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• W1963535341 abstract "S-1153 (AG1549) is perhaps the most promising non-nucleoside inhibitor of HIV-1 reverse transcriptase currently under development as a potential anti-AIDS drug, because it has a favorable profile of resilience to many drug resistance mutations. We have determined the crystal structure of S-1153 in a complex with HIV-1 reverse transcriptase. The complex possesses some novel features, including an extensive network of hydrogen bonds involving the main chain of residues 101, 103, and 236 of the p66 reverse transcriptase subunit. Such interactions are unlikely to be disrupted by side chain mutations. The reverse transcriptase/S-1153 complex suggests different ways in which resilience to mutations in the non-nucleoside inhibitors of reverse transcriptase binding site can be achieved. S-1153 (AG1549) is perhaps the most promising non-nucleoside inhibitor of HIV-1 reverse transcriptase currently under development as a potential anti-AIDS drug, because it has a favorable profile of resilience to many drug resistance mutations. We have determined the crystal structure of S-1153 in a complex with HIV-1 reverse transcriptase. The complex possesses some novel features, including an extensive network of hydrogen bonds involving the main chain of residues 101, 103, and 236 of the p66 reverse transcriptase subunit. Such interactions are unlikely to be disrupted by side chain mutations. The reverse transcriptase/S-1153 complex suggests different ways in which resilience to mutations in the non-nucleoside inhibitors of reverse transcriptase binding site can be achieved. human immunodeficiency virus non-nucleoside reverse transcriptase inhibitor reverse transcriptase 5-(3, 5-dichlorophenyl)thio-4-isopropyl-1-(4-pyridyl)methyl-1H-imidazol-2ylmethyl carbamate root mean square The introduction of highly active antiretroviral therapy involving the use of multidrug combinations has resulted in dramatic falls in death rates from HIV1infection and AIDS for patients receiving such treatment (1.Brettle R.P. Wilson A. Povey S. Morris S. Morgan R. Leen C.L. Hutchinson S. Lewis S. Gore S. Int. J. STD AIDS. 1998; 9: 80-87Crossref PubMed Scopus (35) Google Scholar, 2.Mocroft A. Vella S. Benfield T.L. Chiesi A. Miller V. Gargalianos P. d'Arminio-Monforte A. Yust I. Bruun J.N. Phillips A.N. Lundgren J.D. Lancet. 1998; 352: 1725-1730Abstract Full Text Full Text PDF PubMed Scopus (1194) Google Scholar). However, because of the high replication rate of HIV (3.Ho D.D. Neumann A.U. Perelson A.S. Chen W. Leonard J.M. Markowitz M. Nature. 1995; 373: 123-126Crossref PubMed Scopus (3789) Google Scholar), which allows a rapid selection of escape mutants, these current drug regimens are likely to become increasingly ineffective with time. To be able to effectively treat HIV infection in the future, further new drugs with activity against the emerging drug-resistant viruses will be required. The non-nucleoside inhibitors of reverse transcriptase (NNRTIs) are now established as part of multidrug combinations for treating HIV infection (4.De Clercq E. Antiviral Res. 1998; 38: 153-179Crossref PubMed Scopus (367) Google Scholar, 5.Gazzard B.G. Int. J. Clin. Pract. 1999; 53: 60-64PubMed Google Scholar). So-called “first generation” NNRTI drugs, such as nevirapine and delavirdine (U-90152), are generally very susceptible to the effects of single point resistance mutations within RT (6.Richman D. Shih C.