Title: Phenylethylthiazolylthiourea (PETT) Non-nucleoside Inhibitors of HIV-1 and HIV-2 Reverse Transcriptases
Abstract: Most non-nucleoside reverse transcriptase (RT) inhibitors are specific for HIV-1 RT and demonstrate minimal inhibition of HIV-2 RT. However, we report that members of the phenylethylthiazolylthiourea (PETT) series of non-nucleoside reverse transcriptase inhibitors showing high potency against HIV-1 RT have varying abilities to inhibit HIV-2 RT. Thus, PETT-1 inhibits HIV-1 RT with an IC50 of 6 nm but shows only weak inhibition of HIV-2 RT, whereas PETT-2 retains similar potency against HIV-1 RT (IC50 of 5 nm) and also inhibits HIV-2 RT (IC50 of 2.2 μm). X-ray crystallographic structure determinations of PETT-1 and PETT-2 in complexes with HIV-1 RT reveal the compounds bind in an overall similar conformation albeit with some differences in their interactions with the protein. To investigate whether PETT-2 could be acting at a different site on HIV-2 RT (e.g. the dNTP or template primer binding site), we compared modes of inhibition for PETT-2 against HIV-1 and HIV-2 RT. PETT-2 was a noncompetitive inhibitor with respect to the dGTP substrate for both HIV-1 and HIV-2 RTs. PETT-2 was also a noncompetitive inhibitor with respect to a poly(rC)·(dG) template primer for HIV-2 RT. These results are consistent with PETT-2 binding in corresponding pockets in both HIV-1 and HIV-2 RT with amino acid sequence differences in HIV-2 RT affecting the binding of PETT-2 compared with PETT-1. Most non-nucleoside reverse transcriptase (RT) inhibitors are specific for HIV-1 RT and demonstrate minimal inhibition of HIV-2 RT. However, we report that members of the phenylethylthiazolylthiourea (PETT) series of non-nucleoside reverse transcriptase inhibitors showing high potency against HIV-1 RT have varying abilities to inhibit HIV-2 RT. Thus, PETT-1 inhibits HIV-1 RT with an IC50 of 6 nm but shows only weak inhibition of HIV-2 RT, whereas PETT-2 retains similar potency against HIV-1 RT (IC50 of 5 nm) and also inhibits HIV-2 RT (IC50 of 2.2 μm). X-ray crystallographic structure determinations of PETT-1 and PETT-2 in complexes with HIV-1 RT reveal the compounds bind in an overall similar conformation albeit with some differences in their interactions with the protein. To investigate whether PETT-2 could be acting at a different site on HIV-2 RT (e.g. the dNTP or template primer binding site), we compared modes of inhibition for PETT-2 against HIV-1 and HIV-2 RT. PETT-2 was a noncompetitive inhibitor with respect to the dGTP substrate for both HIV-1 and HIV-2 RTs. PETT-2 was also a noncompetitive inhibitor with respect to a poly(rC)·(dG) template primer for HIV-2 RT. These results are consistent with PETT-2 binding in corresponding pockets in both HIV-1 and HIV-2 RT with amino acid sequence differences in HIV-2 RT affecting the binding of PETT-2 compared with PETT-1. HIV-2, human immunodeficiency virus type 1, type 2 reverse transcriptase non-nucleoside reverse transcriptase inhibitor phenylethylthiazolylthiourea Simian immunodeficiency virus (macaque) root mean square The use of multidrug combination therapy has resulted in a significant improvement in the survival rate of HIV1-infected individuals (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 (1185) Google Scholar). The regimens are based on inhibitors of HIV reverse transcriptase (RT), together with protease inhibitors. RT catalyzes the conversion of genomic RNA to proviral DNA, by RNA-dependent DNA polymerase, RNase H, and DNA-dependent DNA polymerase reactions. The well established nucleoside analogue reverse transcriptase inhibitor-based drugs (e.g.3′-azido-2′,3′-deoxythymidine, 2′,3′-dideoxyinosine, and 2′,3′-dideoxy-3′-thiacytidine) act as terminators of RT catalyzed DNA synthesis in their activated triphosphate forms. Nucleoside analogue reverse transcriptase inhibitors are relatively broad spectrum drugs that inhibit both HIV-1 and HIV-2 serotypes with comparable potency (3.De Clercq E. Med. Res. Rev. 1993; 13: 229-258Crossref PubMed Scopus (189) Google Scholar). More recently non-nucleoside inhibitors (NNRTIs) nevirapine, delavirdine, and efavirenz have established an important role in combination therapy of HIV infection. NNRTIs