12</xref>-<xref ref-type="bibr" rid="B14">14</xref>]. Benzene ring scaffolds minimize the number of chiral centers, potentially simplifying the chemical synthesis and reducing associated expenses [<xref ref-type="bibr" rid="B15">15</xref>-<xref ref-type="bibr" rid="B18">18</xref>]. The challenge is to configure substituents on the ring to optimize the interactions with the active site. The direct contacts with sialic acid are conserved in all influenza NAs, but the subsites that surround the sialic acid—named according to the ring atoms of sialic acid (Figure <xref ref-type="fig" rid="F1">1E</xref>)—show differences in size and shape between strains and sometimes account for large differences in potency between N1, N2 and B NAs [<xref ref-type="bibr" rid="B17">17</xref>,<xref ref-type="bibr" rid="B19">19</xref>]. In addition, inhibitors with a hydrophobic side chain often show differences in potency between the two structural groups of type A NA: group 1 (subtypes N1, N4, N5 and N8) and group 2 (subtypes N2, N3, N6, N7, and N9) [<xref ref-type="bibr" rid="B20">20</xref>].</p><fig id="F1" position="float"><label>Figure 1</label><caption><p><bold>NA inhibitors.</bold> A, Zanamivir; B, oseltamivir; C, Inhibitor 1 (compound 14 of Brouillette et al. [<xref ref-type="bibr" rid="B17">17</xref>]); D, Inhibitor <bold>2</bold>; E, The NA subsites that surround the transition state analog 2-deoxy-2-dehydro-N-acetylneuraminic acid (DANA). Note that in the benzoic acid series, subsite 6 is adjacent to C4 of the benzene ring. Figure made with PDB ID: 1NNB.</p></caption><graphic xlink:href="1472-6807-12-7-1"></graphic></fig><p>X-ray structures of several simple benzoic acid derivatives bound to influenza B NA show that these inhibitors bind to the active site in the same orientation as sialic acid [<xref ref-type="bibr" rid="B15">15</xref>,<xref ref-type="bibr" rid="B16">16</xref>]. The derivatives' common carboxylate substitution at the C2 position maintains the native interaction with the arginine triad (Arg292, Arg118, and Arg371 in N2 numbering), and the N-acetyl substitution at the C5 subsite maintains the native interaction with Arg152.</p><p>In addition, using a benzene ring scaffold, hydrophobic groups of varying lengths fit into the glycerol binding site (C6 subsite) of the sialic acid [<xref ref-type="bibr" rid="B21">21</xref>]. The length and branching of the aliphatic chain have been modified to improve inhibition, but benzoic acids from this structural class have yet to match the inhibitory activities observed for zanamivir and oseltamivir. It is likely that this results from the lack of a basic group (aliphatic amine or guanidine) in the C4 subsite, which forms an important salt bridge with inhibitors in clinical use.</p><p>One of the best compounds from the existing benzoic acid series, inhibitor <bold>1</bold>, (Figure <xref ref-type="fig" rid="F1">1C</xref>) lacks the C4 subsite substituent yet exhibited moderately potent (low to mid nM) activity against NAs of the N2 and N9 subtypes of influenza A virus, with less inhibitory activity against influenza B virus NA, as observed for other compounds with a similar hydrophobic substituent on the benzene ring [<xref ref-type="bibr" rid="B18">18</xref>].</p><p>We are trying to broaden the specificity of the benzoic acids against influenza viruses of different subtypes and to increase potency. The best way to accomplish this goal is likely by adding a basic substituent to form a salt bridge in the C4 subsite. However, attempts to design substituents on the benzene ring that occupy both the negatively charged C4 subsite and the glycerol binding site (C6 subsite) of sialic acid have been difficult because the two subsites are offset from the plane of the benzene ring. The results of molecular modeling studies using <italic>FlexX</italic>[<xref ref-type="bibr" rid="B22">22</xref>] suggested that a different “tilt” of the benzene ring may be needed, and one way to alter the tilt may be to change the size of the hydrophobic substituent, such that an increase or a decrease in steric crowding reorients the benzene ring in the binding site.</p><p>The inhibitor studied here, compound <bold>2</bold>, like compound <bold>1</bold>, contains no chiral centers but has a longer hydrophobic substituent consisting of a 3-heptyl (instead of 3-pentyl) group positioned one atom further from the benzene ring (Figure <xref ref-type="fig" rid="F1">1D</xref>). Enzyme inhibition assays indicate that the affinity of <bold>2</bold> is in the low micromolar range for type A NAs but, compared to <bold>1</bold>, lower for type B NA (Table <xref ref-type="table" rid="T1">1</xref>) [<xref ref-type="bibr" rid="B12">12</xref>,<xref ref-type="bibr" rid="B17">17</xref>]. We have determined the crystal structure of <bold>2</bold> in complex with a type A N9 NA to a resolution of 1.55 Å, and we discuss it in terms of the successful reorientation of the benzene ring and the lack of improvement in binding affinity. The structure of the complex suggests routes to design inhibitors that might show improved affinity.</p><table-wrap position="float" id="T1"><label>Table 1</label><caption><p><bold>IC</bold><sub><bold>50</bold></sub><bold>values of the inhibitor 2 compared with compound 1</bold></p></caption><table frame="hsides" rules="groups" border="1"><colgroup><col align="left"></col><col align="left"></col><col align="left"></col></colgroup><thead><tr><th align="left"><bold>Inhibitor</bold></th><th align="left"><bold>NA</bold></th><th align="left"><bold>IC50 (μM)</bold></th></tr></thead><tbody><tr><td align="left" valign="bottom"><bold>1</bold>[<xref ref-type="bibr" rid="B17">17</xref>]<hr></hr></td><td align="left" valign="bottom">A/Udorn/72 NA (N2)<hr></hr></td><td align="left" valign="bottom">0.49<hr></hr></td></tr><tr><td align="left" valign="bottom"> <hr></hr></td><td align="left" valign="bottom">A/tern/Australia/G70c NA (N9)<hr></hr></td><td align="left" valign="bottom">4.4<hr></hr></td></tr><tr><td align="left" valign="bottom"> <hr></hr></td><td align="left" valign="bottom">B/Lee/40 NA<hr></hr></td><td align="left" valign="bottom">271<hr></hr></td></tr><tr><td align="left" valign="bottom"><bold>2</bold>[<xref ref-type="bibr" rid="B23">23</xref>]<hr></hr></td><td align="left" valign="bottom">H3N2<hr></hr></td><td align="left" valign="bottom">2<hr></hr></td></tr><tr><td align="left" valign="bottom"> <hr></hr></td><td align="left" valign="bottom">H1N1<hr></hr></td><td align="left" valign="bottom">20<hr></hr></td></tr><tr><td align="left" valign="bottom"> <hr></hr></td><td align="left" valign="bottom">A/tern/Australia/G70c NA (N9)<hr></hr></td><td align="left" valign="bottom">9.1 ± 2.2<hr></hr></td></tr><tr><td align="left"> </td><td align="left">B/Lee/40 NA</td><td align="left">180</td></tr></tbody></table></table-wrap></sec><sec><title>Results and discussion</title><sec><title>Overall structure</title><p>N9 NA crystals that had been soaked with inhibitor <bold>2</bold> showed similar unit cell dimensions and space group symmetry as low temperature crystal structures of other N9-inhibitor complexes and the uncomplexed mutant R292K (Protein Data Bank identifier [PDB ID] 2QWA) [<xref ref-type="bibr" rid="B24">24</xref>]. The available uncomplexed native structures (PDB ID, 1NNA and 7NN9) were determined at room temperature and were not as suitable for direct comparison with our low temperature data, so we judged the crystal structure of the uncomplexed mutant R292K to be a less biased starting model for molecular replacement. Inhibitor <bold>2</bold> bound to the active site with nearly full occupancy (refined occupancy factor of 0.68) and did not bind to additional sites such as the second sialic acid binding site [<xref ref-type="bibr" rid="B25">25</xref>,<xref ref-type="bibr" rid="B26">26</xref>] (PDB ID, 2C4A).</p></sec><sec><title>Inhibitor–N9 interactions</title><p>The X-ray data were of high quality and the structure of <bold>2</bold> complexed with N9 NA was refined with 1.55 Å X-ray data. The final model had good geometry (Table <xref ref-type="table" rid="T2">2</xref>) with all amino acid residues in the allowed region of the Ramachandran plot (96.1<italic>%</italic> in the favored region and 0% outliers). Initial F<sub>o</sub>–F<sub>c</sub> maps showed clear electron density of the inhibitor in the active site of NA (Figure <xref ref-type="fig" rid="F2">2A</xref>). The two substituents of the inhibitor's pyrrolidine ring were buried inside the active site cavity (dihedral angle C6-C5-N5-C13 at the atropisomeric center −112°) with no indication of alternative conformers with rotation about the C8-N bond. The F<sub>o</sub>–F<sub>c</sub> maps revealed very good electron density for the propyl group involving C9, C10 and C11, but the other propyl group involving C12, C13 and C14 was disordered (Figure <xref ref-type="fig" rid="F2">2</xref>), showing two tracks of weak electron density. We tried to refine this branch with split occupancy, but the electron density after refinement was not continuous. This result suggested that this branch adopted additional conformations and that each of the two tracks of weak electron density had much less than 50% occupancy. At this point, we decided to model this branch in one partially occupied conformation rather than all of the possible conformations.</p><table-wrap position="float" id="T2"><label>Table 2</label><caption><p>X-ray diffraction data and refinement statistics</p></caption><table frame="hsides" rules="groups" border="1"><colgroup><col align="left"></col><col align="left"></col></colgroup><thead><tr><th align="left"><bold>A. X-ray Data</bold></th><th align="left"> </th></tr></thead><tbody><tr><td align="left" valign="bottom">X-ray Source<hr></hr></td><td align="left" valign="bottom">SSRL<hr></hr></td></tr><tr><td align="left" valign="bottom"> <hr></hr></td><td align="left" valign="bottom">Beam line 7–1<hr></hr></td></tr><tr><td align="left" valign="bottom">Space Group<hr></hr></td><td align="left" valign="bottom"><italic>I</italic>432<hr></hr></td></tr><tr><td align="left" valign="bottom">Cell Dimensions<hr></hr></td><td align="left" valign="bottom"> <hr></hr></td></tr><tr><td align="left" valign="bottom">a = b = c (Å)<hr></hr></td><td align="left" valign="bottom">181.0<hr></hr></td></tr><tr><td align="left" valign="bottom">Asymmetric Unit<hr></hr></td><td align="left" valign="bottom">1 monomer<hr></hr></td></tr><tr><td align="left" valign="bottom">Resolution Range (Å)<hr></hr></td><td align="left" valign="bottom">27.9–1.55<hr></hr></td></tr><tr><td align="left" valign="bottom"> <hr></hr></td><td align="left" valign="bottom">(1.63–1.55)<hr></hr></td></tr><tr><td align="left" valign="bottom"><italic>R</italic><sub>merge</sub><hr></hr></td><td align="left" valign="bottom">0.067<hr></hr></td></tr><tr><td align="left" valign="bottom"> <hr></hr></td><td align="left" valign="bottom">(0.767)*<hr></hr></td></tr><tr><td align="left" valign="bottom"><italic>R</italic><sub><italic>p.i.m.</italic></sub><italic>*</italic><hr></hr></td><td align="left" valign="bottom">0.013<hr></hr></td></tr><tr><td align="left" valign="bottom"> <hr></hr></td><td align="left" valign="bottom">(0.25)<hr></hr></td></tr><tr><td align="left" valign="bottom">mean (I/σ(I))<hr></hr></td><td align="left" valign="bottom">29.4<hr></hr></td></tr><tr><td align="left" valign="bottom"> <hr></hr></td><td align="left" valign="bottom">(3.8)<hr></hr></td></tr><tr><td align="left" valign="bottom">Multiplicity<hr></hr></td><td align="left" valign="bottom">24.7<hr></hr></td></tr><tr><td align="left" valign="bottom"> <hr></hr></td><td align="left" valign="bottom">(17.8)<hr></hr></td></tr><tr><td align="left" valign="bottom">No. Unique Reflections<hr></hr></td><td align="left" valign="bottom">72,486<hr></hr></td></tr><tr><td align="left" valign="bottom"> <hr></hr></td><td align="left" valign="bottom">(10,350)<hr></hr></td></tr><tr><td align="left" valign="bottom">Data Completeness (%)<hr></hr></td><td align="left" valign="bottom">99.9<hr></hr></td></tr><tr><td align="left" valign="bottom"> <hr></hr></td><td align="left" valign="bottom">(99.1)<hr></hr></td></tr><tr><td align="left" valign="bottom">B. Refinement<hr></hr></td><td align="left" valign="bottom"> <hr></hr></td></tr><tr><td align="left" valign="bottom"><italic>R</italic><sub><italic>work</italic></sub><hr></hr></td><td align="left" valign="bottom">0.1267<hr></hr></td></tr><tr><td align="left" valign="bottom"><italic>R</italic><sub><italic>free</italic></sub><hr></hr></td><td align="left" valign="bottom">0.1552<hr></hr></td></tr><tr><td align="left" valign="bottom">Bonds (Å)<hr></hr></td><td align="left" valign="bottom">0.008<hr></hr></td></tr><tr><td align="left" valign="bottom">Angles (˚)<hr></hr></td><td align="left" valign="bottom">1.562<hr></hr></td></tr><tr><td align="left" valign="bottom">No. Amino Acids<hr></hr></td><td align="left" valign="bottom">391<hr></hr></td></tr><tr><td align="left" valign="bottom">No. Glycan monomers<hr></hr></td><td align="left" valign="bottom">15<hr></hr></td></tr><tr><td align="left" valign="bottom">No. D-glucose molecules<hr></hr></td><td align="left" valign="bottom">4<hr></hr></td></tr><tr><td align="left">No. Waters</td><td align="left">458</td></tr></tbody></table><table-wrap-foot><p>(Statistics for the highest resolution shell are in parentheses).