-K. Lowy I. Rose J. Prodanovich P. Goff S. Griffin J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 11241-11245Crossref PubMed Scopus (376) Google Scholar, 7.Schinazi R.F. Larder B.A. Mellors J.W. Int. Antiviral News. 1997; 5: 129-135Google Scholar). In contrast, more recent “second generation” NNRTIs, such as efavirenz (DMP-266) (8.Young S.D. Britcher S.F. Tran L.O. Payne L.S. Lumma W.C. Lyle T.A. Huff J.R. Anderson P.S. Olsen D.B. Carroll S.S. Pettibone D.J. O'Brien J.A. Ball R.G. Balani S.K. Lin J.H. Chen I.-W. Schleif W.A. Sardana V.V. Long W.J. Byrnes V.W. Emini E.A. Antimicrob. Agents Chemother. 1995; 39: 2602-2605Crossref PubMed Scopus (485) Google Scholar), the carboxanlide, UC-781 (9.Balzarini J. Brouwer W.G. Felauer E.E. De Clercq E. Karlsson A. Antiviral Res. 1995; 27: 219-236Crossref PubMed Scopus (42) Google Scholar), and certain quinoxalines (10.Kleim J.-P. Bender R. Billhardt U.-M. Meichsner C. Riess G. Rosner M. Winkler I. Paessens A. Antimicrob. Agents Chemother. 1993; 37: 1659-1664Crossref PubMed Scopus (131) Google Scholar), demonstrate much greater resilience to the presence of such mutations within RT. Previous crystallographic studies have shown a common binding site for many classes of NNRTI, with a high degree of overlap for chemically divergent compounds (11.Kohlstaedt L.A. Wang J. Friedman J.M. Rice P.A. Steitz T.A. Science. 1992; 256: 1783-1790Crossref PubMed Scopus (1757) Google Scholar, 12.Ren J. Esnouf R. Garman E. Somers D. Ross C. Kirby I. Keeling J. Darby G. Jones Y. Stuart D. Stammers D. Nat. Struct. Biol. 1995; 2: 293-302Crossref PubMed Scopus (557) Google Scholar, 13.Ren J. Esnouf R. Hopkins A. Ross C. Jones Y. Stammers D. Stuart D. Structure. 1995; 3: 915-926Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar, 14.Ding J. Das K. Moereels H. Koymans L. Andries K. Janssen P.A.J. Hughes S.H. Arnold E. Nat. Struct. Biol. 1995; 2: 407-415Crossref PubMed Scopus (352) Google Scholar, 15.Hopkins A.L. Ren J. Esnouf R.M. Willcox B.E. Jones E.Y. Ross C. Miyasaka T. Walker R.T. Tanaka H. Stammers D.K. Stuart D.I. J. Med. Chem. 1996; 39: 1589-1600Crossref PubMed Scopus (363) Google Scholar, 16.Esnouf R.M. Ren J. Hopkins A.L. Ross C.K. Jones E.Y. Stammers D.K. Stuart D.I. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3984-3989Crossref PubMed Scopus (195) Google Scholar, 17.Ren J. Esnouf R.M. Hopkins A.L. Stuart D.I. Stammers D.K. J. Med. Chem. 1999; 42: 3845-3851Crossref PubMed Scopus (47) Google Scholar, 18.Ren J. Diprose J. Warren J. Esnouf R.M. Bird L.E. Ikemizu S. Slater M. Milton J. Balzarini J. Stuart D.I. Stammers D.K. J. Biol. Chem. 2000; 275 (in press)Google Scholar). Crystallographic and kinetic studies indicate the mechanism of inhibition of NNRTIs is via a distortion of the catalytic aspartate residues in the polymerase active site (19.Esnouf R. Ren J. Ross C. Jones Y. Stammers D. Stuart D. Nat. Struct. Biol. 1995; 2: 303-308Crossref PubMed Scopus (447) Google Scholar,20.Spence R.A. Kati W.M. Anderson K.S. Johnson K.A. Science. 1995; 267: 988-993Crossref PubMed Scopus (464) Google Scholar). More recently structural studies have revealed some common factors that contribute to the second generation properties of UC-781 (21.Ren J. Esnouf R.M. Hopkins A.L. Warren J. Balzarini J. Stuart D.I. Stammers D.K. Biochemistry. 1998; 37: 14394-14403Crossref PubMed Scopus (105) Google Scholar). Such factors include a combination of a nonaromatic moiety of optimal size that contacts the aromatic residues Tyr-181, Tyr-188, and Trp-229 at the top of the NNRTI pocket. Additionally, the presence of main chain hydrogen bonding and an overall small compound bulk, thereby allowing rearrangement within a mutated drug binding site, appear to be important. A new NNRTI S-1153 (also now referred to as AG1549) has recently been described (Scheme FS1), which appears to have better resilience properties than any previously reported compound (22.Fujiwara T. Sato A. el-Farrash M. Miki S. Abe K. Isaka Y. Kodama M. Wu Y. Chen L.B. Harada H. Sugimoto H. Hatanaka M Y H. Antimicrob. Agents Chemother. 