Vol. 36, No. 5
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, May 1992, p. 1019-1023
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Copyright X 1992, American Society for Microbiology
L-696,229 Specifically Inhibits Human Immunodeficiency Virus Type 1 Reverse Transcriptase and Possesses Antiviral Activity In Vitro MARK E. GOLDMAN,l* JULIE A. O'BRIEN,1 THOMAS L. RUFFING,1 JACK H. NUNBERG,2 WILLIAM A. SCHLEIF,2 JULIO C. QUINTERO,2 PETER K. S. SIEGL,3 JACOB M. HOFFMAN,4 ANTHONY M. SMITH,4 AND EMILIO A. EMINI2 Departments of New Lead Pharmacology, 1 Virus and Cell Biology,2 Pharmacology, 3 and Medicinal Chemistry, Merck Research Laboratories, West Point, Pennsylvania 19486-0004 Received 11 December 1991/Accepted 11 February 1992
L-696,229 {3-[2-(benzoxazol-2-yl)ethylj-5-ethyl-6-methyl-pyridin-2(1H)-one} is a specific inhibitor of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) activity that possesses antiviral activity in cell culture (W. S. Saari, J. M. Hoffman, J. S. Wai, T. E. Fisher, C. S. Rooney, A. M. Smith, C. M. Thomas, M. E. Goldman, J. A. O'Brien, J. H. Nunberg, J. C. Quintero, W. A. Schleif, E. A. Emini, and P. S. Anderson, J. Med. Chem. 34:2922-2925, 1991). In the present study, the RT-inhibitory activity and antiviral properties were characterized in detail. The inhibition of RT activity was template-primer dependent with 50% inhibitory concentrations of 0.018 to 0.50 pM and was noncompetitive with respect to deoxynucleoside triphosphates. L-696,229 inhibited RT activity in a mutually exclusive manner with respect to either phosphonoformate or azidothymidine triphosphate and was a weak partial inhibitor of the RNase H activity associated with HIV-1 RT. The compound did not significantly inhibit other retroviral or cellular polymerases at 300 ,uM. L-696,229 inhibited the spread of HIV-1 infection in cell cultures with all cell types and viral isolates tested, including human peripheral blood mononuclear cells and a virus isolate resistant to azidothymidine. The aminomethyl linker pyridinone derivatives L-697,639 and L-697,661 (Fig. 1) inhibit by at least 95% the progression of human immunodeficiency virus type 1 (HIV-1) infections in cell culture at concentrations of 0.012 to 0.20 ,uM, depending on the host cell type and viral isolate (5). The mechanism of antiviral activity is inhibition of the virally encoded polymerase reverse transcriptase (RT). These compounds inhibit both RNA- and DNA-directed DNA syntheses at 50% inhibitory concentrations of 0.020 to 0.80 p.M. The inhibition is reversible and noncompetitive with respect to all deoxynucleoside triphosphates. It is magnesium dependent and highly specific for HIV-1 RT. Binding (5) and viral cross-resistance (15) studies have demonstrated that these compounds probably inhibit HIV-1 RT activity by the same mechanism as tetrahydroimidazobenzodiazepinone (TIBO) R82150 (16) and nevirapine (13). While phosphonoformate (PFA) can displace [3HJL-697,639 from the enzymesubstrate complex, its mechanism of HIV-1 RT inhibition is different from that of the pyridinones, nevirapine, and TIBO R82150 (5, 15). Both L-697,639 and L-697,661 have entered clinical trials (3, 11). These compounds, however, are highly bound to human plasma and are extensively metabolized (2, 7). L-696,229 is related structurally to L-697,639 and L-697,661 but contains an ethylene linker instead of an aminomethyl linker and is devoid of nuclear substituents on the benzoxazole ring (Fig. 1) (18). This compound prevents the spread of HIV-1 infection in cell culture and inhibits HIV-1 RT activity at concentrations similar to those of L-697,639 and L-697,661 (18). L-696,229, however, binds to human plasma 10- to 20-fold less than do L-697,639 and L-697,661 (8). Unlike L-697,639 and L-697,661, the metabolic cleavage reaction at the ethylene linker is not observed in L-696,229 (1). Given these potential advantages, *
L-696,229 is presently undergoing safety and pharmacokinetic evaluation in humans. The detailed characterization of the RT-inhibitory and antiviral properties of L-696,229 is the subject of this communication. MATERIALS AND METHODS
HIV-1 RT assays. HIV-1 RT assays were performed at 37°C with purified, recombinant HIV-1 RT (NY5 isolate) and appropriate substrates as described previously (5, 6). The globin mRNA-dT assay mixture contained 55 mM Tris-HCI (pH 8.2); 70 mM KCI; 1 mM MgCl2; 1 mM dithiothreitol (DTT); 1 mg of bovine serum albumin (BSA) per ml; 1 ,ug of globin mRNA (Bethesda Research Laboratories, Gaithersburg, Md.) per ml; 1 ,ug of p(dT)12,18 (Pharmacia, Piscataway, N.J.) per ml; 50 ,uM ethylene glycol-bis(,-aminoethyl ether)-N,N,N',N'-tetraacetic acid, 150 ,uM each dATP, dCTP, and dGTP; 0.5 ,uM [3H]TTP (New England Nuclear, Boston, Mass.); 0.0025 U of RNase Block II (Stratagene, La Jolla, Calif.) per ml; 0.01% (vol/vol) Triton X-100; and 0.63
N
R
0
H FIG. 1. Structures of pyridinone HIV-1 RT inhibitors. X and R are CH2 and H, respectively, in L-696,229, NH and CH3, respectively, in L-697,639, and NH and Cl, respectively, in L-697,661.