</p><p>*<italic>R</italic><sub><italic>merge</italic></sub> is high in the highest resolution shell due to the high multiplicity of the data. The <italic>R</italic><sub><italic>p.i.m.</italic></sub> is independent of the data multiplicity and shows that the data in the highest shell have a reasonable discrepancy of 25%. <italic>R</italic><sub><italic>p.i.m.</italic></sub>is the precision indicating <italic>R</italic><sub><italic>merge</italic></sub>[<xref ref-type="bibr" rid="B27">27</xref>].</p></table-wrap-foot></table-wrap><fig id="F2" position="float"><label>Figure 2</label><caption><p><bold>Electron density of inhibitor 2 in the active site. A, 2</bold><bold><italic>F</italic></bold><sub><bold>o</bold></sub><bold>-</bold><bold><italic>F</italic></bold><sub><bold>c</bold></sub><bold>map of inhibitor contoured at 1σ shows inhibitor fitting snugly in the active site cavity; B, 2</bold><bold><italic>F</italic></bold><sub><bold>o</bold></sub><bold>-</bold><bold><italic>F</italic></bold><sub><bold>c</bold></sub><bold>map of inhibitor and interacting amino acids of the NA.</bold></p></caption><graphic xlink:href="1472-6807-12-7-2"></graphic></fig><p>Potential interactions between inhibitor <bold>2</bold> and N9 NA were assessed using <italic>Chimera</italic>[<xref ref-type="bibr" rid="B28">28</xref>]. In addition, potential hydrogen bonds as well as hydrophobic contacts were identified with <italic>HBPLUS</italic>[<xref ref-type="bibr" rid="B29">29</xref>] (see Table <xref ref-type="table" rid="T3">3</xref> for the geometric criteria). Favorable hydrophobic contacts were defined as non-bonded contacts between two carbon atoms at a distance of ≤4 Å [<xref ref-type="bibr" rid="B30">30</xref>].</p><table-wrap position="float" id="T3"><label>Table 3</label><caption><p>Contacts observed between inhibitor 2 and N9 NA; 3LV is the residue name assigned to the inhibitor by the PDB and 488 is the residue number of the inhibitor</p></caption><table frame="hsides" rules="groups" border="1"><colgroup><col align="left"></col><col align="left"></col><col align="left"></col><col align="left"></col><col align="left"></col></colgroup><thead><tr><th align="left"><bold>Inhibitor atom</bold></th><th align="left"><bold>N9 atom</bold></th><th align="left"><bold>distance (Å)</bold></th><th align="left"><bold>Angle(D-A-AA)°</bold></th><th align="left"> </th></tr></thead><tbody><tr><td colspan="5" align="left" valign="bottom">Potential hydrogen bonds<sup>1</sup><hr></hr></td></tr><tr><td align="left" valign="bottom">3LV488-O1<hr></hr></td><td align="left" valign="bottom">Arg118-NH1<hr></hr></td><td align="left" valign="bottom">2.84<hr></hr></td><td align="left" valign="bottom">117.3<hr></hr></td><td align="left" valign="bottom"> <hr></hr></td></tr><tr><td align="left" valign="bottom">3LV488-O1<hr></hr></td><td align="left" valign="bottom">Arg371-NH1<hr></hr></td><td align="left" valign="bottom">2.86<hr></hr></td><td align="left" valign="bottom">112.8<hr></hr></td><td align="left" valign="bottom"> <hr></hr></td></tr><tr><td align="left" valign="bottom">3LV488-O2<hr></hr></td><td align="left" valign="bottom">Arg292-NH1<hr></hr></td><td align="left" valign="bottom">3.34<hr></hr></td><td align="left" valign="bottom">97.1<hr></hr></td><td align="left" valign="bottom"> <hr></hr></td></tr><tr><td align="left" valign="bottom">3LV488-O2<hr></hr></td><td align="left" valign="bottom">Arg292-NH2<hr></hr></td><td align="left" valign="bottom">3.25<hr></hr></td><td align="left" valign="bottom">101.2<hr></hr></td><td align="left" valign="bottom"> <hr></hr></td></tr><tr><td align="left" valign="bottom">3LV488-O2<hr></hr></td><td align="left" valign="bottom">Arg371-NH2<hr></hr></td><td align="left" valign="bottom">2.84<hr></hr></td><td align="left" valign="bottom">124.9<hr></hr></td><td align="left" valign="bottom"> <hr></hr></td></tr><tr><td align="left" valign="bottom">3LV488-O2<hr></hr></td><td align="left" valign="bottom">HOH-747<hr></hr></td><td align="left" valign="bottom">2.85<hr></hr></td><td align="left" valign="bottom"> <hr></hr></td><td align="left" valign="bottom"> <hr></hr></td></tr><tr><td align="left" valign="bottom"> <hr></hr></td><td align="left" valign="bottom">HOH-747<hr></hr></td><td align="left" valign="bottom">3.25<hr></hr></td><td align="left" valign="bottom"> <hr></hr></td><td align="left" valign="bottom">Asn347 OD1<hr></hr></td></tr><tr><td align="left" valign="bottom">3LV488-O15<hr></hr></td><td align="left" valign="bottom">Arg152- NH1<hr></hr></td><td align="left" valign="bottom">2.54<hr></hr></td><td align="left" valign="bottom">145.9<hr></hr></td><td align="left" valign="bottom"> <hr></hr></td></tr><tr><td align="left" valign="bottom">3LV488-O20<hr></hr></td><td align="left" valign="bottom">HOH-612<hr></hr></td><td align="left" valign="bottom">2.72<hr></hr></td><td align="left" valign="bottom"> <hr></hr></td><td align="left" valign="bottom"> <hr></hr></td></tr><tr><td align="left" valign="bottom"> <hr></hr></td><td align="left" valign="bottom">HOH-612<hr></hr></td><td align="left" valign="bottom">2.82, 3.16<hr></hr></td><td align="left" valign="bottom"> <hr></hr></td><td align="left" valign="bottom">Glu227 OE2, Thr225 O<hr></hr></td></tr><tr><td colspan="5" align="left" valign="bottom">Van der Waals contacts<sup>2</sup> (≤4.0 Å)<hr></hr></td></tr><tr><td align="left" valign="bottom">3LV488-C1<hr></hr></td><td align="left" valign="bottom">Tyr406-OH<hr></hr></td><td align="left" valign="bottom">2.97<hr></hr></td><td align="left" valign="bottom">119.4<hr></hr></td><td align="left" valign="bottom"> <hr></hr></td></tr><tr><td align="left" valign="bottom"> <hr></hr></td><td align="left" valign="bottom">Arg371-NH1<hr></hr></td><td align="left" valign="bottom">3.70<hr></hr></td><td align="left" valign="bottom">96.2<hr></hr></td><td align="left" valign="bottom"> <hr></hr></td></tr><tr><td align="left" valign="bottom"> <hr></hr></td><td align="left" valign="bottom">Arg371-NH2<hr></hr></td><td align="left" valign="bottom">3.49<hr></hr></td><td align="left" valign="bottom">106.5<hr></hr></td><td align="left" valign="bottom"> <hr></hr></td></tr><tr><td align="left" valign="bottom"> <hr></hr></td><td align="left" valign="bottom">Tyr406-CZ<hr></hr></td><td align="left" valign="bottom">3.83<hr></hr></td><td align="left" valign="bottom">42.5<hr></hr></td><td align="left" valign="bottom"> <hr></hr></td></tr><tr><td align="left" valign="bottom">3LV488-O1<hr></hr></td><td align="left" valign="bottom">Tyr406-OH<hr></hr></td><td align="left" valign="bottom">3.50<hr></hr></td><td align="left" valign="bottom">99.8<hr></hr></td><td align="left" valign="bottom"> <hr></hr></td></tr><tr><td align="left" valign="bottom"> <hr></hr></td><td align="left" valign="bottom">Arg118-CZ<hr></hr></td><td align="left" valign="bottom">3.65<hr></hr></td><td align="left" valign="bottom">43.7<hr></hr></td><td align="left" valign="bottom"> <hr></hr></td></tr><tr><td align="left" valign="bottom"> <hr></hr></td><td align="left" valign="bottom">Arg118-NH2<hr></hr></td><td align="left" valign="bottom">3.56<hr></hr></td><td align="left" valign="bottom">83.0<hr></hr></td><td align="left" valign="bottom"> <hr></hr></td></tr><tr><td align="left" valign="bottom"> <hr></hr></td><td align="left" valign="bottom">Arg371-CZ<hr></hr></td><td align="left" valign="bottom">3.59<hr></hr></td><td align="left" valign="bottom">47.2<hr></hr></td><td align="left" valign="bottom"> <hr></hr></td></tr><tr><td align="left" valign="bottom"> <hr></hr></td><td align="left" valign="bottom">Arg371-NH2<hr></hr></td><td align="left" valign="bottom">3.45<hr></hr></td><td align="left" valign="bottom">85.5<hr></hr></td><td align="left" valign="bottom"> <hr></hr></td></tr><tr><td align="left" valign="bottom">3LV488-C2<hr></hr></td><td align="left" valign="bottom">Tyr406-OH<hr></hr></td><td align="left" valign="bottom">2.80<hr></hr></td><td align="left" valign="bottom">136.4<hr></hr></td><td align="left" valign="bottom"> <hr></hr></td></tr><tr><td align="left" valign="bottom">3LV488-O2<hr></hr></td><td align="left" valign="bottom">Tyr406-OH<hr></hr></td><td align="left" valign="bottom">3.32<hr></hr></td><td align="left" valign="bottom">123.9<sup>3</sup><hr></hr></td><td align="left" valign="bottom"> <hr></hr></td></tr><tr><td align="left" valign="bottom"> <hr></hr></td><td align="left" valign="bottom">Arg371-CZ<hr></hr></td><td align="left" valign="bottom">3.76<hr></hr></td><td align="left" valign="bottom">38.3<hr></hr></td><td align="left" valign="bottom"> <hr></hr></td></tr><tr><td align="left" valign="bottom"> <hr></hr></td><td align="left" valign="bottom">Arg371-NH1<hr></hr></td><td align="left" valign="bottom">3.82<hr></hr></td><td align="left" valign="bottom">96.2<hr></hr></td><td align="left" valign="bottom"> <hr></hr></td></tr><tr><td align="left" valign="bottom">3LV488-C3<hr></hr></td><td align="left" valign="bottom">Tyr406-OH<hr></hr></td><td align="left" valign="bottom">3.19<hr></hr></td><td align="left" valign="bottom">161.8<hr></hr></td><td align="left" valign="bottom"> <hr></hr></td></tr><tr><td align="left" valign="bottom">3LV488-C6<hr></hr></td><td align="left" valign="bottom">Glu119-OE2<hr></hr></td><td align="left" valign="bottom">3.39<hr></hr></td><td align="left" valign="bottom">119.6<hr></hr></td><td align="left" valign="bottom"> <hr></hr></td></tr><tr><td align="left" valign="bottom"> <hr></hr></td><td align="left" valign="bottom">Asp151-CG<hr></hr></td><td align="left" valign="bottom">3.49<hr></hr></td><td align="left" valign="bottom">74.6<hr></hr></td><td align="left" valign="bottom"> <hr></hr></td></tr><tr><td align="left" valign="bottom"> <hr></hr></td><td align="left" valign="bottom">Asp151-OD1<hr></hr></td><td align="left" valign="bottom">3.37<hr></hr></td><td align="left" valign="bottom">84.4<hr></hr></td><td align="left" valign="bottom"> <hr></hr></td></tr><tr><td align="left" valign="bottom"> <hr></hr></td><td align="left" valign="bottom">Asp151-OD2<hr></hr></td><td align="left" valign="bottom">3.87<hr></hr></td><td align="left" valign="bottom">62.7<hr></hr></td><td align="left" valign="bottom"> <hr></hr></td></tr><tr><td align="left" valign="bottom">3LV488-C7<hr></hr></td><td align="left" valign="bottom">Glu119-OE2<hr></hr></td><td align="left" valign="bottom">3.49<hr></hr></td><td align="left" valign="bottom">136.6<hr></hr></td><td align="left" valign="bottom"> <hr></hr></td></tr><tr><td align="left" valign="bottom"> <hr></hr></td><td align="left" valign="bottom">Asp151-OD1<hr></hr></td><td align="left" valign="bottom">3.52<hr></hr></td><td align="left" valign="bottom">97.7<hr></hr></td><td align="left" valign="bottom"> <hr></hr></td></tr><tr><td align="left" valign="bottom"> <hr></hr></td><td align="left" valign="bottom">Asp151-CG<hr></hr></td><td align="left" valign="bottom">3.89<hr></hr></td><td align="left" valign="bottom">63.8<hr></hr></td><td align="left" valign="bottom"> <hr></hr></td></tr><tr><td align="left" valign="bottom"> <hr></hr></td><td align="left" valign="bottom">Tyr406-OH<hr></hr></td><td align="left" valign="bottom">3.23<hr></hr></td><td align="left" valign="bottom">118.0<hr></hr></td><td align="left" valign="bottom"> <hr></hr></td></tr><tr><td align="left" valign="bottom"> <hr></hr></td><td align="left" valign="bottom">Arg118-NH1<hr></hr></td><td align="left" valign="bottom">3.63<hr></hr></td><td align="left" valign="bottom">113.4<hr></hr></td><td align="left" valign="bottom"> <hr></hr></td></tr><tr><td align="left" valign="bottom">3LV488-C10<hr></hr></td><td align="left" valign="bottom">Ile222-CD1<hr></hr></td><td align="left" valign="bottom">3.63<hr></hr></td><td align="left" valign="bottom">122.8<hr></hr></td><td align="left" valign="bottom"> <hr></hr></td></tr><tr><td align="left" valign="bottom">3LV488-C11<hr></hr></td><td align="left" valign="bottom">Ala246-CB<hr></hr></td><td align="left" valign="bottom">3.90<hr></hr></td><td align="left" valign="bottom">115.5<hr></hr></td><td align="left" valign="bottom"> <hr></hr></td></tr><tr><td align="left" valign="bottom"> <hr></hr></td><td align="left" valign="bottom">Ile222-CD1<hr></hr></td><td