Defining a Molecular Mechanism of Synergy between Nucleoside and Nonnucleoside AIDS Drugs

Combination therapies treating human immunodeficiency virus type 1 (HIV-1) infection delay the emergence of drug-resistant virus and exhibit synergistic inhibition. This synergy is observed within the two classes of inhibitors that target the essential viral reverse transcriptase (RT): the chain-terminating nucleoside analogs (NRTIs) and the allosteric nonnucleosides (NNRTIs) that bind in a pocket distinct from the active site. A general mechanism to define the molecular basis for synergy between these two classes remains to be elucidated. Previous mechanistic studies from our laboratory (Spence, R. A., Kati, W. M., Anderson, K. S., and Johnson, K. A. (1995) Science 267, 988-993) have shown that the natural deoxynucleoside triphosphate and the NNRTI can simultaneously bind to their respective sites. This work also suggests communication between the two sites, since the inhibition of RT by NNRTIs is manifested through a remote effect on the chemical step. This interplay between the two sites offers a plausible hypothesis for understanding synergy in which binding of NNRTIs modulates the chain termination by NRTIs. The present study supports this hypothesis by illustrating that the clinically approved NNRTIs, nevirapine and efavirenz, inhibit the ATP-mediated removal of AZTMP, d4TMP, ddCMP, (-)3TCMP, (-)FTCMP, and (+)3TCMP, thereby prolonging the effectiveness of chain termination. This inhibition is mediated through an effect on both the rate of the chemical step and binding of ATP, resulting in an overall decrease in efficiency of removal. This work substantiates communication between the two binding pockets, the sustained use of combination therapy to treat HIV infection, and a molecular basis for understanding synergy. Combination therapies treating human immunodeficiency virus type 1 (HIV-1) infection delay the emergence of drug-resistant virus and exhibit synergistic inhibition. This synergy is observed within the two classes of inhibitors that target the essential viral reverse transcriptase (RT): the chain-terminating nucleoside analogs (NRTIs) and the allosteric nonnucleosides (NNRTIs) that bind in a pocket distinct from the active site. A general mechanism to define the molecular basis for synergy between these two classes remains to be elucidated. Previous mechanistic studies from our laboratory (Spence, R. A., Kati, W. M., Anderson, K. S., and Johnson, K. A. (1995) Science 267, 988-993) have shown that the natural deoxynucleoside triphosphate and the NNRTI can simultaneously bind to their respective sites. This work also suggests communication between the two sites, since the inhibition of RT by NNRTIs is manifested through a remote effect on the chemical step. This interplay between the two sites offers a plausible hypothesis for understanding synergy in which binding of NNRTIs modulates the chain termination by NRTIs. The present study supports this hypothesis by illustrating that the clinically approved NNRTIs, nevirapine and efavirenz, inhibit the ATP-mediated removal of AZTMP, d4TMP, ddCMP, (-)3TCMP, (-)FTCMP, and (+)3TCMP, thereby prolonging the effectiveness of chain termination. This inhibition is mediated through an effect on both the rate of the chemical step and binding of ATP, resulting in an overall decrease in efficiency of removal. This work substantiates communication between the two binding pockets, the sustained use of combination therapy to treat HIV infection, and a molecular basis for understanding synergy. Monotherapy treatment against HIV-1 infection can be beneficial in decreasing viral load but is ultimately rendered ineffective by the appearance of drug-selected mutations. Combination therapy, however, strives to delay the emergence of drug-selected mutations and viral resistance (1Hammer S.M. Kessler H.A. Saag M.S. J. Acquir. Immune. Defic. 