include a wide range of chemical series and are usually specific for HIV-1 RT, showing minimal inhibition of HIV-2 RT. A series of biaryl acids has been reported that inhibit both HIV-1 and HIV-2 RT, although these appear not to bind at the NNRTI site (4.Milton J. Slater M.J. Bird A.J. Spinks D. Scott G. Price C.E. Downing S. Green D.V. Madar S. Bethell R. Stammers D.K. Bioorg. Med. Chem. Lett. 1998; 8: 2623-2628Crossref PubMed Scopus (29) Google Scholar). NNRTIs such as nevirapine generally act as noncompetitive inhibitors of HIV-1 RT with respect to substrates (5.Merluzzi V.J. Hargrave K.D. Labadia M. Grozinger K. Skoog M. Wu J.C. Shih C.-K. Eckner K. Hattox S. Adams J. Rosenthal A.S. Faanes R. Eckner R.J. Koup R.A. Sullivan J.L. Science. 1990; 250: 1411-1413Crossref PubMed Scopus (730) Google Scholar), binding in a pocket some 10 Å from the polymerase active site (6.Kohlstaedt L.A. Wang J. Friedman J.M. Rice P.A. Steitz T.A. Science. 1992; 256: 1783-1790Crossref PubMed Scopus (1749) Google Scholar, 7.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 (555) Google Scholar, 8.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 (350) Google Scholar). The NNRTI binding site is contained largely within the p66 subunit of the RT heterodimer with only a few residues at the periphery of the site being contributed by the p51 subunit. The mechanism of inhibition for NNRTIs has been shown to be via a distortion of the key catalytic active site aspartyl residues (9.Esnouf R. Ren J. Ross C. Jones Y. Stammers D. Stuart D. Nat. Struct. Biol. 1995; 2: 303-308Crossref PubMed Scopus (446) Google Scholar). One series of NNRTIs previously described is the phenylethylthiazoylthiourea (PETT) series that have been shown to have potent activity against both HIV-1 virus and it's RT (10.Ahgren C. Backro K. Bell F.W. Cantrell A.S. Clemens M. Colacino J.M. Beeter J.B. Engelhardt J.A. Jaskunas S.R. Johansson N.G. Jordan C.L. Kasher J.S. Kinnick M.D. Lind P. Lopez C. Morin Jr., J.M. Muesing M.A. Noreen R. Oberg B. Paget C.J. Palkowitz J.A. Parrish C.A. Pranc P. Rippy M.K. Rydergard C. Sahlberg C. Swanson S. Ternansky R.J. Unge T. Vasilefe R.T. Vrang L. West S.J. Zhang H. Zhou X.X. Antimicrob. Agents Chemother. 1995; 39: 1329-1335Crossref PubMed Scopus (113) Google Scholar, 11.Zhang H. Vrang L. Backbro K. Lind P. Sahlberg C. Unge T. Oberg B. Antiviral Res. 1995; 28: 331-342Crossref PubMed Scopus (54) Google Scholar, 12.Cantrell A.S. Engelhardt P. Hogberg M. Jaskunas S.R. Johansson N.G. Jordan C.L. Kangasmetsa J. Kinnick M.D. Lind P. Morin Jr., J.M. Muesing M.A. Noreen R. Oberg B. Pranc P. Sahlberg C. Ternansky R.J. Vasileff R.T. Vrang L. West S.J. Zhang H. J. Med. Chem. 1996; 39: 4261-4274Crossref PubMed Scopus (128) Google Scholar, 13.Sahlberg C. Noreen R. Engelhardt P. Hogberg M. Kangasmetsa J. Vrang L. Zhang H. Bioorg. Med. Chem. Lett. 1998; 8: 1511-1516Crossref PubMed Scopus (40) Google Scholar). Further PETT analogues have been designed using information from the three-dimensional structure of HIV-1 RT (14.Vig P. Mao C. Venkatachalam T.K. Tuel-Ahlgren L. Sudbeck E.A. Uckun F.M. Bioorg. Med. Chem. 1998; 6: 1789-1797Crossref PubMed Scopus (107) Google Scholar, 15.Mao C. Vig R. Venkatachalam T.K. Sudbeck E.A. Uckun F.M. Bioorg. Med. Chem. Lett. 1998; 8: 2213-2218Crossref PubMed Scopus (74) Google Scholar, 16.Mao C. Sudbeck E.A. Venkatachalam T.K. Uckun F.M. Bioorg. Med. Chem. Lett. 1999; 9: 1593-1598Crossref PubMed Scopus (79) Google Scholar). In this work we report structural and biochemical studies for two members of this series referred to as PETT-1 and PETT-2 (SchemeFS1). Most NNRTIs rapidly select for drug-resistant HIV-1 strains, both in tissue culture and in clinical studies, which has largely precluded their use as monotherapy (17.Richman D.D. Havlir D. Corbeil J. Looney D. Ignacio C. Spector S.A. Sullivan J. Cheeseman S. Barringer K. Pauletti D. Shih C.-K. Myers M. Griffin J. J. Virol. 1994; 68: 1660-1666Crossref PubMed Google Scholar). In contrast to the “first-generation” drugs, nevirapine and delavirdine, the so-called “second-generation” NNRTI drug, efavirenz, demonstrates resilience to the effects of certain common resistance mutations (18.