align="left" valign="bottom">3.54<hr></hr></td><td align="left" valign="bottom">105.5<hr></hr></td><td align="left" valign="bottom"> <hr></hr></td></tr><tr><td align="left" valign="bottom"> <hr></hr></td><td align="left" valign="bottom">Ile222-CB<hr></hr></td><td align="left" valign="bottom">3.88<hr></hr></td><td align="left" valign="bottom">87.6<hr></hr></td><td align="left" valign="bottom"> <hr></hr></td></tr><tr><td align="left" valign="bottom"> <hr></hr></td><td align="left" valign="bottom">Arg224-CZ<hr></hr></td><td align="left" valign="bottom">3.43<hr></hr></td><td align="left" valign="bottom">79.4<hr></hr></td><td align="left" valign="bottom"> <hr></hr></td></tr><tr><td align="left" valign="bottom"> <hr></hr></td><td align="left" valign="bottom">Arg224-NE<hr></hr></td><td align="left" valign="bottom">3.78<hr></hr></td><td align="left" valign="bottom">64.8<hr></hr></td><td align="left" valign="bottom"> <hr></hr></td></tr><tr><td align="left" valign="bottom"> <hr></hr></td><td align="left" valign="bottom">Arg224-NH1<hr></hr></td><td align="left" valign="bottom">3.44<hr></hr></td><td align="left" valign="bottom">78.4<hr></hr></td><td align="left" valign="bottom"> <hr></hr></td></tr><tr><td align="left" valign="bottom"> <hr></hr></td><td align="left" valign="bottom">Arg224-NH2<hr></hr></td><td align="left" valign="bottom">3.81<hr></hr></td><td align="left" valign="bottom">63.4<hr></hr></td><td align="left" valign="bottom"> <hr></hr></td></tr><tr><td align="left" valign="bottom"> <hr></hr></td><td align="left" valign="bottom">HOH-945<hr></hr></td><td align="left" valign="bottom">2.82<hr></hr></td><td align="left" valign="bottom">---<hr></hr></td><td align="left" valign="bottom"> <hr></hr></td></tr><tr><td align="left" valign="bottom">3LV488-C14<hr></hr></td><td align="left" valign="bottom">Arg292-NH2<sup>4</sup><hr></hr></td><td align="left" valign="bottom">3.50<hr></hr></td><td align="left" valign="bottom">140.0<hr></hr></td><td align="left" valign="bottom"> <hr></hr></td></tr><tr><td align="left" valign="bottom">3LV488-O15<hr></hr></td><td align="left" valign="bottom">Asp151-CB<hr></hr></td><td align="left" valign="bottom">3.59<hr></hr></td><td align="left" valign="bottom">100.5<hr></hr></td><td align="left" valign="bottom"> <hr></hr></td></tr><tr><td align="left" valign="bottom"> <hr></hr></td><td align="left" valign="bottom">Arg152-CG<hr></hr></td><td align="left" valign="bottom">3.84<hr></hr></td><td align="left" valign="bottom">73.6<hr></hr></td><td align="left" valign="bottom"> <hr></hr></td></tr><tr><td align="left" valign="bottom"> <hr></hr></td><td align="left" valign="bottom">Arg152-CD<hr></hr></td><td align="left" valign="bottom">3.71<hr></hr></td><td align="left" valign="bottom">83.0<hr></hr></td><td align="left" valign="bottom"> <hr></hr></td></tr><tr><td align="left" valign="bottom"> <hr></hr></td><td align="left" valign="bottom">Arg152-CZ<hr></hr></td><td align="left" valign="bottom">3.70<hr></hr></td><td align="left" valign="bottom">22.6<hr></hr></td><td align="left" valign="bottom"> <hr></hr></td></tr><tr><td align="left" valign="bottom">3LV488-C16<hr></hr></td><td align="left" valign="bottom">Trp178-CE3<hr></hr></td><td align="left" valign="bottom">3.59<hr></hr></td><td align="left" valign="bottom">91.2<hr></hr></td><td align="left" valign="bottom"> <hr></hr></td></tr><tr><td align="left" valign="bottom"> <hr></hr></td><td align="left" valign="bottom">Trp178-CZ3<hr></hr></td><td align="left" valign="bottom">3.88<hr></hr></td><td align="left" valign="bottom">67.8<hr></hr></td><td align="left" valign="bottom"> <hr></hr></td></tr><tr><td align="left" valign="bottom"> <hr></hr></td><td align="left" valign="bottom">Trp178-O<hr></hr></td><td align="left" valign="bottom">3.78<hr></hr></td><td align="left" valign="bottom">135.7<hr></hr></td><td align="left" valign="bottom"> <hr></hr></td></tr><tr><td align="left" valign="bottom"> <hr></hr></td><td align="left" valign="bottom">Arg152-CD<hr></hr></td><td align="left" valign="bottom">3.73<hr></hr></td><td align="left" valign="bottom">78.1<hr></hr></td><td align="left" valign="bottom"> <hr></hr></td></tr><tr><td align="left" valign="bottom"> <hr></hr></td><td align="left" valign="bottom">Arg152-CG<hr></hr></td><td align="left" valign="bottom">3.73<hr></hr></td><td align="left" valign="bottom">78.1<hr></hr></td><td align="left" valign="bottom"> <hr></hr></td></tr><tr><td align="left" valign="bottom">3LV488-C17<hr></hr></td><td align="left" valign="bottom">Trp178-O<hr></hr></td><td align="left" valign="bottom">3.43<hr></hr></td><td align="left" valign="bottom">139.6<hr></hr></td><td align="left" valign="bottom"> <hr></hr></td></tr><tr><td align="left" valign="bottom"> <hr></hr></td><td align="left" valign="bottom">Glu227-OE2<hr></hr></td><td align="left" valign="bottom">3.62<hr></hr></td><td align="left" valign="bottom">135.8<hr></hr></td><td align="left" valign="bottom"> <hr></hr></td></tr><tr><td align="left" valign="bottom">3LV488-C19<hr></hr></td><td align="left" valign="bottom">Trp178-O<hr></hr></td><td align="left" valign="bottom">3.19<hr></hr></td><td align="left" valign="bottom">167.7<hr></hr></td><td align="left" valign="bottom"> <hr></hr></td></tr><tr><td align="left" valign="bottom"> <hr></hr></td><td align="left" valign="bottom">Glu119-CD<hr></hr></td><td align="left" valign="bottom">3.88<hr></hr></td><td align="left" valign="bottom">85.8<hr></hr></td><td align="left" valign="bottom"> <hr></hr></td></tr><tr><td align="left" valign="bottom">3LV488-O19<hr></hr></td><td align="left" valign="bottom">Asp151-O<hr></hr></td><td align="left" valign="bottom">3.26<hr></hr></td><td align="left" valign="bottom">116.7<hr></hr></td><td align="left" valign="bottom"> <hr></hr></td></tr><tr><td align="left" valign="bottom"> <hr></hr></td><td align="left" valign="bottom">Asp151-CB<hr></hr></td><td align="left" valign="bottom">3.73<hr></hr></td><td align="left" valign="bottom">97.2<hr></hr></td><td align="left" valign="bottom"> <hr></hr></td></tr><tr><td align="left" valign="bottom"> <hr></hr></td><td align="left" valign="bottom">Arg156-NH1<hr></hr></td><td align="left" valign="bottom">3.68<hr></hr></td><td align="left" valign="bottom">139.0<hr></hr></td><td align="left" valign="bottom"> <hr></hr></td></tr><tr><td align="left" valign="bottom"> <hr></hr></td><td align="left" valign="bottom">Trp178-O<hr></hr></td><td align="left" valign="bottom">2.82<hr></hr></td><td align="left" valign="bottom">162.5<hr></hr></td><td align="left" valign="bottom"> <hr></hr></td></tr><tr><td align="left" valign="bottom">3LV488-C20<hr></hr></td><td align="left" valign="bottom">Glu227-CG<hr></hr></td><td align="left" valign="bottom">3.88<hr></hr></td><td align="left" valign="bottom">74.3<hr></hr></td><td align="left" valign="bottom"> <hr></hr></td></tr><tr><td align="left" valign="bottom"> <hr></hr></td><td align="left" valign="bottom">Glu227-CD<hr></hr></td><td align="left" valign="bottom">3.77<hr></hr></td><td align="left" valign="bottom">55.7<hr></hr></td><td align="left" valign="bottom"> <hr></hr></td></tr><tr><td align="left" valign="bottom"> <hr></hr></td><td align="left" valign="bottom">Glu227-OE2<hr></hr></td><td align="left" valign="bottom">3.23<hr></hr></td><td align="left" valign="bottom">105.4<hr></hr></td><td align="left" valign="bottom"> <hr></hr></td></tr><tr><td align="left" valign="bottom"> <hr></hr></td><td align="left" valign="bottom">Glu277-OE2<hr></hr></td><td align="left" valign="bottom">3.34<hr></hr></td><td align="left" valign="bottom">140.2<hr></hr></td><td align="left" valign="bottom"> <hr></hr></td></tr><tr><td align="left" valign="bottom">3LV488-O20<hr></hr></td><td align="left" valign="bottom">Glu227-OE2<hr></hr></td><td align="left" valign="bottom">3.68<hr></hr></td><td align="left" valign="bottom">115.2<hr></hr></td><td align="left" valign="bottom"> <hr></hr></td></tr><tr><td align="left" valign="bottom"> <hr></hr></td><td align="left" valign="bottom">Glu277-CG<hr></hr></td><td align="left" valign="bottom">3.80<hr></hr></td><td align="left" valign="bottom">70.4<hr></hr></td><td align="left" valign="bottom"> <hr></hr></td></tr><tr><td align="left" valign="bottom"> <hr></hr></td><td align="left" valign="bottom">Glu277-CD<hr></hr></td><td align="left" valign="bottom">3.59<hr></hr></td><td align="left" valign="bottom">31.9<hr></hr></td><td align="left" valign="bottom"> <hr></hr></td></tr><tr><td align="left" valign="bottom"> <hr></hr></td><td align="left" valign="bottom">Glu277-OE2<hr></hr></td><td align="left" valign="bottom">2.61<hr></hr></td><td align="left" valign="bottom">133.4<hr></hr></td><td align="left" valign="bottom"> <hr></hr></td></tr><tr><td colspan="5" align="left" valign="bottom">Water bridge – inhibitor – Glu276<hr></hr></td></tr><tr><td align="left" valign="bottom">3LV488-O20<hr></hr></td><td align="left" valign="bottom">HOH-612<hr></hr></td><td align="left" valign="bottom">2.72<hr></hr></td><td align="left" valign="bottom"> <hr></hr></td><td align="left" valign="bottom"> <hr></hr></td></tr><tr><td align="left" valign="bottom">612-HOH<hr></hr></td><td align="left" valign="bottom">553-HOH<hr></hr></td><td align="left" valign="bottom">2.64<hr></hr></td><td align="left" valign="bottom"> <hr></hr></td><td align="left" valign="bottom"> <hr></hr></td></tr><tr><td align="left" valign="bottom">553-HOH<hr></hr></td><td align="left" valign="bottom">Glu276-OE1<hr></hr></td><td align="left" valign="bottom">2.82<hr></hr></td><td align="left" valign="bottom">142.2<hr></hr></td><td align="left" valign="bottom"> <hr></hr></td></tr><tr><td colspan="5" align="left" valign="bottom">Glu276 interactions<hr></hr></td></tr><tr><td align="left" valign="bottom">Glu276-OE1<hr></hr></td><td align="left" valign="bottom">Arg224-NE<hr></hr></td><td align="left" valign="bottom">2.72<hr></hr></td><td align="left" valign="bottom">114.2<hr></hr></td><td align="left" valign="bottom"> <hr></hr></td></tr><tr><td align="left" valign="bottom">Glu276-OE2<hr></hr></td><td align="left" valign="bottom">Arg224-NE<hr></hr></td><td align="left" valign="bottom">3.50<hr></hr></td><td align="left" valign="bottom">83.2<hr></hr></td><td align="left" valign="bottom"> <hr></hr></td></tr><tr><td align="left" valign="bottom">Glu276-OE2<hr></hr></td><td align="left" valign="bottom">Arg224-NH2<hr></hr></td><td align="left" valign="bottom">2.81<hr></hr></td><td align="left" valign="bottom">115.9<hr></hr></td><td align="left" valign="bottom"> <hr></hr></td></tr><tr><td align="left">Glu276-OE2</td><td align="left">His274-NE2</td><td align="left">2.73</td><td align="left">119.7</td><td align="left"> </td></tr></tbody></table><table-wrap-foot><p><sup>1</sup>Potential hydrogen bonds were identified using <italic>HBPLUS</italic>[<xref ref-type="bibr" rid="B29">29</xref>]. Potential hydrogen bonds were interactions between donor and acceptor atoms that met the following geometric requirements: (i) a donor acceptor distance (D-A) < 3.5 Å and a hydrogen acceptor distance (H-A) < 2.5 Å and (ii) a donor-acceptor-acceptor antecedent angle (D-A-AA), of > 90° [<xref ref-type="bibr" rid="B31">31</xref>]<bold>.</bold></p><p><sup>2</sup>Favorable hydrophobic contacts were defined as non-bonded contacts between two carbon atoms at a distance of ≤4 Å [28].</p><p><sup>3</sup>Freely rotating hydroxyl hydrogen atoms had the potential to form hydrogen bonds with two alternative acceptor atoms. Only the more probable acceptor atom at the shorter distance from the hydrogen atom was reported by <italic>HBPLUS.</italic></p><p><sup>4</sup>There were several conformations of the propyl chain C12-C14. We used the best, but it is of low occupancy so the interactions involving C14 contribute very little.</p></table-wrap-foot></table-wrap><p>Structural studies of complexes between several benzoic acid leads and type B NA showed that these inhibitors were bound in the active site in a similar fashion to sialic acid [<xref ref-type="bibr" rid="B21">21</xref>,<xref ref-type="bibr" rid="B32">32</xref>], and the same is observed in the new structure (Figure <xref ref-type="fig" rid="F3">3</xref>). The carboxylate group of the inhibitor interacts with the guanidinium groups of arginine residues at positions 118, 292, and 371 as seen in all NA substrate and inhibitor complexes and calculated to be energetically important [<xref ref-type="bibr" rid="B33">33</xref>]. A weak hydrogen bond is seen between Tyr406 and the carbonyl oxygen that interacts with Arg292. The pyrrolidine ring interacts with Arg152 by forming a hydrogen bond with its carbonyl oxygen (O15). The methylene group C16 lies in the hydrophobic pocket formed by Trp178 and Arg152. The methylene group C17 is involved in C-H<sup>…</sup>O bonds with Trp178 carbonyl O and Glu227 OE2. One of the hydroxyl methyl groups (O20) is hydrogen bonded to Glu277. Atom O20 also interacts with Glu276 through two water molecules, HOH553 and HOH612, by a chain of hydrogen bonds. The other hydroxyl methyl group (O19) was directed toward Trp178 O and Glu119 OE2. The one well-ordered propyl chain is anchored by hydrophobic contacts with Ile222, Ala246, and Arg224. The other propyl chain that is disordered is exposed to the solvent and therefore not contributing to binding energy because the one interaction seen (Table <xref ref-type="table" rid="T3">3</xref>) is of low occupancy and at the upper limit of the distances considered as significant.