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Highly active anti-retroviral therapy includes inhibitors of the HIV-1 reverse transcriptase (RT) 1The abbreviations used are: RT, reverse transcriptase; PR, protease; AIDS, acquired immunodeficiency syndrome; HIV-1, human immunodeficiency virus type 1; NRTI, nucleoside RT inhibitor; NNRTI, nonnucleoside RT inhibitor; dNTP, deoxynucleoside triphosphate; AZT, β-d-(+)-3′-azido-3′-deoxythymidine; UC781, N-[4-chloro-3-(3-methyl-2-butenyloxy)phenyl]-2-methyl-3-furancarbothioamide; PPi, pyrophosphate; AZTMP, β-d-(+)-3′-azido-3′-deoxythymidine monophosphate; (-)3TC, β-l-(-)-2′,3′-dideoxy-3′-thiacytidine; WT, wild type; d4T, β-d-(+)-2′,3′-didehydro-3′-deoxythymidine; ddC, β-d-(+)-2′,3′-dideoxycytidine; (-)FTC, β-l-(-)-2′,3′-dideoxy-5-fluoro-3′-thiacytidine; (+)3TC, β-d-(+)-2′,3′-dideoxy-3′-thiacytidine; dNMP, 3′-deoxynucleoside monophosphate. and the protease (PR). While multiple-drug therapies comprising of RT and PR inhibitors delay resistance, cocktails of these inhibitors can also exhibit additive or synergistic inhibition of viral replication (3De Clercq E. Antiviral Res. 1998; 38: 153-179Crossref PubMed Scopus (369) Google Scholar). Intriguingly, synergy is also observed within two classes of RT inhibitors, that is, the nucleoside RT inhibitors (NRTIs), can show synergy with the nonnucleoside RT inhibitors (NNRTIs) in the absence of PR inhibitors (4Zhu Q.Y. Scarborough A. Polsky B. Chou T.C. AIDS Res. Hum. Retroviruses. 1996; 12: 507-517Crossref PubMed Scopus (26) Google Scholar). For example, the NRTI, AZT (zidovudine), and the NNRTI, nevirapine, exhibit synergy when given in combination to cultured cells (5Richman D. Rosenthal A.S. Skoog M. Eckner R.J. Chou T.C. Sabo J.P. Merluzzi V.J. Antimicrob. Agents Chemother. 1991; 35: 305-308Crossref PubMed Scopus (151) Google Scholar). This increase in potency from combination therapy has been observed among other combinations of NRTIs and NNRTIs, both in cultured cells and in patients (6Balzarini J. Pelemans H. Aquaro S. Perno C.F. Witvrouw M. Schols D. De Clercq E. Karlsson A. Mol. Pharmacol. 1996; 50: 394-401PubMed Google Scholar, 7Carr A. Vella S. de Jong M.D. Sorice F. Imrie A. Boucher C.A. Cooper D.A. AIDS. 1996; 10: 635-641Crossref PubMed Scopus (78) Google Scholar, 8Pauwels R. Andries K. Debyser Z. Kukla M.J. Schols D. Breslin H.J. Woestenborghs R. Desmyter J. Janssen M.A. De Clercq E. Janssen P.A.J. Antimicrob. Agents Chemother. 1994; 38: 2863-2870Crossref PubMed Scopus (84) Google Scholar, 9Zembower D.E. Liao S. Flavin M.T. Xu Z.Q. Stup T.L. Buckheit Jr., R.W. Khilevich A. Mar A.A. Sheinkman A.K. J. Med. Chem. 1997; 40: 1005-1017Crossref PubMed Scopus (84) Google Scholar). These two classes of inhibitors are functionally distinct in their mode of action. The nucleoside analog family of inhibitors is prodrug nucleosides, which are transported across host cellular membranes and phosphorylated to the metabolically active nucleotides. These nucleotides serve as substrates for RT, and lacking a 3′-hydroxyl group, they serve to chain-terminate a growing DNA strand. This chain termination and their competition with natural dNTPs yields their therapeutic value (10Goody R.S. Muller B. Restle T. FEBS Lett. 1991; 291: 1-5Crossref PubMed Scopus (78) Google Scholar). However, previous studies have demonstrated that RT can catalyze either ATP or PPi-mediated removal of chain-terminating analogs (11Arion D. Kaushik N. McCormick S. Borkow G. Parniak M.A. Biochemistry. 1998; 37: 15908-15917Crossref PubMed Scopus (313) Google Scholar, 12Meyer P.R. Matsuura S.E. Mian A.M. So A.G. Scott W.A. Mol. Cell. 1999; 4: 35-43Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar, 13Meyer P.R. Matsuura S.E. So A.G. Scott W.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13471-13476Crossref PubMed Scopus (301) Google Scholar). ATP and PPi act as nucleophiles that directly attack the phosphodiester linkage to form either an ATP analog dinucleoside tetraphosphate (14Meyer P.R. Matsuura S.E. Tolun A.A. Pfeifer I. So A.G. Mellors J.W. Scott W.A. Antimicrob. Agents Chemother. 2002; 46: 1540-1545Crossref PubMed Scopus (67) Google Scholar) or the triphosphate of the analog, respectively, leaving behind the primer poised for incorporation once again. Structural evidence shows that the allosteric nonnucleoside inhibitors bind to a pocket ∼10 Å away from the polymerase site (15Tantillo C. Ding J. Jacobo-Molina A. Nanni R.G. Boyer P.L. Hughes S.H. Pauwels R. Andries K. Janssen P.A. Arnold E. J. Mol. Biol. 1994; 243: 369-387Crossref PubMed Scopus (498) Google Scholar, 16Kohlstaedt L.A. Wang J. Friedman J.M. Rice P.A. Steitz T.A. 