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 (480) Google Scholar). However resistance to such compounds is emerging, meaning that there is a continued need for the development of new drugs to combat such mutant viruses. The HIV-2 serotype was isolated three years after the identification of HIV-1 (19.Clavel F. Guyader M. Guetard D. Salle M. Montagnier L. Alizon M. Nature. 1986; 324: 691-695Crossref PubMed Scopus (208) Google Scholar, 20.Guyader M. Emerman M. Sonigo P. Clavel F. Montagnier L. Alizon M. Nature. 1987; 326: 662-669Crossref PubMed Scopus (568) Google Scholar) and was shown to be closely related to SIVmac. HIV-2 RT has approximately 60% overall amino acid sequence identity with HIV-1 RT and has comparable polymerase activity (21.Hizi A. Tal R. Shaharabany M. Loya S. J. Biol. Chem. 1991; 266: 6230-6239Abstract Full Text PDF PubMed Google Scholar). There are, however, significant sequence changes for amino acids lining the equivalent region to the HIV-1 NNRTI binding pocket within HIV-2 RT, which presumably explains the poor binding of NNRTIs for this latter enzyme. Thus, the tyrosine residues at 181 and 188 in HIV-1 RT, whose side chains are important in aromatic ring-stacking interactions with many NNRTIs (6.Kohlstaedt L.A. Wang J. Friedman J.M. Rice P.A. Steitz T.A. Science. 1992; 256: 1783-1790Crossref PubMed Scopus (1749) Google Scholar, 7.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 (555) Google Scholar, 22.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 (210) Google Scholar, 23.Ding J. Das K. Tantillo C. Zhang W. Clark A.D.J. Jessen S. Lu X. Hsiou Y. Jacobo-Molina A. Andries K. Pauwels R. Moereels H. Koymans L. Janssen P.A.J. Smith R.H.J. Kroeger Koepke R. Michejda C.J. Hughes S.H. Arnold E. Structure. 1995; 3: 365-379Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar, 24.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 (360) Google Scholar, 25.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, 26.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, 27.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) are replaced in HIV-2 RT by aliphatic leucine and isoleucine residues, respectively. The direct role of amino acid sequence changes is confirmed by studies of chimeric HIV-1/HIV-2 RTs where several residues from HIV-1 have to be incorporated into an HIV-2 chimera to give sensitivity to NNRTIs approaching that for wild-type HIV-1 RT (28.Shih C.-O. Rose J.M. Hansen G.L. Wu J.C. Bacolla A. Griffin J.A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9878-9882Crossref PubMed Scopus (98) Google Scholar, 29.Yang G. Song Q. Charles M. Drosopoulos W.C. Arnold E. Prasad V.R. J. Acquired Immune Defic. Syndr. Hum. Retrovirol. 1996; 11: 326-333Crossref PubMed Scopus (19) Google Scholar). We report studies on two NNRTIs of the PETT series and show that they vary in their ability to inhibit HIV-2 RT. Additionally we have determined the crystal structures of these inhibitors complexed with HIV-1 RT. We also describe kinetic studies comparing the modes of inhibition for the PETT compounds with HIV-1 and HIV-2 RTs to address the possibility of alternative binding sites on HIV-2 RT for PETT-2. Crystals of the complex of HIV-1 RT with PETT-2 were grown as described previously (30.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). Crystals of RT with PETT-1 were obtained by inhibitor exchange with a weak binding inhibitor, HEPT, in the crystal (7.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 (555) Google Scholar, 9.Esnouf R. Ren J. Ross C. Jones Y. Stammers D. Stuart D. Nat. Struct. Biol. 1995; 2: 303-308Crossref PubMed Scopus (446) Google Scholar). Crystals were equilibrated in 50% polyethylene glycol 3400 prior to data collection as described previously (25.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, 30.