</p><fig id="F3" position="float"><label>Figure 3</label><caption><p><bold>Interactions between compound 2 and NA.</bold><bold><italic>Ligplot +</italic></bold> [<xref ref-type="bibr" rid="B34">34</xref>]<bold>diagram showing interactions.</bold></p></caption><graphic xlink:href="1472-6807-12-7-3"></graphic></fig></sec><sec><title>Comparative analysis</title><p>The model of <bold>2</bold> complexed with N9 NA was compared to sialic acid complexes with A/Tokyo/3/1967 N2 NA, PDB ID: 2BAT [<xref ref-type="bibr" rid="B35">35</xref>] and A/tern/Australia/G70c/75 N9 NA, PDB ID: 1MWE [<xref ref-type="bibr" rid="B26">26</xref>]. When our new structure was fitted with CCP4's <italic>SUPERPOSE</italic>[<xref ref-type="bibr" rid="B36">36</xref>] using the 11 active site residues, the root mean square deviations (RMSDs) were 0.7 Å to N2 and 0.4 Å to N9. The model gave a RMSD of 1.65 Å when superposed on the complex of compound <bold>1</bold> with B/Lee/40 NA [<xref ref-type="bibr" rid="B21">21</xref>] using <italic>COOT</italic> secondary structure matching [<xref ref-type="bibr" rid="B37">37</xref>]. These small RMSDs suggest that replacing the sialic acid ligand with the inhibitor did not disturb the orientation of the active site residues of NA. The larger deviation between N9 and B NAs was expected given that there is less than 30% sequence identity. In all the above comparisons, most of the active site residues (Asn151, Arg152, Glu227, Arg371, Arg292 and Arg118—numbering as in the current complex) superposed well in the two molecules and a maximum shift of 0.2 to 0.5 Å was observed. However, the side chain of Glu276 showed significant conformational change in the current complex when compared to NA-sialic acid or NA-zanamivir complexes. The two oxygen atoms OE1 and OE2 of Glu227 in the current complex moved toward the solvent and away from the active site by 1 Å. In this position, the carboxyl group interacted with NE of Arg224 and NH2 of His274. Hence, Glu276 did not form the direct hydrogen bonds with the inhibitor hydroxyl oxygen O20 analogous to those that Glu276 formed with the glycerol side chain of sialic acid and its transition state mimics. However, O20 of the inhibitor was linked to Glu276 through the water molecules HOH552 and HOH611. The C14 atom of the inhibitor is seen to make a hydrophobic contact with Glu276 but the low occupancy of the C12-C14 chain precludes a significant contribution to binding.</p><p>In the compound <bold>1</bold> complex with influenza B NA [<xref ref-type="bibr" rid="B21">21</xref>], the aliphatic chain forms van der Waals contacts with the side chains of Arg292, Asn294 and Glu275 while the hydroxymethyl groups interact with Glu117, Trp177 and Glu276. The rotation of the Glu276 side chain towards Arg224 observed in our complex was noted in the other structures where the inhibitor carries a hydrophobic side chain [<xref ref-type="bibr" rid="B21">21</xref>]. N1 NAs have additional flexibility compared to N9 in the 150 loop but binding of oseltamivir to wild-type N1 NA involves a conformational change in the side chain of Glu276 relative to the ligand free enzyme [<xref ref-type="bibr" rid="B20">20</xref>,<xref ref-type="bibr" rid="B38">38</xref>] similar to that seen in N9 NAs.</p><p>We compared the NA and inhibitor contacts with previously reported benzoic acid inhibitor-NA structures using <italic>Chimera</italic> with the relatively stringent constraint of distance ≤3.5 Å and including both polar and hydrophobic contacts. In the BANA 113-B NA complex [<xref ref-type="bibr" rid="B15">15</xref>][<xref ref-type="bibr" rid="B39">39</xref>], 12 drug atom made 21 contacts ≤3.5 Å with 10 amino acids of NA. In <bold>1</bold>-B NA [<xref ref-type="bibr" rid="B21">21</xref>], 14 drug atoms make 23 contacts with 13 amino acids. Inhibitor <bold>2</bold> shows a small increase to 15 drug atoms making 28 contacts with 12 amino acids. The benzene ring of <bold>2</bold> is tilted by 8.9° relative to compound <bold>1</bold> (Figure <xref ref-type="fig" rid="F4">4</xref>), increasing the number of contacts as was predicted in the design. However, one branch of the 3-heptyl group makes no significant contacts due to multiple conformations, which may be why the IC50 is no better than the previous compounds (Table <xref ref-type="table" rid="T1">1</xref>).</p><fig id="F4" position="float"><label>Figure 4</label><caption><p><bold>Bound configurations of Compound 1 (PDB ID 1B9V; cyan) compared to compound 2 (magenta).</bold> The proteins of the complex structures were aligned using <italic>DaliLite</italic>[<xref ref-type="bibr" rid="B40">40</xref>]. The view is in the plane of the benzene ring of <bold>1</bold>, showing that the benzene ring of <bold>2</bold> is slightly rotated as well as tilted 8.9° compared to <bold>1</bold>.</p></caption><graphic xlink:href="1472-6807-12-7-4"></graphic></fig><p>Inhibitor <bold>2</bold> did not bind to the second sialic acid binding site observed in the structure of N9 complexed with sialic acid at low temperature [<xref ref-type="bibr" rid="B26">26</xref>] (PDB ID, 1MWE) or at room temperature (PDB ID, 2CML).</p></sec><sec><title>Glycan structures</title><p>When compared to the structure of the N9 mutant R292K (PDB ID, 2QWA) and the 1.4 Å resolution structures of native N9 in complex with other inhibitors (PDB IDs, 1F8D and1F8E), our structure has four additional sugar units in the glycan chains attached to the delta nitrogen atoms of Asn146 and Asn200 (N2 numbering) (Figure <xref ref-type="fig" rid="F5">5</xref>). At site 146, we found a second NAG residue and evidence for a β-D-mannose (BMA). At site 200, we found two additional mannose residues. One mannose (Man477) is bonded to the O6 oxygen of Man475G. The second mannose (Man476H) is linked to the O3 oxygen atom of Man475G, resulting in a GlcNAc<sub>2</sub>-Man<sub>7</sub> structure (Figure <xref ref-type="fig" rid="F5">5B</xref>). This glycan attached to Asn200 contacts 11 amino acids of the neighboring subunit when the tetramer is built by symmetry (Table <xref ref-type="table" rid="T4">4</xref> and Figure <xref ref-type="fig" rid="F5">5C</xref>). A high-mannose glycan at Asn200 is also present in N2 (A/Tokyo/67) NA, where it interacts with the adjacent symmetry-related subunit at amino acids 391–394 and 453–455 (PDB ID, 1NN2). This glycan forms part of the Mem5 monoclonal antibody epitope on the 1998 N2 NA, and its interactions with the adjacent subunit are not disturbed by the presence of the antibody [<xref ref-type="bibr" rid="B41">41</xref>], suggesting the subunit interactions are energetically significant. In N9 there is experimental evidence that the glycan attached to Asn200 contributes to folding or oligomerization because the mutation N200L in N9 NA resulted in 80% reduction in enzyme activity although the expression level was the same as wild type [<xref ref-type="bibr" rid="B42">42</xref>]. In both N2 and N9 this subunit-spanning glycan is of the high mannose type, as is the glycan at 86 in N2 [<xref ref-type="bibr" rid="B43">43</xref>]. N6 NA (PDB ID 1V0Z) crystallized with the tetramer as the asymmetric unit, so there is direct evidence for the inter-subunit interaction of the glycan attached to Asn200 in N6 NA. N1 NAs do not have a predicted glycosylation site at 200, but a study of N1 NA assembly suggested that tetramerization requires high-mannose glycans. When all the glycans were processed to complex structures then the resulting dimers and monomers did not assemble into tetramers [<xref ref-type="bibr" rid="B44">44</xref>]. The PDB structures of N1 NA have no density for sugar residues, so it is not known if one of the other glycans is a high-mannose structure that spans the subunit interface in the same way as the glycan at Asn200 in N9, N2 and N6.</p><fig id="F5" position="float"><label>Figure 5</label><caption><p><bold>A. Carbohydrates in the N9-compound 2 model at 1.55 Å resolution.</bold> Carbon atoms are green for N9 NA and the N-linked glycans attached at positions 86, 146 and 200 that are in common with other structures. Mannose residues not previously seen in N9 structures (blue) and the four glucose molecules from the cryoprotectant (yellow) are shown. Compound <bold>2</bold> is colored gray. <bold>B.</bold> Electron density (2<italic>F</italic><sub>o</sub><italic>F</italic><sub>c</sub> at 1σ) for the glycan at Asn200. Also shown is the cartoon of the glycan structure using standard Consortium for Functional Glycomics symbols (green circle, mannose; blue square, <italic>N</italic>-acetylglucosamine) made with Glycan Builder [<xref ref-type="bibr" rid="B45">45</xref>]. <bold>C.</bold> Interaction of the glycan attached to Asn200 (green) with the neighboring subunit (gray) built by applying crystallographic symmetry.</p></caption><graphic xlink:href="1472-6807-12-7-5"></graphic></fig><table-wrap position="float" id="T4"><label>Table 4</label><caption><p>Contacts <3.5 Å between the high mannose glycan at Asn200 and the adjacent subunit built as a symmetry-related monomer</p></caption><table frame="hsides" rules="groups" border="1"><colgroup><col align="left"></col><col align="left"></col><col align="left"></col><col align="left"></col><col align="left"></col></colgroup><thead><tr><th align="left"><bold>Sugar</bold></th><th align="left"><bold>Sugar atom</bold></th><th align="left"><bold>Amino acid</bold></th><th align="left"><bold>Atom</bold></th><th align="left"><bold>Distance (Å)</bold></th></tr></thead><tbody><tr><td align="left" valign="bottom">NAG469A<hr></hr></td><td align="left" valign="bottom">O5<hr></hr></td><td align="left" valign="bottom">G454<hr></hr></td><td align="left" valign="bottom">CA<hr></hr></td><td align="left" valign="bottom">3.39<hr></hr></td></tr><tr><td align="left" valign="bottom"> <hr></hr></td><td align="left" valign="bottom"> <hr></hr></td><td align="left" valign="bottom">Q455<hr></hr></td><td align="left" valign="bottom">N<hr></hr></td><td align="left" valign="bottom">3.35<hr></hr></td></tr><tr><td align="left" valign="bottom"> <hr></hr></td><td align="left" valign="bottom">C6<hr></hr></td><td align="left" valign="bottom">L453<hr></hr></td><td align="left" valign="bottom">O<hr></hr></td><td align="left" valign="bottom">3.43<hr></hr></td></tr><tr><td align="left" valign="bottom"> <hr></hr></td><td align="left" valign="bottom">O6<hr></hr></td><td align="left" valign="bottom">Q455<hr></hr></td><td align="left" valign="bottom">CG<hr></hr></td><td align="left" valign="bottom">3.25<hr></hr></td></tr><tr><td align="left" valign="bottom">NAG 470B<hr></hr></td><td align="left" valign="bottom">O3<hr></hr></td><td align="left" valign="bottom">G394<hr></hr></td><td align="left" valign="bottom">N<hr></hr></td><td align="left" valign="bottom">2.96<hr></hr></td></tr><tr><td align="left" valign="bottom"> <hr></hr></td><td align="left" valign="bottom">C3<hr></hr></td><td align="left" valign="bottom">G394<hr></hr></td><td align="left" valign="bottom">O<hr></hr></td><td align="left" valign="bottom">3.16<hr></hr></td></tr><tr><td align="left" valign="bottom">BMA 471 C<hr></hr></td><td align="left" valign="bottom">O2<hr></hr></td><td align="left" valign="bottom">G394<hr></hr></td><td align="left" valign="bottom">CA<hr></hr></td><td align="left" valign="bottom">3.21<hr></hr></td></tr><tr><td align="left" valign="bottom">MAN 472D<hr></hr></td><td align="left" valign="bottom">O3<hr></hr></td><td align="left" valign="bottom">R364<hr></hr></td><td align="left" valign="bottom">NH2<hr></hr></td><td align="left" valign="bottom">3.02<hr></hr></td></tr><tr><td align="left" valign="bottom"> <hr></hr></td><td align="left" valign="bottom">C3<hr></hr></td><td align="left" valign="bottom">E375<hr></hr></td><td align="left" valign="bottom">OE1<hr></hr></td><td align="left" valign="bottom">3.39<hr></hr></td></tr><tr><td align="left" valign="bottom"> <hr></hr></td><td align="left" valign="bottom">O3<hr></hr></td><td align="left" valign="bottom">E375<hr></hr></td><td align="left" valign="bottom">OE2<hr></hr></td><td