1998; 42: 1340-1345Crossref PubMed Google Scholar). S-1153 is also structurally distinct from other second generation inhibitors such as efavirenz (8.Young S.D. Britcher S.F. Tran L.O. Payne L.S. Lumma W.C. Lyle T.A. Huff J.R. Anderson P.S. Olsen D.B. Carroll S.S. Pettibone D.J. O'Brien J.A. Ball R.G. Balani S.K. Lin J.H. Chen I.-W. Schleif W.A. Sardana V.V. Long W.J. Byrnes V.W. Emini E.A. Antimicrob. Agents Chemother. 1995; 39: 2602-2605Crossref PubMed Scopus (485) Google Scholar) and UC-781 (9.Balzarini J. Brouwer W.G. Felauer E.E. De Clercq E. Karlsson A. Antiviral Res. 1995; 27: 219-236Crossref PubMed Scopus (42) Google Scholar) (Scheme FS1). S-1153 retains full activity against the clinically most common NNRTI resistance mutation, Lys-103 → Asn, compared with the 47-fold loss of activity seen for nevirapine (Table I). Almost full activity is also retained for mutations at codons 106, 188, 190, and 227, whereas nevirapine shows ≫74-fold weaker binding to these mutants (22.Fujiwara T. Sato A. el-Farrash M. Miki S. Abe K. Isaka Y. Kodama M. Wu Y. Chen L.B. Harada H. Sugimoto H. Hatanaka M Y H. Antimicrob. Agents Chemother. 1998; 42: 1340-1345Crossref PubMed Google Scholar). High level resistance to S-1153 requires at least two mutations (e.g. Val-106 → Ala and Phe-227 → Leu, or Lys-103 → Thr, Val-106 → Ala and Leu-234 → Ile (22.Fujiwara T. Sato A. el-Farrash M. Miki S. Abe K. Isaka Y. Kodama M. Wu Y. Chen L.B. Harada H. Sugimoto H. Hatanaka M Y H. Antimicrob. Agents Chemother. 1998; 42: 1340-1345Crossref PubMed Google Scholar)). Indeed even some double mutations have only a limited effect, thus Val-106 → Ala and Tyr-181 → Cys result in only an 8-fold loss of activity (TableI).Table IComparison of sensitivities of HIV-I containing mutations within the reverse transcriptase to NNRTIs S-1153 and nevirapineHIV-1 mutantEC50 (mutant)/EC50(wild type)aEC50 is defined as the concentration of compound required to inhibit syncytia formation in HIV-infected cells (adapted from Ref. 22).S-1153NevirapineLeu-100 → Ile3.02.1Lys-103 → Asn1.044.1Val-106 → Ala4.5≫74Tyr-181 → Cys13.5≫74Tyr-188 → Cys1.5≫74Gly-190 → Ala1.1≫74Phc-227 → Leu1.54.1Leu-234 → Ile21.90.5Pro-236 → Leu3.53.1Val-106 → Ala & Tyr-181 → Cys8.1≫74a EC50 is defined as the concentration of compound required to inhibit syncytia formation in HIV-infected cells (adapted from Ref. 22.Fujiwara T. Sato A. el-Farrash M. Miki S. Abe K. Isaka Y. Kodama M. Wu Y. Chen L.B. Harada H. Sugimoto H. Hatanaka M Y H. Antimicrob. Agents Chemother. 1998; 42: 1340-1345Crossref PubMed Google Scholar). Open table in a new tab To investigate the structural basis of this remarkable resilience, we have determined a crystal structure of S-1153 in complex with its molecular receptor HIV-1 RT. Knowledge derived from this study should be of value in the design of novel inhibitors that are active against drug-resistant HIV strains. Further, such drugs will be of importance for the continued effective treatment of HIV infection and AIDS. Crystals of the complex of HIV-1 RT with S-1153 (synthesized at Shionogi and Co. Ltd.) were grown and treated prior to data collection as described previously (23.Stammers D.K. Somers D.O.N. Ross C.K. Kirby I. Ray P.H. Wilson J.E. Norman M. Ren J.S. Esnouf R.M. Garman E.F. Jones E.Y. Stuart D.I. J. Mol. Biol. 1994; 242: 586-588Crossref PubMed Scopus (64) Google Scholar). X-ray data were collected at the Synchrotron Radiation Source (Daresbury, UK) on station PX9.6 using an ADSC Quantum-4 CCD detector. A crystal was initially frozen in liquid propane and then maintained at 100 K in a stream of nitrogen gas during data collection. Indexing and integration of data images were carried out with DENZO, and data were merged with SCALEPACK (24.Otwinowski Z. Minor W. Methods Enzymol. 