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 either beamline PX7.2 (SRS Daresbury Laboratory UK) using an oscillation camera or at beamline BL-6A (KEK, Photon Factory, Japan) using a Weissenberg camera (31.Sakabe N. Nucl. Instr. Methods Phys. Res. Sect. A. 1991; 303: 448-463Crossref Scopus (320) Google Scholar, 32.Stuart D.I. Jones E.Y. Curr. Opin. Struct. Biol. 1993; 3: 737-740Crossref Scopus (12) Google Scholar) (see Table I for further details). Crystals were either frozen in liquid propane and maintained at 100 K during data collection (RT/PETT-1) or maintained at 288 K (RT/PETT-2). Data frames of 1.5° oscillations were collected on a MAR image plate with exposure times of 90 s for RT/PETT-1. For RT/PETT-2, 3.5° frames with a coupling constant 1.5°/mm and an exposure time of 112 s were recorded on pairs of 200 × 400 mm Fuji Bas-IIIB imaging plates positioned 429.7 mm from the crystal on a cylindrical cassette. To reduce background noise, the collimator, crystal enclosure, and camera cassette were flooded with helium. The imaging plates were scanned off-line using Fuji BA 100 IP scanners. Indexing and integration of data images were carried out with DENZO, and the data were merged with SCALEPACK (33.Otwinowski Z. Minor W. Methods Enzymol. 1996; 276: 307-326Crossref Scopus (38252) Google Scholar). Details of the x-ray data statistics are given in Table I.Table IStatistics for crystallographic structure determinationsData collection details Data setRT/PETT-1RT/PETT-2 Data collection siteSRS PX7.2KEK BL-6A2 Wavelength (Å)1.4881.000 Collimation (mm)0.200.10 Unit cell dimensions (a,b,c in Å)137.1, 115.0, 65.6140.7, 110.8, 73.4(cell form F)(cell form C) Resolution range (Å)30.0–2.830.0–3.0 Observations8916586903 Unique reflections2584720940 Completeness (%)95.988.5 Reflections with F/ς(F)>31862216702 R merge (%)aR merge = Σ‖I − <I>‖/Σ<I>.9.89.5Outer resolution shell Resolution range (Å)2.9–2.83.1–3.0 Unique reflections22031771 Completeness (%)82.681.7 Reflections with F/ς(F)>3795740Refinement statistics Resolution range (Å)30.0–2.830.0–3.0 No. of reflections (working/test)23731/120519927/1013 R factor (R work/R free)bR factor = Σ‖F o −F c‖/ΣF o0.224/0.2950.199/0.276 No. of atoms (protein/inhibitor/water)7637/24/267827/23/− r.m.s. bond length deviation (Å)0.0080.008 r.m.s. bond angle deviation (°)1.41.4 Mean B-factor (Å2)cMean B factor for main chain, side chain, inhibitor and water atoms, respectively.61/66/53/4383/89/52/− r.m.s. backbone B-factor deviation (Å2)2.54.1a R merge = Σ‖I − <I>‖/Σ<I>.b R factor = Σ‖F o −F c‖/ΣF oc Mean B factor for main chain, side chain, inhibitor and water atoms, respectively. Open table in a new tab The orientation and position of RT in the unit cell were determined using rigid body refinement with X-PLOR (34.Brunger A.T. X-PLOR Manual, Version 3.1 Ed. Yale University Press, New Haven, CT1992Google Scholar). Coordinates from RT/9-Cl-TIBO (22.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 (210) Google Scholar) and RT/1051U91 (7.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 (555) Google Scholar) complexes were used as initial models for RT/PETT-1 and RT/PETT-2, respectively. The structures were first refined with X-PLOR (34.Brunger A.T. X-PLOR Manual, Version 3.1 Ed. Yale University Press, New Haven, CT1992Google Scholar) and then with CNS (35.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. Sec. D. 1998; 54: 905-921Crossref PubMed Scopus (16918) Google Scholar) using positional, simulated annealing and individual B-factor refinements with bulk solvent correction together with anisotropic B-factor scaling. Model rebuilding was carried out with FRODO (36.Jones T.A. Methods Enzymol. 1985; 115: 157-171Crossref PubMed Scopus (934) Google Scholar) on an Evans and Sutherland ESV workstation. The structure of RT/PETT-1 has been refined to an R factor of 0.224 (R free of 0.295) for all data in the range of 30.0–2.8 Å resolution. The r.m.s. deviations of bond lengths and bond angles from ideality were 0.008 Å and 1.4°, respectively, for a model containing 7637 protein atoms, the inhibitor, and 26 water molecules. The current model of the RT/PETT-2 complex containing 7827 protein atoms, the inhibitor and no water molecules, has an R factor of 0.199 (R free of 0.276) for all data in the range 30.0–3.0 Å resolution with r.m.s. deviations of 0.008 Å and 1.4° from ideal bond lengths and bond angles, respectively. Table Isummarizes these refinement statistics. The coordinates and structure factors for the RT/PETT-1 and RT/PETT-2 complexes have been deposited with the Protein Data Bank. HIV-2 RT (pROD) was expressed and purified using methods described previously for HIV-1 RT (37.Stammers D.K. Tisdale M. Court S. Parmar V. Bradley C. Ross C.K. FEBS Lett. 1991; 283: 298-302Crossref PubMed Scopus (44) Google Scholar, 38.Stammers D.K. Ross C.K. Idriss H. Lowe D.M. Eur. J. Biochem. 