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Desmyter J. Kukla M. Janssen P.A. De Clercq E. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 1451-1455Crossref PubMed Scopus (157) Google Scholar). Despite much published work on the modes of inhibition by NRTIs and NNRTIs, how they mechanistically display synergy observed in vivo and in vitro has largely remained unknown. It is unclear if a general mechanism exists that may account for synergy observed. Earlier mechanistic studies from our lab and others (22Spence R.A. Kati W.M. Anderson K.S. Johnson K.A. Science. 1995; 267: 988-993Crossref PubMed Scopus (470) Google Scholar, 23Rittinger K. Divita G. Goody R.S. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8046-8049Crossref PubMed Scopus (188) Google Scholar) have illustrated that the natural deoxynucleoside triphosphate dNTP and the NNRTI can simultaneously occupy their respective sites. Moreover, this work also suggests that communication occurs between the active and allosteric sites, since the inhibition of RT by NNRTIs is manifested through a remote effect on the chemical step. At the molecular level, transient kinetic studies have shown that nonnucleoside inhibitors change the rate-limiting step of catalysis, resulting in chemistry as the slowest step (22Spence R.A. Kati W.M. Anderson K.S. Johnson K.A. Science. 1995; 267: 988-993Crossref PubMed Scopus (470) Google Scholar, 23Rittinger K. Divita G. Goody R.S. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8046-8049Crossref PubMed Scopus (188) Google Scholar). This interplay between the two sites offers a plausible hypothesis for understanding the molecular mechanism of synergy in which binding of NNRTIs modulates the chain termination by NRTIs. Recent work showing that synergy may be mediated between the nucleoside analog AZT and the nonnucleoside UC781 through a nucleotide removal pathway (24Borkow G. Arion D. Wainberg M.A. Parniak M.A. Antimicrob. Agents Chemother. 1999; 43: 259-263Crossref PubMed Google Scholar) lends support for this hypothesis. In this study, it was proposed that UC781 resensitizes AZT-resistant virus to AZT as a result of decreased PPi-mediated removal of AZTMP from primer ends with the addition of UC781. A similar study with AZT and NNRTIs nevirapine and 9-chlorotetrahydroimidazobenzodiazepinone (9-Cl-TIBO) also implicates this pathway in the context of AZT resistance (25Odriozola L. Cruchaga C. Andreola M. Dolle V. Nguyen C.H. Tarrago-Litvak L. Perez-Mediavilla A. Martinez-Irujo J.J. J. Biol. Chem. 2003; 278: 42710-42716Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Still it remains unanswered if these results may account for synergy with other combinations of NRTIs and NNRTIs. Using a transient kinetic approach that has been previously been employed in our lab to study nucleoside analog incorporation and the mechanism of NNRTI inhibition, the present work was aimed to study clinically relevant combinations to define a general molecular mechanism of synergy between RT inhibitors. The NRTIs AZT, d4T, ddC, (-)3TC, newly approved (-)FTC, (+)3TC, and the NNRTIs nevirapine and efavirenz were used in this study to examine a wide panel of RT inhibitors. We show that nonnucleosides inhibit the ability of HIV-1 RT to remove nucleoside analogs mediated by a physiological concentration of ATP. The inhibition is a result of modulating both the binding of ATP and the rate of the chemical step. This work substantiates communication between the two inhibitory pockets and a molecular basis for understanding synergy. Expression and Purification of HIV-1 RT—The RTWT clone was generously provided by Stephen Hughes, Paul Boyer, and Andrea Ferris (Frederick Cancer Research and Development Center, Frederick, MD). N-terminal histidine-tagged heterodimeric p66/p51 reverse transcriptase was purified as described previously (26Kerr S.G. Anderson K.S. Biochemistry. 1997; 36: 14064-14070Crossref PubMed Scopus (103) Google Scholar). Materials—AZTTP was acquired from Moravek Biochemicals. d4T was purchased from Sigma, and phosphorylation to the triphosphate was performed as described previously (27Ray A.S. Yang Z. Shi J. Hobbs A. Schinazi R.F. Chu C.K. Anderson K.S. Biochemistry. 