1996; 276: 307-326Crossref Scopus (38526) Google Scholar). The unit cell dimensions are closest to our F crystal form (25.Esnouf R.M. Ren J. Garman E.F. Somers D.O.N. Ross C.K. Jones E.Y. Stammers D.K. Stuart D.I. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 938-954Crossref PubMed Scopus (73) Google Scholar). Details of the x-ray data statistics are given in Table II. The data are reasonably complete and reliable to 2.5 Å resolution.Table IIStatistics for crystallographic structure determinationsData collection details Data collection sitePX9.6 SRS Wavelength (Å)0.87 Collimation (mm)0.2 × 0.2 Unit cell (a,b,c in Å)135.7, 118.0, 67.3 Resolution range (Å)30.0–2.5 Observations10,8379 Unique reflections35,008 Completeness (%)90.9 Reflections withI/ς(I)>229,394 R merge (%)aR merge = Σ‖I-<I>‖/Σ<I>.5.1Outer resolution shell Resolution range (Å)2.59–2.5 Unique reflections3261 Completeness (%)86.2 Reflections withI/ς(I)>21859Refinement statistics Resolution range (Å)30.0–2.5 Unique reflections (working/test)32,992/1704 (F>0) R factor (R-working/R-free)bR factor = Σ‖F o −F c‖/Σ F o.0.254/0.330 R factor (all data)0.242 Number of atomscProtein atoms/water molecules/inhibitor atoms. Residues 1–3, 65–70, 137–141, 219–220, and 540–560 are disordered in the p66 subunit. Residues 1–4, 89–95, 216–232, 357–361, and 428–440 of the p51 subunit are also not defined in the electron density map.7564/197/29 r.m.s. bond length deviation (Å)0.0095 r.m.s. bond angle deviation (°)1.54 Mean B factor (Å3)dMean B-factor for main chain, side chain, water, and inhibitor atoms, respectively.69/72/57/52 r.m.s. backbone B-factor deviationer.m.s. deviation between B-factors for bonded main chain atoms.2.5a R merge = Σ‖I-<I>‖/Σ<I>.b R factor = Σ‖F o −F c‖/Σ F o.c Protein atoms/water molecules/inhibitor atoms. Residues 1–3, 65–70, 137–141, 219–220, and 540–560 are disordered in the p66 subunit. Residues 1–4, 89–95, 216–232, 357–361, and 428–440 of the p51 subunit are also not defined in the electron density map.d Mean B-factor for main chain, side chain, water, and inhibitor atoms, respectively.e r.m.s. deviation between B-factors for bonded main chain atoms. Open table in a new tab The orientation and position of HIV-1 RT in the unit cell were determined using rigid body refinement with XPLOR (26.Brunger A.T. X-PLOR Manual, Version 3.1. Yale University Press, New Haven, CT1992Google Scholar), using the RT/9-Cl-TIBO complex (1rev) (13.Ren J. Esnouf R. Hopkins A. Ross C. Jones Y. Stammers D. Stuart D. Structure. 1995; 3: 915-926Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar) as an initial model. The structure was first refined with XPLOR (26.Brunger A.T. X-PLOR Manual, Version 3.1. Yale University Press, New Haven, CT1992Google Scholar) and then with CNS (27.Brunger A.T. Adams P.D. Clore G.M. Delano W.L. Gros P. Grosse K.R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16957) Google Scholar), using positional, simulated annealing and individual B-factor refinement with bulk solvent correction and anisotropic B-factor scaling. Model rebuilding was carried out on an Evans and Sutherland ESV workstation using FRODO (28.Jones T.A. Methods Enzymol. 