1992; 206: 437-440Crossref PubMed Scopus (9) Google Scholar). Assays for both HIV-1 and HIV-2 RTs were carried out using the methods described previously (39.Lowe D.M. Aitken A. Bradley C. Darby G.K. Larder B.A. Powell K.L. Purifoy D.J.M. Tisdale M. Stammers D.K. Biochemistry. 1988; 27: 8884-8889Crossref PubMed Scopus (126) Google Scholar), but with poly(rC)·(dG)12–18 as the template primer and with the incorporation of [3H]dGTP into DNA to follow the reaction. IC50 values for PETT-1 and PETT-2 were determined at template-primer and substrate concentrations of 50 μg/ml poly(rC)·(dG) and 5 μmdGTP, respectively, with variation of inhibitor concentrations on a log scale. Inhibition curves were fitted by nonlinear regression methods using ORIGIN (Microcal Software Inc.). The mode of inhibition studies and K i determinations were carried out using a protocol involving a 4 × 5 matrix of varying substrate and inhibitor concentrations over ranges of [S]/[K m] from 0.5 to 7 and [I]/[K i] from 0.5 to 10 (40.Stammers D.K. Dann J.G. Harris C.J. Smith D.R. Arch. Biochem. Biophys. 1987; 258: 413-420Crossref PubMed Scopus (20) Google Scholar). In each case when either dGTP or template primer was not being varied in concentration, then it was held at saturating levels (i.e. 30 μm dGTP or 50 μg/ml poly(rC)·(dG)). Duplicate measurements were made for each experimental point. Between 2 and 4 replicate experiments were performed, and these were scaled together prior to curve fitting. Data were fitted to standard enzyme inhibition models (i.e.competitive, noncompetitive, uncompetitive, and mixed) using GRAFIT (Erithacus Software). The best fit was judged from the residual χ2 values, the run of signs of the residuals, and percentage errors in the fitted parameters. A summary of the refinement statistics for the RT/PETT complexes is shown in Table I. Both structures have excellent stereochemistry. The electron density for each structure is of good quality particularly in the region of the well ordered NNRTI binding site. Omit electron density maps for the inhibitors are very clear (Fig. 1). The orientation of PETT-1 and PETT-2 within the NNRTI binding site could be determined unambiguously due to the presence of clear electron density for the thioether and the two pyridinyl rings. The ether substituent of ring A, which is common to both compounds, could be clearly distinguished from the less bulky nitrile or chlorine group of the B rings (Fig. 1). Both compounds adopt similar conformations when bound to RT such that their aromatic rings are positioned approximately at right angles, whereas the linking group is folded in a somewhat constrained nonextended conformation (Fig.2). There is an intramolecular hydrogen bond between the ring B pyridinyl nitrogen and one of the nitrogen atoms of the thiourea group. Comparison of PETT inhibitors with other examples of NNRTIs, viz. nevirapine and TIBO, reveals that these inhibitors adopt conformations that occupy similar volumes of space within the drug pocket despite their diverse chemical structures (Fig. 3).Figure 3. Stereo diagram showing the relative orientations and positions of nevirapine (cyan), PETT-1 (red), and Cl-TIBO (yellow). The superimposition was carried out as described previously (7.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 (555) Google Scholar).View Large Image Figure ViewerDownload Hi-res image Download (PPT) The interactions of PETT-1 and PETT-2 with HIV-1 RT are shown in Fig.4. These are predominantly hydrophobic in nature but with some polar interactions also present. The larger nitrile substituent on ring B of PETT-1 compared with the chloro of PETT-2 alters the contacts in several places. Thus although ring A of both compounds is positioned at the “top” of the NNRTI pocket forming ring-stacking interactions with the side chains of both Tyr-181 and Tyr-188, the latter has more contacts with PETT-2 than with PETT-1. PETT-2 also makes a number of contacts with the side chain of Leu-100. The nitrile group of PETT-1 has extensive contacts with residues Leu-234, His-235, Pro-236, and Tyr-318, whereas the equivalent chlorine group of PETT-2 has few contacts with this region. The side chain of Glu-138 from the p51 subunit is positioned closer to the NNRTI pocket than for other inhibitor complexes (7.