2002; 41: 5150-5162Crossref PubMed Scopus (42) Google Scholar). The triphosphates of (-)3TC, (-)FTC, and (+)3TC were kindly provided by Dr. R. F. Schinazi (Emory University, Atlanta, GA). Efavirenz was purchased from Toronto Research Chemicals Inc., North York, Ontario. NNRTIs were diluted in dimethyl sulfoxide. ATP was purchased from Sigma and treated with thermostable pyrophosphatase (Roche Applied Science) to degrade any contaminating pyrophosphate. Labeling and Annealing of Oligonucleotides—Primers and templates were synthesized at the Keck Facility at Yale University and purified with 20% polyacrylamide denaturing gel electrophoresis. The sequences of primers and templates used in this study are: D23A (5′-*TCA GGT CCC TGT TCG GGC GCC AC-3′), D23B (5′-*GCC TCG CAG CCG TCC AAC CAA CT-3′), D22A (5′-*GCC TCG CAG CCG TCC AAC CAA C-3′), D22B (5′-*TCA GGT CCC TGT TCG GGC GCC A-3′), D36 (5′-TCT CTA GCA GTG GCG CCC GAA CAG GGA CCT GAA AGC-3′), and D45 (5′-GGA CGG CAT TGG ATC GAG GTT GAG TTG GTT GGA CGG CTG CGA GGC-3′). D23A-AZTMP, D22A-d4TMP, D23B-(-)3TCMP, D23B-(-)FTC, and D23B-(+)3TC primers were made with previously reported methods (28Johnson A.A. Ray A.S. Hanes J. Suo Z. Colacino J.M. Anderson K.S. Johnson K.A. J. Biol. Chem. 2001; 276: 40847-40857Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar). D22B-ddCMP was synthesized by the Keck Facility and purified as stated above. Primer/templates were labeled and annealed as described previously (29Murakami E. Feng J.Y. Lee H. Hanes J. Johnson K.A. Anderson K.S. J. Biol. Chem. 2003; 278: 36403-36409Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). Primer/templates combinations studied were D23A-AZTMP/D36, D22A-d4TMP/D45, D22B-ddCMP/D36, D23B-(-)3TCMP/D45, D23B-(-)FTCMP/D45, and D23B-(+)3TCMP/D45. Pre-steady State Removal Assays—ATP-mediated removal was studied as described previously (30Ray A.S. Murakami E. Basavapathruni A. Vaccaro J.A. Ulrich D. Chu C.K. Schinazi R.F. Anderson K.S. Biochemistry. 2003; 42: 8831-8841Crossref PubMed Scopus (70) Google Scholar) by quantitating the disappearance of substrate in single turnover experiments, in which each reaction mixture contained 250 nm wild-type RT, 50 nm primer/template, 10 mm Mg2+, and the appropriate concentration of ATP (see figures). All concentrations represent final concentrations, and enzyme concentration is based on a pre-steady-state active site determination. In removal experiments lacking NNRTIs, dimethyl sulfoxide was used as a control (representing less than 2% of total reaction mixture). Products were separated on a 20% polyacrylamide gel and quantitated on a Bio-Rad Molecular Imager FX. Data Analysis—Data were quantitated and fit using previously reported methods (30Ray A.S. Murakami E. Basavapathruni A. Vaccaro J.A. Ulrich D. Chu C.K. Schinazi R.F. Anderson K.S. Biochemistry. 2003; 42: 8831-8841Crossref PubMed Scopus (70) Google Scholar). Many combinations of NRTIs and NNRTIs inhibit viral growth synergistically, including AZT and efavirenz, d4T and nevirapine, and (-)3TC and efavirenz (3De Clercq E. Antiviral Res. 1998; 38: 153-179Crossref PubMed Scopus (369) Google Scholar, 31King R.W. Klabe R.M. Reid C.D. Erickson-Viitanen S.K. Antimicrob. Agents Chemother. 2002; 46: 1640-1646Crossref PubMed Scopus (56) Google Scholar, 32Merrill D.P. Moonis M. Chou T.C. Hirsch M.S. J. Infect. Dis. 1996; 173: 355-364Crossref PubMed Scopus (95) Google Scholar). An understanding of this phenomenon improves the quality of treatment for current patients and provides a platform for developing new therapies, but a general mechanism that accounts for this synergy is unknown. In the context of the ability of RT to remove chain terminating nucleoside analogs in the presence of PPi or ATP (11Arion D. Kaushik N. McCormick S. Borkow G. Parniak M.A. Biochemistry. 1998; 37: 15908-15917Crossref PubMed Scopus (313) Google Scholar, 12Meyer P.R. Matsuura S.E. Mian A.M. So A.G. Scott W.A. Mol. Cell. 1999; 4: 35-43Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar, 13Meyer P.R. Matsuura S.E. So A.G. Scott W.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13471-13476Crossref PubMed Scopus (301) Google Scholar,30Ray A.S. Murakami E. Basavapathruni A. Vaccaro J.A. Ulrich D. Chu C.K. Schinazi R.F. Anderson K.S. Biochemistry. 