align="left" valign="bottom">2.58<hr></hr></td></tr><tr><td align="left" valign="bottom"> <hr></hr></td><td align="left" valign="bottom">O4<hr></hr></td><td align="left" valign="bottom">E375<hr></hr></td><td align="left" valign="bottom">OE1<hr></hr></td><td align="left" valign="bottom">2.75<hr></hr></td></tr><tr><td align="left" valign="bottom">MAN 473E<hr></hr></td><td align="left" valign="bottom">O5<hr></hr></td><td align="left" valign="bottom">R364<hr></hr></td><td align="left" valign="bottom">NH2<hr></hr></td><td align="left" valign="bottom">3.15<hr></hr></td></tr><tr><td align="left" valign="bottom"> <hr></hr></td><td align="left" valign="bottom">O6<hr></hr></td><td align="left" valign="bottom">K389<hr></hr></td><td align="left" valign="bottom">NZ<hr></hr></td><td align="left" valign="bottom">2.82<hr></hr></td></tr><tr><td align="left" valign="bottom"> <hr></hr></td><td align="left" valign="bottom">O6<hr></hr></td><td align="left" valign="bottom">D330<hr></hr></td><td align="left" valign="bottom">OD2<hr></hr></td><td align="left" valign="bottom">2.54<hr></hr></td></tr><tr><td align="left" valign="bottom">MAN 473 F<hr></hr></td><td align="left" valign="bottom">O2<hr></hr></td><td align="left" valign="bottom">N329<hr></hr></td><td align="left" valign="bottom">OD1<hr></hr></td><td align="left" valign="bottom">3.10<hr></hr></td></tr><tr><td align="left" valign="bottom"> <hr></hr></td><td align="left" valign="bottom">O3<hr></hr></td><td align="left" valign="bottom">D330<hr></hr></td><td align="left" valign="bottom">N<hr></hr></td><td align="left" valign="bottom">2.93<hr></hr></td></tr><tr><td align="left" valign="bottom"> <hr></hr></td><td align="left" valign="bottom">O3<hr></hr></td><td align="left" valign="bottom">N329<hr></hr></td><td align="left" valign="bottom">OD1<hr></hr></td><td align="left" valign="bottom">2.74<hr></hr></td></tr><tr><td align="left" valign="bottom"> <hr></hr></td><td align="left" valign="bottom">O3<hr></hr></td><td align="left" valign="bottom">N329<hr></hr></td><td align="left" valign="bottom">CA<hr></hr></td><td align="left" valign="bottom">2.99<hr></hr></td></tr><tr><td align="left" valign="bottom"> <hr></hr></td><td align="left" valign="bottom">O4<hr></hr></td><td align="left" valign="bottom">R327<hr></hr></td><td align="left" valign="bottom">NH2<hr></hr></td><td align="left" valign="bottom">3.11<hr></hr></td></tr><tr><td align="left"> </td><td align="left">O6</td><td align="left">I366</td><td align="left">O</td><td align="left">2.75</td></tr></tbody></table></table-wrap></sec><sec><title>Glucose molecules</title><p>We used 49% (w/v) glucose as cryoprotectant, and we observed four glucose molecules bound with greater than 50% occupancy in the structure of the inhibitor complex (Figure <xref ref-type="fig" rid="F4">4A</xref>). The glucose molecules are in a mix of alpha and beta configurations about the anomeric carbon atom. The O3 oxygen atom of Glc487 interacts with the ND2 nitrogen atom of Arg141 through a hydrogen bond. O3 and O1 of Glc486 forms hydrogen bonds with NE2 of Gln315 and ND2 of Asn338 respectively.</p></sec></sec><sec sec-type="conclusions"><title>Conclusions</title><p>Our aim in designing inhibitor <bold>2</bold> was to extend the substituent at C4, corresponding to C6 of sialic acid, to (i) increase the contact surface in the C6-subsite and (ii) force the benzene ring to tilt to maximize these interactions while retaining the interactions of the carboxylate and the pyrolidinone substituents. The crystal structure at 1.55 Å shows that we were partially successful in that the ring in <bold>2</bold> is tilted relative to compound <bold>1</bold> (Figure <xref ref-type="fig" rid="F4">4</xref>) and the overall number of contacts is increased. The IC<sub>50</sub> did not decrease, and the reason became clear when we solved the crystal structure. The second propyl group is not making any contacts but is freely waving above the surface of the NA. Future design efforts will include unequal branches that may be accommodated in the C6-subsite.</p><p>The N9 NA that was crystallized for this experiment was purified from virus grown in embryonated chicken eggs, so it contains a full complement of vertebrate processed N-linked glycans, in contrast to NA expressed in insect cells which has truncated glycan structures. We therefore refined the glycans as far as we could see electron density. Most glycans are flexible and only the first few sugars are seen, but the glycan attached to Asn200 is well resolved due to interaction with the adjacent subunit and is seen to contain GlcNAc<sub>2</sub>Man<sub>7</sub>. Mutation data suggests this glycan plays a role in stabilizing the NA tetramer, which is a significant property because, for reasons that are not understood [<xref ref-type="bibr" rid="B9">9</xref>], only the NA tetramer has enzymatic activity.</p></sec><sec sec-type="materials|methods"><title>Materials and methods</title><sec><title>Inhibitor synthesis</title><p>The synthesis, purification and evaluation of the in vitro inhibitory activity of <bold>2</bold> {4-[2,2-bis(hydroxymethyl)-5-oxo-pyrrolidin-1-yl]-3-[(dipropylamino)methyl]benzoic acid} will be described elsewhere [<xref ref-type="bibr" rid="B23">23</xref>].</p></sec><sec><title>Protein preparation and crystallization</title><p>The reassortant virus A<bold>/</bold>NWS/33<sub>H</sub>-A/tern/Australia/G70C/75<sub>N</sub> (H1N9) was grown and purified as previously described [<xref ref-type="bibr" rid="B25">25</xref>]. The purified virus was digested with Pronase (Sigma) at a concentration of 6 mg/ml at 37 C for 16 hours. The cores were removed by centrifugation, and the released heads were pooled, concentrated, and purified by gel filtration using FPLC [<xref ref-type="bibr" rid="B41">41</xref>]. The purified protein was concentrated to 10 mg/ml. Single crystals of N9 NA were grown by vapor diffusion using the hanging drop method containing equal volumes of N9 and the reservoir solution of 1.9 M potassium phosphate buffer pH 6.9 [<xref ref-type="bibr" rid="B8">8</xref>].</p></sec><sec><title>Inhibitor soaking, cryoprotection and X-ray data collection</title><p>The successful flash cooling of large (0.2 − 0.4 mm per edge) cubic crystals in liquid nitrogen required serial equilibration by vapor diffusion. The estimated osmolality for 1.9 M K-phosphate buffer (pH 6.9) in the reservoir solution was matched to that of glucose using standard tables [<xref ref-type="bibr" rid="B46">46</xref>,<xref ref-type="bibr" rid="B47">47</xref>]. Crystals were placed over wells with 200 mM K-phosphate buffer (pH 6.9) containing 45 g/100 ml (45% w/v) glucose for 6 hours to two days and then placed over a well with 46% glucose. This procedure was repeated until the crystals were over wells that contained 49% glucose. After the final equilibration, the crystals were soaked in 49% glucose and 200 mM K-phosphate with 25 mM inhibitor for 5 minutes before they were mounted in a rayon cryoloop and vitrified at 100 K in a nitrogen cold-stream.</p><p>X-ray data were collected at 100 K at SSRL beam line 7–1 on an ADSC Quantum 315 CCD detector using monochromatic radiation with a wavelength of 0.9537 Å. The X-ray data were collected at distances of 300 and 220 mm with oscillation angles of 0.5° and 0.35° respectively and with 2 s exposures at both distances.</p><p>The X-ray data were indexed and integrated with <italic>XDS</italic>[<xref ref-type="bibr" rid="B48">48</xref>]. The integrated intensities were scaled and merged with the <italic>CCP4</italic>[<xref ref-type="bibr" rid="B49">49</xref>] program <italic>SCALA</italic>[<xref ref-type="bibr" rid="B50">50</xref>] and converted to structure factors with <italic>TRUNCATE</italic>[<xref ref-type="bibr" rid="B51">51</xref>].</p></sec><sec><title>Structure determination and analysis</title><p>The structure was solved by molecular substitution. The 1.7 Å crystal structure of the R292K mutant of tern N9 influenza virus NA [<xref ref-type="bibr" rid="B24">24</xref>] (2QWA) without the glycans or solvent molecules was used as the starting model in rigid body and coordinate refinement in <italic>REFMAC</italic>[<xref ref-type="bibr" rid="B52">52</xref>]. Refinement was continued using <italic>PHENIX</italic>[<xref ref-type="bibr" rid="B53">53</xref>] together with iterative rounds of model rebuilding using the molecular graphic package <italic>COOT</italic>[<xref ref-type="bibr" rid="B37">37</xref>]. The <italic>PRODRG</italic> webserver [<xref ref-type="bibr" rid="B54">54</xref>] was used to build the initial coordinates and stereochemical restraints of inhibitor <bold>2</bold>. The restraints for three β-D-mannose monomers (BMA) were taken from the <italic>REFMAC</italic> library [<xref ref-type="bibr" rid="B34">34</xref>]. Riding hydrogen atoms were added with the program <italic>REDUCE</italic>[<xref ref-type="bibr" rid="B55">55</xref>].</p><p>The structure was analyzed with <italic>HBPLUS</italic>[<xref ref-type="bibr" rid="B29">29</xref>] and <italic>Chimera</italic>[<xref ref-type="bibr" rid="B28">28</xref>]. The figures were made using <italic>PyMOL</italic> (Schrödinger LLC) and Liglot+ [<xref ref-type="bibr" rid="B56">56</xref>].</p></sec><sec><title>Accession codes</title><p>The atomic coordinates and structure factors were deposited in the protein data bank under accession code 4DGR for the inhibitor complex.</p></sec></sec><sec><title>Competing interests</title><p>The authors declare that they have no competing interests.</p></sec><sec><title>Authors’ contributions</title><p>WJB, BHMM and GMA designed the research, analyzed the results and wrote the manuscript. WJB, ESJ and GK designed and synthesized the inhibitor and GMA tested it. BHMM and LV crystallized the complex, solved and refined the structure, analyzed the structure and drafted the manuscript. All authors read and approved the final manuscript.</p></sec></body><back><sec><title>Acknowledgements</title><p>We thank Dr Tzanko Doukov for help with data collection at Stanford Synchrotron Radiation Lightsource (SSRL) beam line 7–1. The Structural Molecular Biology Program at SSRL is supported by DOE-OBER, NIH-NCRR (P41RR001209), and NIH-NIGMS. Some refinements with <italic>PHENIX</italic> were run remotely at the OU Supercomputing Center for Education & Research (OSCER) at the University of Oklahoma. We thank OSCER Director Dr Henry Neeman and Joshua Alexander for valuable technical assistance and Shelly Gulati for growth and purification of virus and determination of the IC50. This work was supported by NIAID grant AI 062950 to WJB.</p></sec><ref-list><ref id="B1"><mixed-citation publication-type="journal"><name><surname>Palese</surname><given-names>P</given-names></name><name><surname>Tobita</surname><given-names>K</given-names></name><name><surname>Ueda</surname><given-names>M</given-names></name><name><surname>Compans</surname><given-names>RW</given-names></name><article-title>Characterization of temperature-sensitive influenza virus mutants defective in neuraminidase</article-title><source>Virology</source><year>1974</year><volume>61</volume><fpage>397</fpage><lpage>410</lpage><pub-id pub-id-type="doi">10.1016/0042-6822(74)90276-1</pub-id><pub-id pub-id-type="pmid">4472498</pub-id></mixed-citation></ref><ref id="B2"><mixed-citation publication-type="journal"><name><surname>Liu</surname><given-names>C</given-names></name><name><surname>Eichelberger</surname><given-names>MC</given-names></name><name><surname>Compans</surname><given-names>RW</given-names></name><name><surname>Air</surname><given-names>GM</given-names></name><article-title>Influenza type A virus neuraminidase does not play a role in viral entry, replication, assembly, or budding</article-title><source>J Virol</source><year>1995</year><volume>69</volume><issue>2</issue><fpage>1099</fpage><lpage>1106</lpage><pub-id pub-id-type="pmid">7815489</pub-id></mixed-citation></ref><ref id="B3"><mixed-citation publication-type="journal"><name><surname>Webster</surname><given-names>RG</given-names></name><name><surname>Govorkova</surname><given-names>EA</given-names></name><article-title>H5N1 influenza–continuing evolution and spread</article-title><source>New Engl J Med</source><year>2006</year><volume>355</volume><issue>21</issue><fpage>2174</fpage><lpage>2177</lpage><pub-id pub-id-type="doi">10.1056/NEJMp068205</pub-id><pub-id pub-id-type="pmid">17124014</pub-id></mixed-citation></ref><ref id="B4"><mixed-citation publication-type="journal"><name><surname>Rota</surname><given-names>P</given-names></name><article-title>Cocirculation of two distinct evolutionary lineages of influenza type B virus since 1983</article-title><source>Virology</source><year>1990</year><volume>175</volume><issue>1</issue><fpage>59</fpage><lpage>68</lpage><pub-id