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 (555) Google Scholar), allowing contacts with ring A for both PETT-1 and PETT-2. The sulfur atom of the thiourea group makes a number of van der Waals contacts with the backbone of Lys-101. There is a single hydrogen bond to the main chain, from one of the thiourea nitrogens to the carbonyl of Lys-101. The results of the IC50 determinations for PETT inhibition of the RTs are shown in Fig. 5. Both PETT-1 and PETT-2 inhibit HIV-1 RT with similar IC50 values in the low nm range (6 and 5 nm, respectively) (Fig.5). For HIV-2 RT, PETT-2 gave an IC50 of 2.2 μm, whereas for PETT-1 a maximum of 20% inhibition at 50 μm was observed, meaning that it was not possible to determine an IC50 value. Results of studies to determine the mode of inhibition of PETT-2 against HIV-1 and HIV-2 RTs are shown in Tables II and III and in Figs.6 and 7. A comparison of χ2 values for different models shows that with dGTP as the varying substrate, the noncompetitive model was strongly preferred over the competitive model for both HIV-1 and HIV-2 RT (Table II). For poly(rC)·(dG) as the varying substrate the discrimination between the different models was lower, although noncompetitive inhibition was preferred for HIV-2 RT in all experiments performed, whereas for HIV-1 RT mixed or uncompetitive modes were preferred (Table II). The K i values (Table III) generally agree with the corresponding IC50 values (Fig. 5).Table IIComparison of models for PETT-2 inhibition of HIV-1 RT or HIV-2 RT with either dGTP or poly(rC) · oligo(dG12–18) as varying substrate from non-linear regression analysisReduced χ2(×10−3)Varying substratedGTPPoly(rC) · (dG)12–18ModelHIV-1 RTHIV-2 RTHIV-1 RTHIV-2 RTCompetitive24.579.32.70.89Mixed0.668.50.300.49Noncompetitive0.627.90.430.46Uncompetitive6.416.50.290.53χ2 is defined as Σ (Δγi/ςi)2, where Δγi is the difference between calculated and observed data points and ςi is the variance of data point i. Open table in a new tab Table IIIKinetic parameters for PETT-2, dGTP, and rC-dG derived for different inhibition modelsK mS.E.K iS.E.V maxS.E.χ2(×10−3)K i1S.E.dGTP and PETT-2aVarying dGTP and PETT-2. HIV-1 competitive2.40.700.340.0975.86.1424.5 HIV-1 mixed2.80.132.260.2979.81.090.662.160.10 HIV-1 noncompetitive2.80.112.180.0679.70.960.62 HIV-1 uncompetitive3.40.441.530.1483.23.546.37 HIV-2 competitive2.61.070.300.1193.18.2479.3 HIV-2 mixed3.50.422.740.92101.23.068.42.90.31 HIV-2 noncompetitive3.50.352.900.19101.42.717.9 HIV-2 uncompetitive4.30.632.220.22104.84.4116.5rC-dG and PETT-2bVarying rC-dG and PETT-2. HIV-1 competitive0.130.080.370.2112.81.772.7 HIV-1 mixed0.200.0333.457.914.80.720.302.060.28 HIV-1 noncompetitive0.160.032.80.3113.90.710.43 HIV-1 uncompetitive0.210.032.00.2014.80.680.29 HIV-2 competitive0.290.110.410.1311.91.440.89 HIV-2 mixed0.360.092.82.1712.81.180.491.840.54 HIV-2 noncompetitive0.340.082.040.3212.61.020.46 HIV-2 uncompetitive0.410.101.310.2513.31.270.53a Varying dGTP and PETT-2.b Varying rC-dG and PETT-2. Open table in a new tab Figure 7Fitted curves for PETT-2 inhibition of HIV-2 RT (noncompetitive model) and HIV-1 RT (uncompetitive model) with poly(rC)·(dG)12–18 as varying substrate. a, HIV-2 RT was incubated for 30 min at 25 °C with the various concentrations of PETT-2 and poly (rC)-(dG)12–18indicated. ●, 0 μm PETT-2; ○, 2 μmPETT-2; X, 6 μm PETT-2; ▪, 18 μm PETT-2; ■, 54 μm PETT-2. b, HIV-1 RT was incubated for 30 min at 25 °C with the various concentrations of PETT-2 and poly (rC)-(dG)12–18 indicated. ●, 0 nmPETT-2; ○, 2 nm PETT-2; X, 6 nm PETT-2; ▪, 18 nm PETT-2; ■, 54 nm PETT-2.View Large Image Figure ViewerDownload Hi-res image Download (PPT) χ2 is defined as Σ (Δγi/ςi)2, where Δγi is the difference between calculated and observed data points and ςi is the variance of data point i. The work reported here shows that, in line with reports for other members of this series, PETT-1 and PETT-2 are potent inhibitors of HIV-1 RT (10.Ahgren C. Backro K. Bell F.W. Cantrell A.S. Clemens M. Colacino J.M. Beeter J.B. Engelhardt J.A. Jaskunas S.R. Johansson N.G. Jordan C.L. Kasher J.S. Kinnick M.D. Lind P. Lopez C. Morin Jr., J.M. Muesing M.A. Noreen R. Oberg B. Paget C.J. Palkowitz J.A. Parrish C.A. Pranc P. Rippy M.K. Rydergard C. Sahlberg C. Swanson S. Ternansky R.J. Unge T. Vasilefe R.T. Vrang L. West S.J. Zhang H. Zhou X.X. Antimicrob. Agents Chemother. 