1985; 115: 157-171Crossref PubMed Scopus (936) Google Scholar). The R factor for the final model of RT/S-1153 was 0.242, for all data in the range of 30.0–2.5 Å resolution with r.m.s. deviations from ideality of 0.0095 Å and 1.54° for bond lengths and bond angles, respectively. Table II shows the statistics on the refinement and quality of the structure. All figures were produced using BOBSCRIPT (29.Esnouf R.M. J. Mol. Graphics. 1997; 15: 132-134Crossref Scopus (1794) Google Scholar), a modified version of MOLSCRIPT (30.Kraulis P.J. J. Appl. Crystallogr. 1991; 24: 946-950Crossref Google Scholar), and rendered with RASTER3D (31.Merritt E.A. Murphy M.E.P. Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 869-873Crossref PubMed Scopus (2857) Google Scholar). An “omit” electron-density map for S-1153 unambiguously revealed the orientation and conformation of the inhibitor in the NNRTI binding site (Fig. 1). A comparison of the bound conformation of S-1153 with the first generation NNRTI, nevirapine, is shown in Fig. 2 a, whereas an overlap of S-1153 with the second generation NNRTI, UC-781, is shown in Fig. 2 b. There is mutual overlap between the cyclopropyl group of nevirapine, part of the furanyl ring of UC-781, and the isopropyl group of S-1153. These structural features position the side chain of Tyr-181 so as to give tight binding (15.Hopkins A.L. Ren J. Esnouf R.M. Willcox B.E. Jones E.Y. Ross C. Miyasaka T. Walker R.T. Tanaka H. Stammers D.K. Stuart D.I. J. Med. Chem. 1996; 39: 1589-1600Crossref PubMed Scopus (363) Google Scholar). For S-1153, the 3,5-dichlorophenyl ring is situated further toward the “top” of the NNRTI pocket than the equivalent aromatic ring for nevirapine, TNK-651 (15.Hopkins A.L. Ren J. Esnouf R.M. Willcox B.E. Jones E.Y. Ross C. Miyasaka T. Walker R.T. Tanaka H. Stammers D.K. Stuart D.I. J. Med. Chem. 1996; 39: 1589-1600Crossref PubMed Scopus (363) Google Scholar), or GCA-186 (32.Hopkins A.L. Ren J. Tanaka H. Baba M. Okamato M. Stuart D.I. Stammers D.K. J. Med. Chem. 1999; 42: 4500-4505Crossref PubMed Scopus (132) Google Scholar). This reflects the close association of the 3,5-dichlorophenyl ring with the side chain of Trp-229, which appears to be repositioned higher in the pocket because of a conformational change in the β9-β10-β11 strands, caused by the interaction between the pyridyl and carbamate groups of S-1153 with Pro-236 in the β10-β11 loop (Fig. 2 a). As a result the β9-β10-β11 strands are pushed away from the pocket giving a more open pocket entrance (16.Esnouf R.M. Ren J. Hopkins A.L. Ross C.K. Jones E.Y. Stammers D.K. Stuart D.I. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3984-3989Crossref PubMed Scopus (195) Google Scholar) and allowing the side chain of Trp-229 to sit higher. The magnitude of this conformational change is exemplified by the mean displacement of α-carbon positions for residues 227–241 in different RT/NNRTI complexes. For RT/nevirapine and RT/UC-781 this is 1.3 Å, whereas for RT/S-1153 and RT·nevirapine this displacement is 3.2 Å, and for RT/S-1153 and RT/UC-781 it is 3.6 Å. The carbamoyloxymethyl group of S-1153 has little overlap with groups in other RT/NNRTIs complexes except for delavirdine, where the carbonyl oxygen is in a similar position (16.Esnouf R.M. Ren J. Hopkins A.L. Ross C.K. Jones E.Y. Stammers D.K. Stuart D.I. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3984-3989Crossref PubMed Scopus (195) Google Scholar).Figure 2Stereo diagrams showing the relative orientations and positions of NNRTIs when bound to HIV-1 RT (a) S-1153 (orange), nevirapine (cyan) and (b) S-1153 (orange), UC-781 (cyan). The Cα backbone and side chains of RT/nevirapine and RT/UC-781 are shown in light gray, and those of RT/S-1153 are in dark gray. The superimpositions were carried out using residues 94–118, 156–215, and 317–319 of the p66 subunit and 137–139 of the p51 subunit. Because of the large movements of residues in the β9-β11 sheet, these were not used in the superposition.