Corresponding author. 1019
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nM recombinant HIV-1 RT. The rRNA-15-mer assay mixture contained 55 mM Tris-HCl (pH 8.2); 50 mM KCI; 10 mM MgCl2; 1 mM DTT; 1 mg of BSA per ml; 3 jig of 16S rRNA (Bethesda Research Laboratories) per ml annealed to 5'-TAACCrTGCGGCCGT-3' (1:1 molar ratio; 21); 100 ,uM each dATP, dCTP, and dGTP; 0.5 PM [3H]TTP; 0.025 U of RNase Block II per ml; 0.01% (vol/vol) Triton X-100; and 3.15 nM recombinant HIV-1 RT. The dC-dG assay mixture contained 55 mM Tris-HCl (pH 8.2), 30 mM MgCl2, 1 mg of BSA per ml, 1.38 ,ug of dC-dG (Pharmacia) per ml, 30 ,uM [3H]dGTP, 0.01% (vol/vol) Triton X-100, and 0.63 nM recombinant HIV-1 RT. HIV-1 RNase H assay. A modification of the procedure of Starnes and Cheng (20) was used to prepare the [3H]rG-dC substrate and to perform the nuclease assay. In brief, the hybrid was prepared with Eschenichia coli RNA polymerase (100 U/500 ,u; Pharmacia), poly(dC) (1 A260 unit; Pharmacia), and 0.5 mM [3H]GTP in a buffer composed of 40 mM Tris-HCI (pH 8.0), 100 mM KCI, 8 mM MgCl2, and 2 mM DTT. Incubation was performed at 37°C, and hybrid formation was monitored by 7% perchloric acid precipitation on glass fiber filters and was typically complete in 60 to 90 min. The HIV-1 RNase H assay was optimized to yield assay component concentrations of 50 mM Tris-HCl (pH 8.0), 30 mM KCI, 2 mM MgCl2, 2 mM DTT, 0.001% (vo/vol) Triton X-100, 1 mg of BSA per ml, 20,000 cpm of ['H]rG-dC, and purified HIV-1 RT (0.1 nM) in a volume of 50 ,ul. Following incubation at 37°C for 45 min in Skatron tube strips, racks were transferred to an ice-water bath. Ten microliters of a 10-mg/ml solution of bakers' yeast RNA type III (Sigma Chemical Co., St. Louis, Mo.) was added, and then 300 ,u of 7% perchloric acid was added. The tube strips were vortexed and incubated for 30 min. The racks were centrifuged at 1,100 x g for 15 min, and 200 pl of supernatant was transferred to scintillation vials with the aid of a Tecan robotic sample processor. Two milliliters of scintillation cocktail was added to each vial, and radioactivity was determined. Antiviral assays. The antiviral properties of L-696,229 in cell culture were determined as described previously (5, 15). The cell culture 95% inhibitory concentration (CIC95) was defined as the concentration of compound that prevented the spread of HIV-1 infection in the culture by at least 95%, as measured by p24 core antigen production (enzyme-linked immunosorbent assay; Coulter Immunology, Hialeah, Fla.). Cytotoxicity was assessed by microscopic evaluation of cell viability. Compounds. L-696,229 was synthesized as described previously (18). Erie violet {5,5'[(1,1'-biphenyl)-4,4'diylbis(azo)I bis(6-amino-4-hydroxy-2-naphthalenesulfonic acid)} was purchased from Allied Chemical and Dye Corp. (New York, N.Y.). Azidothymidine (AZT) triphosphate (AZT-TP) was custom synthesized by Sierra Bioresearch (Tucson, Ariz.). Data analysis. Enzyme inhibition assays were performed in duplicate, and results were expressed as the mean + standard error of at least three experiments. In some experiments, the results were analyzed by the procedures of Lineweaver and Burk (12) or Yonetani and Theorell (22). RESULTS Inhibition of HIV-1 RT activity. L-696,229 inhibited wildtype HIV-1 RT activity in a concentration-dependent manner, and its 50% inhibitory concentration was a function of the template-primer substrate. With rC-dG, dA-dT, rA-dT, dC-dG, globin mRNA-dT, and rRNA-15-mer, the 50% in-
c
0
cJ
50-
a1)
2a)
a.