pub-id-type="doi">10.1016/0042-6822(90)90186-U</pub-id><pub-id pub-id-type="pmid">2309452</pub-id></mixed-citation></ref><ref id="B5"><mixed-citation publication-type="book"><name><surname>Air</surname><given-names>GM</given-names></name><name><surname>Brouillette</surname><given-names>WJ</given-names></name><person-group person-group-type="editor">LaFemina R</person-group><article-title>Influenza virus antiviral targets</article-title><source>Antiviral Res</source><year>2009</year><publisher-name>ASM Press, Washington, DC</publisher-name><fpage>187</fpage><lpage>207</lpage></mixed-citation></ref><ref id="B6"><mixed-citation publication-type="journal"><name><surname>Colman</surname><given-names>PM</given-names></name><name><surname>Varghese</surname><given-names>JN</given-names></name><name><surname>Laver</surname><given-names>WG</given-names></name><article-title>Structure of the catalytic and antigenic sites in influenza virus neuraminidase</article-title><source>Nature</source><year>1983</year><volume>303</volume><fpage>41</fpage><lpage>44</lpage><pub-id pub-id-type="doi">10.1038/303041a0</pub-id><pub-id pub-id-type="pmid">6188957</pub-id></mixed-citation></ref><ref id="B7"><mixed-citation publication-type="journal"><name><surname>Burmeister</surname><given-names>WP</given-names></name><name><surname>Ruigrok</surname><given-names>RW</given-names></name><name><surname>Cusack</surname><given-names>S</given-names></name><article-title>The 2.2 A resolution crystal structure of influenza B neuraminidase and its complex with sialic acid</article-title><source>EMBO J</source><year>1992</year><volume>11</volume><issue>1</issue><fpage>49</fpage><lpage>56</lpage><pub-id pub-id-type="pmid">1740114</pub-id></mixed-citation></ref><ref id="B8"><mixed-citation publication-type="journal"><name><surname>Bossart-Whitaker</surname><given-names>P</given-names></name><name><surname>Carson</surname><given-names>M</given-names></name><name><surname>Babu</surname><given-names>YS</given-names></name><name><surname>Smith</surname><given-names>CD</given-names></name><name><surname>Laver</surname><given-names>WG</given-names></name><name><surname>Air</surname><given-names>GM</given-names></name><article-title>Three-dimensional structure of influenza A N9 neuraminidase and its complex with the inhibitor 2-deoxy 2,3-dehydro-N-acetyl neuraminic acid</article-title><source>J Mol Biol</source><year>1993</year><volume>232</volume><issue>4</issue><fpage>1069</fpage><lpage>1083</lpage><pub-id pub-id-type="doi">10.1006/jmbi.1993.1461</pub-id><pub-id pub-id-type="pmid">8371267</pub-id></mixed-citation></ref><ref id="B9"><mixed-citation publication-type="other"><name><surname>Air</surname><given-names>GM</given-names></name><article-title>Influenza neuraminidase</article-title><source>Influenza Other Respir Viruses</source><year>2011</year><comment>Nov 16,</comment><pub-id pub-id-type="doi">10.1111/j.1750-2659.2011.00304.x</pub-id></mixed-citation></ref><ref id="B10"><mixed-citation publication-type="journal"><name><surname>von Itzstein</surname><given-names>M</given-names></name><name><surname>Wu</surname><given-names>WY</given-names></name><name><surname>Kok</surname><given-names>GB</given-names></name><name><surname>Pegg</surname><given-names>MS</given-names></name><name><surname>Dyason</surname><given-names>JC</given-names></name><name><surname>Jin</surname><given-names>B</given-names></name><name><surname>Van Phan</surname><given-names>T</given-names></name><name><surname>Smythe</surname><given-names>ML</given-names></name><name><surname>White</surname><given-names>HF</given-names></name><name><surname>Oliver</surname><given-names>SW</given-names></name><etal></etal><article-title>Rational design of potent sialidase-based inhibitors of influenza virus replication</article-title><source>Nature</source><year>1993</year><volume>363</volume><issue>6428</issue><fpage>418</fpage><lpage>423</lpage><pub-id pub-id-type="doi">10.1038/363418a0</pub-id><pub-id pub-id-type="pmid">8502295</pub-id></mixed-citation></ref><ref id="B11"><mixed-citation publication-type="journal"><name><surname>Kim</surname><given-names>CU</given-names></name><name><surname>Lew</surname><given-names>W</given-names></name><name><surname>Williams</surname><given-names>MA</given-names></name><name><surname>Liu</surname><given-names>H</given-names></name><name><surname>Zhang</surname><given-names>L</given-names></name><name><surname>Swaminathan</surname><given-names>S</given-names></name><name><surname>Bischofberger</surname><given-names>N</given-names></name><name><surname>Chen</surname><given-names>MS</given-names></name><name><surname>Mendel</surname><given-names>DB</given-names></name><name><surname>Tai</surname><given-names>CY</given-names></name><etal></etal><article-title>Influenza neuraminidase inhibitors possessing a novel hydrophobic interaction in the enzyme active site: design, synthesis, and structural analysis of carbocyclic sialic acid analogues with potent anti-influenza activity</article-title><source>J Am Chem Soc</source><year>1997</year><volume>119</volume><issue>4</issue><fpage>681</fpage><lpage>690</lpage><pub-id pub-id-type="doi">10.1021/ja963036t</pub-id><pub-id pub-id-type="pmid">16526129</pub-id></mixed-citation></ref><ref id="B12"><mixed-citation publication-type="journal"><name><surname>Brouillette</surname><given-names>WJ</given-names></name><name><surname>Atigadda</surname><given-names>VR</given-names></name><name><surname>Luo</surname><given-names>M</given-names></name><name><surname>Air</surname><given-names>GM</given-names></name><name><surname>Babu</surname><given-names>YS</given-names></name><name><surname>Bantia</surname><given-names>S</given-names></name><article-title>Design of benzoic acid inhibitors of influenza neuraminidase containing a cyclic substitution for the N-acetyl grouping</article-title><source>Bioorg Med Chem Lett</source><year>1999</year><volume>9</volume><issue>14</issue><fpage>1901</fpage><lpage>1906</lpage><pub-id pub-id-type="doi">10.1016/S0960-894X(99)00318-2</pub-id><pub-id pub-id-type="pmid">10450950</pub-id></mixed-citation></ref><ref id="B13"><mixed-citation publication-type="journal"><name><surname>Kim</surname><given-names>CU</given-names></name><name><surname>Lew</surname><given-names>W</given-names></name><name><surname>Williams</surname><given-names>MA</given-names></name><name><surname>Wu</surname><given-names>H</given-names></name><name><surname>Zhang</surname><given-names>L</given-names></name><name><surname>Chen</surname><given-names>X</given-names></name><name><surname>Escarpe</surname><given-names>PA</given-names></name><name><surname>Mendel</surname><given-names>DB</given-names></name><name><surname>Laver</surname><given-names>WG</given-names></name><name><surname>Stevens</surname><given-names>RC</given-names></name><article-title>Structure-activity relationship studies of novel carbocyclic influenza neuraminidase inhibitors</article-title><source>J Med Chem</source><year>1998</year><volume>41</volume><issue>14</issue><fpage>2451</fpage><lpage>2460</lpage><pub-id pub-id-type="doi">10.1021/jm980162u</pub-id><pub-id pub-id-type="pmid">9651151</pub-id></mixed-citation></ref><ref id="B14"><mixed-citation publication-type="journal"><name><surname>Babu</surname><given-names>YS</given-names></name><name><surname>Chand</surname><given-names>P</given-names></name><name><surname>Bantia</surname><given-names>S</given-names></name><name><surname>Kotian</surname><given-names>P</given-names></name><name><surname>Dehghani</surname><given-names>A</given-names></name><name><surname>El-Kattan</surname><given-names>Y</given-names></name><name><surname>Lin</surname><given-names>TH</given-names></name><name><surname>Hutchison</surname><given-names>TL</given-names></name><name><surname>Elliott</surname><given-names>AJ</given-names></name><name><surname>Parker</surname><given-names>CD</given-names></name><etal></etal><article-title>BCX-1812 (RWJ-270201): discovery of a novel, highly potent, orally active, and selective influenza neuraminidase inhibitor through structure-based drug design</article-title><source>J Med Chem</source><year>2000</year><volume>43</volume><issue>19</issue><fpage>3482</fpage><lpage>3486</lpage><pub-id pub-id-type="doi">10.1021/jm0002679</pub-id><pub-id pub-id-type="pmid">11000002</pub-id></mixed-citation></ref><ref id="B15"><mixed-citation publication-type="journal"><name><surname>Jedrzejas</surname><given-names>MJ</given-names></name><name><surname>Singh</surname><given-names>S</given-names></name><name><surname>Brouillette</surname><given-names>WJ</given-names></name><name><surname>Air</surname><given-names>GM</given-names></name><name><surname>Luo</surname><given-names>M</given-names></name><article-title>A strategy for theoretical binding constant, Ki, calculations for neuraminidase aromatic inhibitors designed on the basis of the active site structure of influenza virus neuraminidase</article-title><source>Proteins</source><year>1995</year><volume>23</volume><issue>2</issue><fpage>264</fpage><lpage>277</lpage><pub-id pub-id-type="doi">10.1002/prot.340230215</pub-id><pub-id pub-id-type="pmid">8592707</pub-id></mixed-citation></ref><ref id="B16"><mixed-citation publication-type="journal"><name><surname>Singh</surname><given-names>S</given-names></name><name><surname>Jedrzejas</surname><given-names>MJ</given-names></name><name><surname>Air</surname><given-names>GM</given-names></name><name><surname>Luo</surname><given-names>M</given-names></name><name><surname>Laver</surname><given-names>WG</given-names></name><name><surname>Brouillette</surname><given-names>WJ</given-names></name><article-title>Structure-based inhibitors of influenza virus sialidase. A benzoic acid lead with novel interaction</article-title><source>J Med Chem</source><year>1995</year><volume>38</volume><issue>17</issue><fpage>3217</fpage><lpage>3225</lpage><pub-id pub-id-type="doi">10.1021/jm00017a005</pub-id><pub-id pub-id-type="pmid">7650674</pub-id></mixed-citation></ref><ref id="B17"><mixed-citation publication-type="journal"><name><surname>Brouillette</surname><given-names>WJ</given-names></name><name><surname>Bajpai</surname><given-names>SN</given-names></name><name><surname>Ali</surname><given-names>SM</given-names></name><name><surname>Velu</surname><given-names>SE</given-names></name><name><surname>Atigadda</surname><given-names>VR</given-names></name><name><surname>Lommer</surname><given-names>BS</given-names></name><name><surname>Finley</surname><given-names>JB</given-names></name><name><surname>Luo</surname><given-names>M</given-names></name><name><surname>Air</surname><given-names>GM</given-names></name><article-title>Pyrrolidinobenzoic acid inhibitors of influenza virus neuraminidase: modifications of essential pyrrolidinone ring substituents</article-title><source>Bioorg Med Chem</source><year>2003</year><volume>11</volume><issue>13</issue><fpage>2739</fpage><lpage>2749</lpage><pub-id pub-id-type="doi">10.1016/S0968-0896(03)00271-2</pub-id><pub-id pub-id-type="pmid">12788348</pub-id></mixed-citation></ref><ref id="B18"><mixed-citation publication-type="journal"><name><surname>Atigadda</surname><given-names>VR</given-names></name><name><surname>Brouillette</surname><given-names>WJ</given-names></name><name><surname>Duarte</surname><given-names>F</given-names></name><name><surname>Ali</surname><given-names>SM</given-names></name><name><surname>Babu</surname><given-names>YS</given-names></name><name><surname>Bantia</surname><given-names>S</given-names></name><name><surname>Chand</surname><given-names>P</given-names></name><name><surname>Chu</surname><given-names>N</given-names></name><name><surname>Montgomery</surname><given-names>JA</given-names></name><name><surname>Walsh</surname><given-names>DA</given-names></name><etal></etal><article-title>Potent inhibition of influenza sialidase by a benzoic acid containing a 2-pyrrolidinone substituent</article-title><source>J Med Chem</source><year>1999</year><volume>42</volume><issue>13</issue><fpage>2332</fpage><lpage>2343</lpage><pub-id pub-id-type="doi">10.1021/jm980707k</pub-id><pub-id pub-id-type="pmid">10395473</pub-id></mixed-citation></ref><ref id="B19"><mixed-citation publication-type="journal"><name><surname>Smith</surname><given-names>PW</given-names></name><name><surname>Robinson</surname><given-names>JE</given-names></name><name><surname>Evans</surname><given-names>DN</given-names></name><name><surname>Sollis</surname><given-names>SL</given-names></name><name><surname>Howes</surname><given-names>PD</given-names></name><name><surname>Trivedi</surname><given-names>N</given-names></name><name><surname>Bethell</surname><given-names>RC</given-names></name><article-title>Sialidase