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The interactions of S-1153 with HIV-1 RT are shown in Figs.3 and 4. As with all other NNRTIs there are a wide range of hydrophobic contacts between inhibitor and protein. The 3,5-dichlorophenyl group is positioned edge on to the plane of the indole ring of Trp-229, making extensive favorable contacts with a closest approach of 3.7 Å. There are also contacts between the 3,5-dichlorophenyl group and the aromatic rings of Tyr-181 and Tyr-188 (the latter also contacts the sulfur atom of the inhibitor). The other substituents of the imidazole ring make many interactions with the protein. The pyridyl group contacts the side chain of Phe-227 and the main chain of His-235; this group is positioned parallel with the side chain of Pro-236 but approaches no closer than 4.5 Å. The isopropyl group contacts the main chain of Tyr-188 and in addition the side chain of Val-106. The methyl carbamoyloxymethyl group contacts a number of residues at the base of the pocket including Lys-101, Lys-102, and Lys-103 (Fig. 3.).Figure 4Stereo view of S-1153 positioned in the NNRTI binding pocket. Hydrogen bonds that form interactions between S-1153 and the RT main chain either directly or via a water molecule are shown as dashed yellow lines. The inhibitor is shown in standard atom colors. The protein main chain backbone and side chains are shown in cyan and gray, respectively. Water molecules are represented as magenta spheres.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The most surprising feature of the interaction of S-1153 with the NNRTI binding site is the series of three hydrogen bonds with the protein main chain of RT (Figs. 3 and 4). The carbamoyloxymethyl group of S-1153 forms two direct hydrogen bonds with the protein main chain: from its nitrogen to the main chain carbonyl of Pro-236 and from the terminal oxygen to the main chain nitrogen of Lys-103. The conformational rearrangement of the β9-β10-β11 strands described above results in Pro-236 shifting by about 3.5 Å, breaking a main chain hydrogen bond from its carbonyl to Lys-103, and positioning it instead to hydrogen bond to the carbamate nitrogen of S-1153. A final hydrogen bond is mediated via a water molecule from an imidazole nitrogen of S-1153 to the main chain carbonyl of Lys-101 (Fig. 4). Given the substantial data base of structures of RT/NNRTI complexes, we are in a position to dissect out structural features responsible for the resilience of S-1153 to drug resistance mutations within RT. The most remarkable feature of the interaction of S-1153 with HIV RT is the network of three hydrogen-bonding interactions with the main chain, two of which are direct to the protein, whereas one is mediated by a water molecule. The presence of an appropriately positioned carbamate group provides two of these hydrogen bonds (to residues 103 and 236). This is entirely unprecedented for an RT/NNRTI complex. Some inhibitors for example, nevirapine, α-APA (12.Ren J. Esnouf R. Garman E. Somers D. Ross C. Kirby I. Keeling J. Darby G. Jones Y. Stuart D. Stammers D. Nat. Struct. Biol. 1995; 2: 293-302Crossref PubMed Scopus (557) Google Scholar), and BM+21.1326(17) bind to RT without any main chain hydrogen bonding. There are several examples of NNRTIs with single hydrogen-bonding interactions with the main chain. Thus in the case of Cl-TIBO, emivirine (MKC-442), TNK-651, UC-10, UC-38, UC-84, UC-781, each inhibitor has an interaction with the main chain carbonyl of Lys-101 (13.Ren J. Esnouf R. Hopkins A. Ross C. Jones Y. Stammers D. Stuart D. Structure. 