2003; 42: 8831-8841Crossref PubMed Scopus (70) Google Scholar), previous reports have suggested that NNRTIs may inhibit removal of AZT monophosphate, consequently maintaining chain termination at the primer end (24Borkow G. Arion D. Wainberg M.A. Parniak M.A. Antimicrob. Agents Chemother. 1999; 43: 259-263Crossref PubMed Google Scholar, 25Odriozola L. Cruchaga C. Andreola M. Dolle V. Nguyen C.H. Tarrago-Litvak L. Perez-Mediavilla A. Martinez-Irujo J.J. J. Biol. Chem. 2003; 278: 42710-42716Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). The of in removal is as our laboratory and others have shown RT AZTMP with a rate than that of AZT-resistant RT at a physiological concentration of which against PPi-mediated removal as a mechanism of AZT resistance (14Meyer P.R. Matsuura S.E. Tolun A.A. Pfeifer I. So A.G. Mellors J.W. Scott W.A. Antimicrob. Agents Chemother. 2002; 46: 1540-1545Crossref PubMed Scopus (67) Google Scholar, A.S. Murakami E. Basavapathruni A. Vaccaro J.A. Ulrich D. Chu C.K. Schinazi R.F. Anderson K.S. Biochemistry. 2003; 42: 8831-8841Crossref PubMed Scopus (70) Google Scholar). AZT-resistant RT AZTMP than RT at a physiological concentration of ATP (30Ray A.S. Murakami E. Basavapathruni A. Vaccaro J.A. Ulrich D. Chu C.K. Schinazi R.F. Anderson K.S. Biochemistry. 2003; 42: 8831-8841Crossref PubMed Scopus (70) Google Scholar). It has been that ATP directly to RT at a site to the active site S.G. Jr., K. S. J.J. J.M. Boyer P.L. Hughes S.H. Arnold E. J. 2002; PubMed Scopus Google Scholar) with the of ATP, as a directly the phosphodiester linkage between AZTMP and the The of this attack are the primer to and the dinucleoside tetraphosphate these suggest that ATP, and PPi, to AZTMP removal in the context of AZT We have to study ATP-mediated removal in this a host of and to a general mechanism of synergy shown in show that the addition of 10 nevirapine or efavirenz ATP-mediated removal of a wide panel of nucleoside including AZT, d4T, ddC, (-)3TC, (+)3TC study an and approved These results the ability of RT to remove chain-terminating analogs and that between the polymerase active site and nonnucleoside binding pocket (22Spence R.A. Kati W.M. Anderson K.S. Johnson K.A. Science. 1995; 267: 988-993Crossref PubMed Scopus (470) Google Scholar). In efavirenz removal than nevirapine and the observed in the The between NNRTI inhibition of nucleotide removal and was by primers with the or In each the of NNRTI inhibition on the of removal for (+)3TC were with that of (-)3TC, showing that is at the site of chain termination. the inhibition of removal when either nevirapine or efavirenz is to the in that both the rate and of AZTMP removal are decreased when an NNRTI is It is also to that in removal by wild-type RT in the absence of NNRTIs, that analogs are than the analogs for removal and In the mode of inhibition by which NNRTIs may ATP-mediated removal of the nonnucleoside inhibitors may have a effect on ATP binding an effect on the chemical step. this the ATP-mediated removal of AZTMP was in the presence of concentrations of ATP in the absence or presence of a concentration of At a physiological concentration of ATP, nevirapine removal Using this concentration of nevirapine, the rate of removal at ATP concentrations was shows that the addition of nevirapine both a change in the rate of chemistry of ATP-mediated removal and in the of ATP The efficiency of removal by from to with the addition of This a molecular communication between the two binding pockets in which binding of an NNRTI the polymerase active site and the of ATP in chain termination. Earlier studies have that nonnucleosides the active site of RT and incorporation of nucleoside analogs (22Spence R.A. Kati W.M. Anderson K.S. Johnson K.A. Science. 1995; 267: 988-993Crossref PubMed Scopus (470) Google Scholar). The current study a new for the of site communication at the enzyme and in of understanding combination These also support for the that a the of a and NNRTI bind and to RT and be in the treatment of AIDS. In this provides a mechanistic for NRTIs and NNRTIs in combination therapy, understanding the molecular basis of inhibition and and offers new for and We F. Schinazi for and Stephen Hughes, Paul Boyer, and Andrea Ferris for the HIV-1 RTWT and A. Scott for ATP-mediated nucleotide removal

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