inhibitors related to zanamivir: synthesis and biological evaluation of 4 H-pyran 6-ether and ketone</article-title><source>Bioorg Med Chem Lett</source><year>1999</year><volume>9</volume><issue>4</issue><fpage>601</fpage><lpage>604</lpage><pub-id pub-id-type="doi">10.1016/S0960-894X(99)00031-1</pub-id><pub-id pub-id-type="pmid">10098672</pub-id></mixed-citation></ref><ref id="B20"><mixed-citation publication-type="journal"><name><surname>Russell</surname><given-names>RJ</given-names></name><name><surname>Haire</surname><given-names>LF</given-names></name><name><surname>Stevens</surname><given-names>DJ</given-names></name><name><surname>Collins</surname><given-names>PJ</given-names></name><name><surname>Lin</surname><given-names>YP</given-names></name><name><surname>Blackburn</surname><given-names>GM</given-names></name><name><surname>Hay</surname><given-names>AJ</given-names></name><name><surname>Gamblin</surname><given-names>SJ</given-names></name><name><surname>Skehel</surname><given-names>JJ</given-names></name><article-title>The structure of H5N1 avian influenza neuraminidase suggests new opportunities for drug design</article-title><source>Nature</source><year>2006</year><volume>443</volume><issue>7107</issue><fpage>45</fpage><lpage>49</lpage><pub-id pub-id-type="doi">10.1038/nature05114</pub-id><pub-id pub-id-type="pmid">16915235</pub-id></mixed-citation></ref><ref id="B21"><mixed-citation publication-type="journal"><name><surname>Finley</surname><given-names>JB</given-names></name><name><surname>Atigadda</surname><given-names>VR</given-names></name><name><surname>Duarte</surname><given-names>F</given-names></name><name><surname>Zhao</surname><given-names>JJ</given-names></name><name><surname>Brouillette</surname><given-names>WJ</given-names></name><name><surname>Air</surname><given-names>GM</given-names></name><name><surname>Luo</surname><given-names>M</given-names></name><article-title>Novel aromatic inhibitors of influenza virus neuraminidase make selective interactions with conserved residues and water molecules in the active site</article-title><source>J Mol Biol</source><year>1999</year><volume>293</volume><issue>5</issue><fpage>1107</fpage><lpage>1119</lpage><pub-id pub-id-type="doi">10.1006/jmbi.1999.3180</pub-id><pub-id pub-id-type="pmid">10547289</pub-id></mixed-citation></ref><ref id="B22"><mixed-citation publication-type="journal"><name><surname>Rarey</surname><given-names>M</given-names></name><name><surname>Kramer</surname><given-names>B</given-names></name><name><surname>Lengauer</surname><given-names>T</given-names></name><name><surname>Klebe</surname><given-names>G</given-names></name><article-title>A fast flexible docking method using an incremental construction algorithm</article-title><source>J Mol Biol</source><year>1996</year><volume>261</volume><issue>3</issue><fpage>470</fpage><lpage>489</lpage><pub-id pub-id-type="doi">10.1006/jmbi.1996.0477</pub-id><pub-id pub-id-type="pmid">8780787</pub-id></mixed-citation></ref><ref id="B23"><mixed-citation publication-type="other"><name><surname>Kolavi</surname><given-names>G</given-names></name><name><surname>Li</surname><given-names>Y</given-names></name><name><surname>Johnson</surname><given-names>ES</given-names></name><name><surname>Gulati</surname><given-names>S</given-names></name><name><surname>Air</surname><given-names>GM</given-names></name><name><surname>Brouillette</surname><given-names>WJ</given-names></name><comment>in preparation</comment></mixed-citation></ref><ref id="B24"><mixed-citation publication-type="journal"><name><surname>Varghese</surname><given-names>JN</given-names></name><name><surname>Smith</surname><given-names>PW</given-names></name><name><surname>Sollis</surname><given-names>SL</given-names></name><name><surname>Blick</surname><given-names>TJ</given-names></name><name><surname>Sahasrabudhe</surname><given-names>A</given-names></name><name><surname>McKimm-Breschkin</surname><given-names>JL</given-names></name><name><surname>Colman</surname><given-names>PM</given-names></name><article-title>Drug design against a shifting target: a structural basis for resistance to inhibitors in a variant of influenza virus neuraminidase</article-title><source>Structure</source><year>1998</year><volume>6</volume><issue>6</issue><fpage>735</fpage><lpage>746</lpage><pub-id pub-id-type="doi">10.1016/S0969-2126(98)00075-6</pub-id><pub-id pub-id-type="pmid">9655825</pub-id></mixed-citation></ref><ref id="B25"><mixed-citation publication-type="journal"><name><surname>Laver</surname><given-names>WG</given-names></name><name><surname>Colman</surname><given-names>PM</given-names></name><name><surname>Webster</surname><given-names>RG</given-names></name><name><surname>Hinshaw</surname><given-names>VS</given-names></name><name><surname>Air</surname><given-names>GM</given-names></name><article-title>Influenza virus neuraminidase with hemagglutinin activity</article-title><source>Virology</source><year>1984</year><volume>137</volume><fpage>314</fpage><lpage>323</lpage><pub-id pub-id-type="doi">10.1016/0042-6822(84)90223-X</pub-id><pub-id pub-id-type="pmid">6485252</pub-id></mixed-citation></ref><ref id="B26"><mixed-citation publication-type="journal"><name><surname>Varghese</surname><given-names>JN</given-names></name><name><surname>Colman</surname><given-names>PM</given-names></name><name><surname>van Donkelaar</surname><given-names>A</given-names></name><name><surname>Blick</surname><given-names>TJ</given-names></name><name><surname>Sahasrabudhe</surname><given-names>A</given-names></name><name><surname>McKimm-Breschkin</surname><given-names>JL</given-names></name><article-title>Structural evidence for a second sialic acid binding site in avian influenza virus neuraminidases</article-title><source>Proc Natl Acad Sci USA</source><year>1997</year><volume>94</volume><issue>22</issue><fpage>11808</fpage><lpage>11812</lpage><pub-id pub-id-type="doi">10.1073/pnas.94.22.11808</pub-id><pub-id pub-id-type="pmid">9342319</pub-id></mixed-citation></ref><ref id="B27"><mixed-citation publication-type="journal"><name><surname>Weiss</surname><given-names>MS</given-names></name><article-title>Global indicators of X-ray data quality</article-title><source>J Appl Cryst</source><year>2001</year><volume>34</volume><fpage>130</fpage><lpage>135</lpage><pub-id pub-id-type="doi">10.1107/S0021889800018227</pub-id></mixed-citation></ref><ref id="B28"><mixed-citation publication-type="journal"><name><surname>Pettersen</surname><given-names>EF</given-names></name><name><surname>Goddard</surname><given-names>TD</given-names></name><name><surname>Huang</surname><given-names>CC</given-names></name><name><surname>Couch</surname><given-names>GS</given-names></name><name><surname>Greenblatt</surname><given-names>DM</given-names></name><name><surname>Meng</surname><given-names>EC</given-names></name><name><surname>Ferrin</surname><given-names>TE</given-names></name><article-title>UCSF Chimera–a visualization system for exploratory research and analysis</article-title><source>J Comput Chem</source><year>2004</year><volume>25</volume><issue>13</issue><fpage>1605</fpage><lpage>1612</lpage><pub-id pub-id-type="doi">10.1002/jcc.20084</pub-id><pub-id pub-id-type="pmid">15264254</pub-id></mixed-citation></ref><ref id="B29"><mixed-citation publication-type="journal"><name><surname>McDonald</surname><given-names>I</given-names></name><name><surname>Thornton</surname><given-names>J</given-names></name><article-title>Satisfying hydrogen bonding potential in proteins</article-title><source>J Mol Biol</source><year>1994</year><volume>238</volume><fpage>777</fpage><lpage>793</lpage><pub-id pub-id-type="doi">10.1006/jmbi.1994.1334</pub-id><pub-id pub-id-type="pmid">8182748</pub-id></mixed-citation></ref><ref id="B30"><mixed-citation publication-type="book"><name><surname>Copeland</surname><given-names>RA</given-names></name><source>Enzymes: A Practical Introduction to Structure, Mechanism, and Data Analysis</source><year>2000</year><edition>2</edition><publisher-name>Wiley-VCH, New York</publisher-name></mixed-citation></ref><ref id="B31"><mixed-citation publication-type="journal"><name><surname>Baker</surname><given-names>EN</given-names></name><name><surname>Hubbard</surname><given-names>RE</given-names></name><article-title>Hydrogen bonding in globular proteins</article-title><source>Prog Biophys Mol Biol</source><year>1984</year><volume>44</volume><issue>2</issue><fpage>97</fpage><lpage>179</lpage><pub-id pub-id-type="doi">10.1016/0079-6107(84)90007-5</pub-id><pub-id pub-id-type="pmid">6385134</pub-id></mixed-citation></ref><ref id="B32"><mixed-citation publication-type="journal"><name><surname>Lommer</surname><given-names>BS</given-names></name><name><surname>Ali</surname><given-names>SM</given-names></name><name><surname>Bajpai</surname><given-names>SN</given-names></name><name><surname>Brouillette</surname><given-names>WJ</given-names></name><name><surname>Air</surname><given-names>GM</given-names></name><name><surname>Luo</surname><given-names>M</given-names></name><article-title>A benzoic acid inhibitor induces a novel conformational change in the active site of Influenza B virus neuraminidase</article-title><source>Acta Crystallogr D Biol Crystallogr</source><year>2004</year><volume>60</volume><issue>Pt 6</issue><fpage>1017</fpage><lpage>1023</lpage><pub-id pub-id-type="pmid">15159560</pub-id></mixed-citation></ref><ref id="B33"><mixed-citation publication-type="journal"><name><surname>Wang</surname><given-names>T</given-names></name><name><surname>Wade</surname><given-names>RC</given-names></name><article-title>Comparative binding energy (COMBINE) analysis of influenza neuraminidase-inhibitor complexes</article-title><source>J Med Chem</source><year>2001</year><volume>44</volume><issue>6</issue><fpage>961</fpage><lpage>971</lpage><pub-id pub-id-type="doi">10.1021/jm001070j</pub-id><pub-id pub-id-type="pmid">11300878</pub-id></mixed-citation></ref><ref id="B34"><mixed-citation publication-type="journal"><name><surname>Vagin</surname><given-names>AA</given-names></name><name><surname>Steiner</surname><given-names>RA</given-names></name><name><surname>Lebedev</surname><given-names>AA</given-names></name><name><surname>Potterton</surname><given-names>L</given-names></name><name><surname>McNicholas</surname><given-names>S</given-names></name><name><surname>Long</surname><given-names>F</given-names></name><name><surname>Murshudov</surname><given-names>GN</given-names></name><article-title>REFMAC5 dictionary: organization of prior chemical knowledge and guidelines for its use</article-title><source>Acta Crystallogr D Biol Crystallogr</source><year>2004</year><volume>60</volume><issue>Pt 12 Pt 1</issue><fpage>2184</fpage><lpage>2195</lpage><pub-id pub-id-type="pmid">15572771</pub-id></mixed-citation></ref><ref id="B35"><mixed-citation publication-type="journal"><name><surname>Varghese</surname><given-names>JN</given-names></name><name><surname>McKimm-Breschkin</surname><given-names>JL</given-names></name><name><surname>Caldwell</surname><given-names>JB</given-names></name><name><surname>Kortt</surname><given-names>AA</given-names></name><name><surname>Colman</surname><given-names>PM</given-names></name><article-title>The structure of the complex between influenza virus neuraminidase and sialic acid, the viral receptor</article-title><source>Proteins</source><year>1992</year><volume>14</volume><issue>3</issue><fpage>327</fpage><lpage>332</lpage><pub-id pub-id-type="doi">10.1002/prot.340140302</pub-id><pub-id pub-id-type="pmid">1438172</pub-id></mixed-citation></ref><ref id="B36"><mixed-citation publication-type="journal"><name><surname>Krissinel</surname><given-names>E</given-names></name><name><surname>Henrick</surname><given-names>K</given-names></name><article-title>Secondary-structure matching (SSM), a new tool for fast protein structure alignment in three dimensions</article-title><source>Acta Crystallogr D Biol Crystallogr</source><year>2004</year><volume>60</volume><issue>Pt 12 Pt 1</issue><fpage>2256</fpage><lpage>2268</lpage><pub-id pub-id-type="pmid">15572779</pub-id></mixed-citation></ref><ref id="B37"><mixed-citation publication-type="journal"><name><surname>Emsley</surname><given-names>P</given-names></name><name><surname>Lohkamp</surname><given-names>B</given-names></name><name><surname>Scott</surname><given-names>WG</given-names></name><name><surname>Cowtan</surname><given-names>K</given-names></name><article-title>Features and development of Coot</article-title><source>Acta Crystallogr D Biol Crystallogr</source><year>2010</year><volume>66</volume><issue>Pt 4</issue><fpage>486</fpage><lpage>501</lpage><pub-id pub-id-type="pmid">20383002</pub-id></mixed-citation></ref><ref