1995; 3: 915-926Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar, 15.Hopkins A.L. Ren J. Esnouf R.M. Willcox B.E. Jones E.Y. Ross C. Miyasaka T. Walker R.T. Tanaka H. Stammers D.K. Stuart D.I. J. Med. Chem. 1996; 39: 1589-1600Crossref PubMed Scopus (363) Google Scholar, 21.Ren J. Esnouf R.M. Hopkins A.L. Warren J. Balzarini J. Stuart D.I. Stammers D.K. Biochemistry. 1998; 37: 14394-14403Crossref PubMed Scopus (105) Google Scholar). Delavirdine (U-90152) has a pair of hydrogen bonds from its carbonyl oxygen and indole nitrogen groups to the main chain of Lys-103 (16.Esnouf R.M. Ren J. Hopkins A.L. Ross C.K. Jones E.Y. Stammers D.K. Stuart D.I. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3984-3989Crossref PubMed Scopus (195) Google Scholar). Hydrogen bonding to the main chain of Pro-236 as observed here for S-1153 has not been previously reported for any other NNRTI. The rearrangement of the β9-β10-β11 sheet reported here, which is necessary for the Pro-236 main chain hydrogen bonding interaction with S-1153, is facilitated by the great flexibility of this part of the NNRTI pocket that we have previously characterized through the substantial differences in conformation observed for complexes with TNK-651 (15.Hopkins A.L. Ren J. Esnouf R.M. Willcox B.E. Jones E.Y. Ross C. Miyasaka T. Walker R.T. Tanaka H. Stammers D.K. Stuart D.I. J. Med. Chem. 1996; 39: 1589-1600Crossref PubMed Scopus (363) Google Scholar) and delavirdine (16.Esnouf R.M. Ren J. Hopkins A.L. Ross C.K. Jones E.Y. Stammers D.K. Stuart D.I. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3984-3989Crossref PubMed Scopus (195) Google Scholar). Such flexibility presents additional opportunities for favorable interactions but also makes the job of predicting inhibitor interactions with RT from modeling studies problematic. It seems likely that the extensive network of main chain inhibitor hydrogen bonds observed contribute substantially to the free energy of binding for the inhibitor. In this case we would expect them to confer favorable resilience properties to mutation within the protein, because side chain changes will generally have no direct effect on such interactions. Indeed tissue culture experiments show that for HIV to escape S-1153, multiple mutations in RT are required (22.Fujiwara T. Sato A. el-Farrash M. Miki S. Abe K. Isaka Y. Kodama M. Wu Y. Chen L.B. Harada H. Sugimoto H. Hatanaka M Y H. Antimicrob. Agents Chemother. 1998; 42: 1340-1345Crossref PubMed Google Scholar). Examples of this include the double mutation of Val-106 → Ala and Phe-227 → Leu (both of these side chains make significant contacts with the inhibitor) and a triple mutant with Lys-103 → Thr, Val-106 → Ala, and Leu-234 → Ile. This second clone has Val-106 → Ala in common with the double mutant and this change presumably acts directly. Model building studies indicate that the Leu-234 → Ile mutation gives unfavorable contacts with the S-1153 pyridyl group as a result of the close approach of the CG2 methyl moiety of the isoleucine side chain. The mutation at 103 is, however, more intriguing; a change at codon 103 from lysine to asparagine is the most commonly observed NNRTI resistance mutation in the clinic, giving wide cross-resistance to most NNRTIs (7.Schinazi R.F. Larder B.A. Mellors J.W. Int. Antiviral News. 1997; 5: 129-135Google Scholar). We have suggested that this may be because of the stabilization of the unliganded enzyme by the formation of a hydrogen bond between the asparagine side chain and the hydroxyl group of Tyr-188 (19.Esnouf R. Ren J. Ross C. Jones Y. Stammers D. Stuart D. Nat. Struct. Biol. 1995; 2: 303-308Crossref PubMed Scopus (447) Google Scholar). S-1153, however, shows no loss of potency for the Lys-103 → Asn mutation (22.Fujiwara T. Sato A. el-Farrash M. Miki S. Abe K. Isaka Y. Kodama M. Wu Y. Chen L.B. Harada H. Sugimoto H. Hatanaka M Y H. Antimicrob. Agents Chemother. 