1995; 39: 1329-1335Crossref PubMed Scopus (113) Google Scholar, 11.Zhang H. Vrang L. Backbro K. Lind P. Sahlberg C. Unge T. Oberg B. Antiviral Res. 1995; 28: 331-342Crossref PubMed Scopus (54) Google Scholar, 12.Cantrell A.S. Engelhardt P. Hogberg M. Jaskunas S.R. Johansson N.G. Jordan C.L. Kangasmetsa J. Kinnick M.D. Lind P. Morin Jr., J.M. Muesing M.A. Noreen R. Oberg B. Pranc P. Sahlberg C. Ternansky R.J. Vasileff R.T. Vrang L. West S.J. Zhang H. J. Med. Chem. 1996; 39: 4261-4274Crossref PubMed Scopus (128) Google Scholar). PETT-1 and PETT-2 differ only at the paraposition of the B ring (nitrile or chlorine, respectively), yet they show differing ability to inhibit HIV-2 RT. Although PETT-2 is a much weaker inhibitor of HIV-2 RT than it is of HIV-1 RT, such a relatively high level of inhibition of HIV-2 RT by an NNRTI is unusual. Recently the inhibitory activity of the NNRTIs delavirdine and emivirine, with IC50 values in the low micromolar range, have been reported against HIV-2 (EHO strain) in MT-4 cell cultures, although their activities against HIV-2 RT appeared minimal (41.Witvrouw M. Pannecouque C. Van Laethem K. Desmyter J. De Clercq E. Vandamme A.M. AIDS. 1999; 13: 1477-1483Crossref PubMed Scopus (73) Google Scholar). Crystallographic structure determinations of complexes of PETT-1 and PETT-2 with HIV-1 RT reveal that the overall mode of binding of both inhibitors adheres to the “two ring” conformation observed for a range of other chemically diverse NNRTI series (7.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 (555) Google Scholar, 22.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 (210) Google Scholar, 23.Ding J. Das K. Tantillo C. Zhang W. Clark A.D.J. Jessen S. Lu X. Hsiou Y. Jacobo-Molina A. Andries K. Pauwels R. Moereels H. Koymans L. Janssen P.A.J. Smith R.H.J. Kroeger Koepke R. Michejda C.J. Hughes S.H. Arnold E. Structure. 1995; 3: 365-379Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar, 24.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 (360) Google Scholar, 25.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, 26.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, 27.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). There are some differences between the interactions of the two PETT compounds with the NNRTI site. These appear to be related to the larger nitrile substituent on the B ring of PETT-1, which results in closer contacts with the main chain of residues 234 to 236, whereas PETT-2 makes slightly closer contacts with Tyr-188. The PETT conformations described here are generally in agreement with modeling studies, where the hydrogen bonding to the main chain of Lys-101 was correctly predicted (15.Mao C. Vig R. Venkatachalam T.K. Sudbeck E.A. Uckun F.M. Bioorg. Med. Chem. Lett. 1998; 8: 2213-2218Crossref PubMed Scopus (74) Google Scholar, 16.Mao C. Sudbeck E.A. Venkatachalam T.K. Uckun F.M. Bioorg. Med. Chem. Lett. 1999; 9: 1593-1598Crossref PubMed Scopus (79) Google Scholar). Previously published resistance data for a PETT compound trovirdine (11.Zhang H. Vrang L. Backbro K. Lind P. Sahlberg C. Unge T. Oberg B. Antiviral Res. 1995; 28: 331-342Crossref PubMed Scopus (54) Google Scholar), show that mutations of Leu-100→Ile, Glu-138→Arg, and Tyr-188→His give 10–20-fold resistance when compared with wild-type HIV-1 RT. These three residues all make contacts with structural features of PETT-1 and PETT-2 that are also common to trovirdine. As outlined previously residues 100 and 188 make van der Waals contact with ring A. From modeling studies it is clear that these interactions would be disrupted by mutations to Ile and His, respectively. The side chain of Glu-138 from the p51 subunit forms contacts with ring A, which would be disrupted by the mutation to a bulkier side chain such as arginine. What are the particular features of a compound such as PETT-2 compared with most NNRTIs that allows it to bind to HIV-2 RT? One possibility