id="B38"><mixed-citation publication-type="journal"><name><surname>Collins</surname><given-names>PJ</given-names></name><name><surname>Haire</surname><given-names>LF</given-names></name><name><surname>Lin</surname><given-names>YP</given-names></name><name><surname>Liu</surname><given-names>J</given-names></name><name><surname>Russell</surname><given-names>RJ</given-names></name><name><surname>Walker</surname><given-names>PA</given-names></name><name><surname>Skehel</surname><given-names>JJ</given-names></name><name><surname>Martin</surname><given-names>SR</given-names></name><name><surname>Hay</surname><given-names>AJ</given-names></name><name><surname>Gamblin</surname><given-names>SJ</given-names></name><article-title>Crystal structures of oseltamivir-resistant influenza virus neuraminidase mutants</article-title><source>Nature</source><year>2008</year><volume>453</volume><issue>7199</issue><fpage>1258</fpage><lpage>1261</lpage><pub-id pub-id-type="doi">10.1038/nature06956</pub-id><pub-id pub-id-type="pmid">18480754</pub-id></mixed-citation></ref><ref id="B39"><mixed-citation publication-type="journal"><name><surname>Jedrzejas</surname><given-names>MJ</given-names></name><name><surname>Singh</surname><given-names>S</given-names></name><name><surname>Brouillette</surname><given-names>WJ</given-names></name><name><surname>Laver</surname><given-names>WG</given-names></name><name><surname>Air</surname><given-names>GM</given-names></name><name><surname>Luo</surname><given-names>M</given-names></name><article-title>Structures of aromatic inhibitors of influenza virus neuraminidase</article-title><source>Biochemistry</source><year>1995</year><volume>34</volume><fpage>3144</fpage><lpage>3151</lpage><pub-id pub-id-type="doi">10.1021/bi00010a003</pub-id><pub-id pub-id-type="pmid">7880809</pub-id></mixed-citation></ref><ref id="B40"><mixed-citation publication-type="journal"><name><surname>Holm</surname><given-names>L</given-names></name><name><surname>Park</surname><given-names>J</given-names></name><article-title>DaliLite workbench for protein structure comparison</article-title><source>Bioinformatics</source><year>2000</year><volume>16</volume><issue>6</issue><fpage>566</fpage><lpage>567</lpage><pub-id pub-id-type="doi">10.1093/bioinformatics/16.6.566</pub-id><pub-id pub-id-type="pmid">10980157</pub-id></mixed-citation></ref><ref id="B41"><mixed-citation publication-type="journal"><name><surname>Venkatramani</surname><given-names>L</given-names></name><name><surname>Bochkareva</surname><given-names>E</given-names></name><name><surname>Lee</surname><given-names>JT</given-names></name><name><surname>Gulati</surname><given-names>U</given-names></name><name><surname>Graeme Laver</surname><given-names>W</given-names></name><name><surname>Bochkarev</surname><given-names>A</given-names></name><name><surname>Air</surname><given-names>GM</given-names></name><article-title>An epidemiologically significant epitope of a 1998 human influenza virus neuraminidase forms a highly hydrated interface in the NA-antibody complex</article-title><source>J Mol Biol</source><year>2006</year><volume>356</volume><issue>3</issue><fpage>651</fpage><lpage>663</lpage><comment>and cover picture</comment><pub-id pub-id-type="doi">10.1016/j.jmb.2005.11.061</pub-id><pub-id pub-id-type="pmid">16384583</pub-id></mixed-citation></ref><ref id="B42"><mixed-citation publication-type="journal"><name><surname>Lee</surname><given-names>JT</given-names></name><name><surname>Air</surname><given-names>GM</given-names></name><article-title>Contacts between influenza N9 neuraminidase and monoclonal antibody NC10</article-title><source>Virology</source><year>2002</year><volume>300</volume><fpage>255</fpage><lpage>268</lpage><pub-id pub-id-type="doi">10.1006/viro.2002.1564</pub-id><pub-id pub-id-type="pmid">12350356</pub-id></mixed-citation></ref><ref id="B43"><mixed-citation publication-type="journal"><name><surname>Ward</surname><given-names>CW</given-names></name><name><surname>Murray</surname><given-names>JM</given-names></name><name><surname>Roxburgh</surname><given-names>CM</given-names></name><name><surname>Jackson</surname><given-names>DC</given-names></name><article-title>Chemical and antigenic characterization of the carbohydrate side chains of an Asian (N2) influenza virus neuraminidase</article-title><source>Virology</source><year>1983</year><volume>126</volume><issue>1</issue><fpage>370</fpage><lpage>375</lpage><pub-id pub-id-type="doi">10.1016/0042-6822(83)90486-5</pub-id><pub-id pub-id-type="pmid">6189288</pub-id></mixed-citation></ref><ref id="B44"><mixed-citation publication-type="journal"><name><surname>Wu</surname><given-names>Z</given-names></name><name><surname>Ethen</surname><given-names>C</given-names></name><name><surname>Hickey</surname><given-names>G</given-names></name><name><surname>Jiang</surname><given-names>W</given-names></name><article-title>Active 1918 pandemic flu viral neuraminidase has distinct N-glycan profile and is resistant to trypsin digestion</article-title><source>Biochem Biophys Res Commun</source><year>2009</year><volume>379</volume><fpage>749</fpage><lpage>753</lpage><pub-id pub-id-type="doi">10.1016/j.bbrc.2008.12.139</pub-id><pub-id pub-id-type="pmid">19133226</pub-id></mixed-citation></ref><ref id="B45"><mixed-citation publication-type="journal"><name><surname>Ceroni</surname><given-names>A</given-names></name><name><surname>Dell</surname><given-names>A</given-names></name><name><surname>Haslam</surname><given-names>S</given-names></name><article-title>The GlycanBuilder: a fast, intuitive and flexible software tool for building and displaying glycan structures</article-title><source>Source Code Biol Med</source><year>2007</year><volume>2</volume><fpage>3</fpage><pub-id pub-id-type="doi">10.1186/1751-0473-2-3</pub-id><pub-id pub-id-type="pmid">17683623</pub-id></mixed-citation></ref><ref id="B46"><mixed-citation publication-type="journal"><name><surname>Garman</surname><given-names>E</given-names></name><article-title>Cool data: quantity AND quality</article-title><source>Acta Crystallogr D Biol Crystallogr</source><year>1999</year><volume>55</volume><issue>Pt 10</issue><fpage>1641</fpage><lpage>1653</lpage><pub-id pub-id-type="pmid">10531512</pub-id></mixed-citation></ref><ref id="B47"><mixed-citation publication-type="book"><person-group person-group-type="editor">Weast RC</person-group><source>Handbook of Chemistry and Physics</source><year>1988</year><edition>69</edition><publisher-name>CRC PRess, Boca Raton, FL</publisher-name></mixed-citation></ref><ref id="B48"><mixed-citation publication-type="journal"><name><surname>Kabsch</surname><given-names>W</given-names></name><article-title>XDS</article-title><source>Acta Crystallogr D Biol Crystallogr</source><year>2010</year><volume>66</volume><issue>2</issue><fpage>125</fpage><lpage>132</lpage><pub-id pub-id-type="doi">10.1107/S0907444909047337</pub-id><pub-id pub-id-type="pmid">20124692</pub-id></mixed-citation></ref><ref id="B49"><mixed-citation publication-type="journal"><name><surname>Winn</surname><given-names>MD</given-names></name><name><surname>Ballard</surname><given-names>CC</given-names></name><name><surname>Cowtan</surname><given-names>KD</given-names></name><name><surname>Dodson</surname><given-names>EJ</given-names></name><name><surname>Emsley</surname><given-names>P</given-names></name><name><surname>Evans</surname><given-names>PR</given-names></name><name><surname>Keegan</surname><given-names>RM</given-names></name><name><surname>Krissinel</surname><given-names>EB</given-names></name><name><surname>Leslie</surname><given-names>AG</given-names></name><name><surname>McCoy</surname><given-names>A</given-names></name><etal></etal><article-title>Overview of the CCP4 suite and current developments</article-title><source>Acta Crystallogr D Biol Crystallogr</source><year>2011</year><volume>67</volume><issue>Pt 4</issue><fpage>235</fpage><lpage>242</lpage><pub-id pub-id-type="pmid">21460441</pub-id></mixed-citation></ref><ref id="B50"><mixed-citation publication-type="journal"><name><surname>Evans</surname><given-names>P</given-names></name><article-title>Scaling and assessment of data quality</article-title><source>Acta Crystallogr D Biol Crystallogr</source><year>2006</year><volume>62</volume><issue>Pt 1</issue><fpage>72</fpage><lpage>82</lpage><pub-id pub-id-type="pmid">16369096</pub-id></mixed-citation></ref><ref id="B51"><mixed-citation publication-type="journal"><name><surname>French</surname><given-names>S</given-names></name><name><surname>Wilson</surname><given-names>K</given-names></name><article-title>On the treatment of negative intensity observations</article-title><source>Acta Crystallogr A</source><year>1978</year><volume>34</volume><fpage>517</fpage><lpage>525</lpage></mixed-citation></ref><ref id="B52"><mixed-citation publication-type="journal"><name><surname>Murshudov</surname><given-names>GN</given-names></name><name><surname>Vagin</surname><given-names>AA</given-names></name><name><surname>Dodson</surname><given-names>EJ</given-names></name><article-title>Refinement of macromolecular structures by the maximum-likelihood method</article-title><source>Acta Crystallogr D Biol Crystallogr</source><year>1997</year><volume>53</volume><issue>Pt 3</issue><fpage>240</fpage><lpage>255</lpage><pub-id pub-id-type="pmid">15299926</pub-id></mixed-citation></ref><ref id="B53"><mixed-citation publication-type="journal"><name><surname>Adams</surname><given-names>PD</given-names></name><name><surname>Afonine</surname><given-names>PV</given-names></name><name><surname>Bunkoczi</surname><given-names>G</given-names></name><name><surname>Chen</surname><given-names>VB</given-names></name><name><surname>Davis</surname><given-names>IW</given-names></name><name><surname>Echols</surname><given-names>N</given-names></name><name><surname>Headd</surname><given-names>JJ</given-names></name><name><surname>Hung</surname><given-names>LW</given-names></name><name><surname>Kapral</surname><given-names>GJ</given-names></name><name><surname>Grosse-Kunstleve</surname><given-names>RW</given-names></name><etal></etal><article-title>PHENIX: a comprehensive Python-based system for macromolecular structure solution</article-title><source>Acta Crystallogr D Biol Crystallogr</source><year>2010</year><volume>66</volume><issue>Pt 2</issue><fpage>213</fpage><lpage>221</lpage><pub-id pub-id-type="pmid">20124702</pub-id></mixed-citation></ref><ref id="B54"><mixed-citation publication-type="journal"><name><surname>Schuttelkopf</surname><given-names>AW</given-names></name><name><surname>van Aalten</surname><given-names>DM</given-names></name><article-title>PRODRG: a tool for high-throughput crystallography of protein-ligand complexes</article-title><source>Acta Crystallogr D Biol Crystallogr</source><year>2004</year><volume>60</volume><issue>Pt 8</issue><fpage>1355</fpage><lpage>1363</lpage><pub-id pub-id-type="pmid">15272157</pub-id></mixed-citation></ref><ref id="B55"><mixed-citation publication-type="journal"><name><surname>Word</surname><given-names>JM</given-names></name><name><surname>Lovell</surname><given-names>SC</given-names></name><name><surname>Richardson</surname><given-names>JS</given-names></name><name><surname>Richardson</surname><given-names>DC</given-names></name><article-title>Asparagine and glutamine: using hydrogen atom contacts in the choice of side-chain amide orientation</article-title><source>J Mol Biol</source><year>1999</year><volume>285</volume><issue>4</issue><fpage>1735</fpage><lpage>1747</lpage><pub-id pub-id-type="doi">10.1006/jmbi.1998.2401</pub-id><pub-id pub-id-type="pmid">9917408</pub-id></mixed-citation></ref><ref id="B56"><mixed-citation publication-type="journal"><name><surname>Laskowski</surname><given-names>RA</given-names></name><name><surname>Swindells</surname><given-names>MB</given-names></name><article-title>LigPlot+: multiple ligand-protein interaction diagrams for drug discovery</article-title><source>J Chem Inf Model</source><year>2011</year><volume>51</volume><issue>10</issue><fpage>2778</fpage><lpage>2786</lpage><pub-id pub-id-type="doi">10.1021/ci200227u</pub-id><pub-id pub-id-type="pmid">21919503</pub-id></mixed-citation></ref></ref-list></back></pmc></record>Pour manipuler ce document sous Unix (Dilib)
EXPLOR_STEP=$WICRI_ROOT/Sante/explor/H2N2V1/Data/Pmc/Corpus
HfdSelect -h $EXPLOR_STEP/biblio.hfd -nk 000592 | SxmlIndent | more
Ou
HfdSelect -h $EXPLOR_AREA/Data/Pmc/Corpus/biblio.hfd -nk 000592 | SxmlIndent | more
Pour mettre un lien sur cette page dans le réseau Wicri
{{Explor lien
|wiki= Sante
|area= H2N2V1
|flux= Pmc
|étape= Corpus
|type= RBID
|clé=
|texte=
}}
| This area was generated with Dilib version V0.6.33. |  |