1998; 42: 1340-1345Crossref PubMed Google Scholar), and modeling indicates that a hydrogen bond might be formed between the carbonyl moiety of the asparagine side chain and the imidazole nitrogen of S-1153, canceling out the effect of stabilization of the unliganded enzyme. It is not entirely clear why, in the presence of S-1153, threonine is selected as a mutation at codon 103 rather than asparagine, because its shorter side chain would be unable to make this interaction, and it is unclear that it could stabilize the unliganded enzyme. A second possible source of resilience in the RT/S-1153 interactions may be the relative flexibility of the inhibitor itself, which might allow it to adapt to a mutated drug pocket more readily than, for example, the rigid fused ring structure of the first generation compound nevirapine. Further structural work on mutant RT/S-1153 complexes is required to test this possibility. Other features in the interactions of S-1153 with RT set it aside from second generation NNRTIs such as UC-781. Thus UC-781 has a small nonaromatic dimethylallyl group occupying the “top” of the NNRTI pocket, whereas S-1153 has a more bulky phenyl ring, reminiscent of many first generation compounds. However, the presence of the 3,5-dichloro substituents results in more extensive contacts with the highly conserved Trp-229 side chain, when compared with the unsubstituted phenyl ring of a first generation compound such as TNK-651 (15.Hopkins A.L. Ren J. Esnouf R.M. Willcox B.E. Jones E.Y. Ross C. Miyasaka T. Walker R.T. Tanaka H. Stammers D.K. Stuart D.I. J. Med. Chem. 1996; 39: 1589-1600Crossref PubMed Scopus (363) Google Scholar). Indeed direct support for this explanation comes from a study where 3,5-dimethyl substitution of the phenyl ring of the emivirine analogue GCA-186 results in the compound being substantially less affected by the Tyr-181 → Cys mutation (32.Hopkins A.L. Ren J. Tanaka H. Baba M. Okamato M. Stuart D.I. Stammers D.K. J. Med. Chem. 1999; 42: 4500-4505Crossref PubMed Scopus (132) Google Scholar). Thus an appropriately substituted phenyl ring provides an alternative route to resilience to this mutation compared with the small nonaromatic moieties of other second generation NNRTIs. Second generation NNRTIs can employ alternative strategies to minimize the effects of many drug resistance mutations within RT. S-1153 has distinctive features, when compared with second generation compounds such as UC-781, in its interactions with RT. This leads to the hope that a range of further NNRTIs can be designed to progressively counter newly emerging resistant HIV strains resulting from the strong selective pressure of effective chemotherapy. We thank the staff of the Synchrotron Radiation Source, Daresbury Laboratory, United Kingdom, for their help with data collection and Richard Bryan and Robert Esnonf for computer support." @default.
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• W1963535341 title "Binding of the Second Generation Non-nucleoside Inhibitor S-1153 to HIV-1 Reverse Transcriptase Involves Extensive Main Chain Hydrogen Bonding" @default.
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