we considered, given the significant amino acid sequence changes both within the NNRTI site and more generally, was whether PETT-2 could be binding at an entirely different site. The kinetic studies reported here attempt to address this question. The mode of inhibition experiments show that PETT-2 is noncompetitive with respect to dGTP for both HIV-1 and HIV-2 RT, thus indicating it is not binding at the dNTP site (Table II). For the equivalent experiments with template primer and HIV-2 RT, the level of discrimination for the different inhibition models is not so marked, nevertheless noncompetitive inhibition is the preferred model in each experiment (Table II). For HIV-1 RT with template primer and PETT-2 varied there was a large discrimination against the competitive model, whereas mixed and uncompetitive modes were difficult to distinguish (Table II). Our data indicate that PETT-2 is neither competing for template primer nor for dNTP substrate sites on HIV-2 RT but are consistent with PETT-2 interacting at an HIV-2 equivalent of the HIV-1 NNRTI site. In considering this possibility and in attempting to explain the varying potency of PETT-1 and PETT-2, we examined both differences in the compounds and the sequence differences between HIV-1 and HIV-2 RTs, particularly in the region where the PETT B ring interacts. First we considered whether the pK a of the B ring pyridinyl nitrogen varied due to differing electron withdrawing/donating properties of nitrile and chlorine substituents, which could potentially modulate the hydrogen bonding capability to the main chain of Lys-101. In fact, the measured pK a of these nitrogens is very similar (4.25 and 4.32 for PETT-1 and PETT-2, respectively), and this means there will be minimal protonation of the pyridinyl nitrogen at physiological pH in either case. Hence, this cannot account for the different binding strength of the two compounds to HIV-2 RT. The nitrile group of PETT-1 makes van der Waals interactions with the main chain of Leu-234 and His-235 and with the side chain of Tyr-318. In HIV-2 RT, Leu-234 and Tyr-318 are conserved, but His-235 is replaced by a Trp residue. It is conceivable that the larger side chain of this Trp residue causes an alteration in the main chain position resulting in a slight reduction in the volume of the NNRTI site in this region, although still allowing accommodation of the smaller chlorine of PETT-2 but not the bulkier nitrile group of PETT-1. Certain members of the PETT series of NNRTIs may be better able to bind to HIV-2 RT due to a greater flexibility compared with the fused ring systems found in other NNRTIs such as nevirapine and TIBO. The linker region joining the two PETT pyridine rings could have enough flexibility to allow significant rearrangement and thereby be better accommodated within the HIV-2 RT “NNRTI” binding site. If this is the case then there are still clearly significant constraints on PETT binding to HIV-2 RT as exemplified by the effect of different B-ring substituents on potency of inhibition for this enzyme. To further define the binding of PETT-2 to HIV-2 RT in detail, a crystal structure of this complex will be required. Given the success of using NNRTIs to produce crystals of HIV-1 RT diffracting from medium to high resolution (6.Kohlstaedt L.A. Wang J. Friedman J.M. Rice P.A. Steitz T.A. Science. 1992; 256: 1783-1790Crossref PubMed Scopus (1749) Google Scholar, 30.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), it is hoped that NNRTIs that bind to HIV-2 RT could provide a similar means for obtaining a high resolution structure of this latter enzyme. A high resolution HIV-2 RT structure might provide insights into ways of inhibiting widely variant RTs including different serotypes as well as drug-resistant forms. The rational design of potent non-nucleoside inhibitors with broad spectrum activity against a wide range of RTs should be of importance in the continued fight against AIDS. We thank the staffs of the Synchotron Radiation Source, Daresbury Laboratory, UK and the Photon Factory, High Energy Accelerator Research Organization, Japan for their help with data collection; Richard Bryan and Kathryn Measures for computing support; and Stephen Lee for photographic support.