25-
9
8 7 6 5 4 Concentration (-Log M)
3
FIG. 2. Inhibition of HIV-1 RNase H activity by L-696,229 (0) or Erie violet (A). Although Erie violet inhibited HIV-1 RNase H activity, it did not inhibit E. coli RNase H activity (at up to 300 ,uM). In the spread assay, Erie violet had a CIC95 of 25 p.M.
hibitory concentrations were 0.018 + 0.002, 0.02 + 0.006, 0.50 ± 0.11, 0.10 t 0.03, 0.37 ± 0.20, and 0.030 ± 0.01 ,uM, respectively. As noted previously with other members of this class, L-696,229 inhibited HIV-1 RT activity most effectively with the homogeneous templates-primers rC-dG and dA-dT and least effectively with rA-dT. The effectiveness of the compound with rRNA-15-mer in the assay was similar to those with rC-dG and dA-dT. Effectiveness was intermediate with the other templates-primers tested, including dC-dG and globin mRNA-dT. In addition, LineweaverBurk analysis as a function of dGTP (with rC-dG) at various L-696,229 concentrations demonstrated that the inhibition was noncompetitive with respect to dGTP (results not shown). Specificity. While L-696,229 was a potent, submicromolar inhibitor of HIV-1 RT activity, it did not significantly inhibit, at 300 ,uM, other polymerases of retroviral, bacterial, or mammalian origin (results not shown). L-696,229 was a partial inhibitor of the RNase H activity associated with HIV-1 RT (Fig. 2). Unlike the inhibition of RT activity, the inhibition of RNase H activity yielded a very shallow doseresponse curve. In contrast, Erie violet caused 100% inhibition of HIV-1 RNase H activity (Fig. 2). Mutual exclusivity. V/Vi was plotted against various concentrations of L-696,229 at fixed concentrations of PFA (Fig. 3) or AZT-TP (Fig. 4). The slope of L-696,229 inhibition of HIV-1 RT activity at each PFA or AZT-TP concentration was independent of that of the second inhibitor (PFA or AZT-TP), providing evidence for mutually exclusive inhibition between pairs of compounds. Since the concentration of L-696,229 was varied by the same factor as that of the second compound, the Yagi-Ozawa plot (broken lines in Fig. 3 and 4) (22) was determined and found to be linear, providing further evidence for mutually exclusive interactions between L-696,229 and PFA or AZT-TP. Antiviral activity. L-696,229 prevented the spread of HIV-1 infection in cell culture (Table 1); at concentrations of up to 200 ,uM, however, cytotoxicity was not evident. When L-696,229 was added to H9 human T-lymphoid cells before HIV-lIIIb infection, the CIC95 value was 0.20 ,uM. Similar
INHIBITION OF HIV-1 RT AND REPLICATION BY L-696,229
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TABLE 1. Comparison of antiviral properties of L-696,229 in cell cultures with those of AZT CIC95 (piM) of:
4.0-
3.0-
HIV-1
isolatea
IIIbb
H9 H9 MT4 IlIb PBMCC IIIb MT4 MN MT4 RF MT4 WMJ-2 MT4 RUTZ MT4 rA17 A018A/H112-2 CEM A018C/G910-6 CEM IIIb
0
0.2
0.4
0.6
0.8
1.0
L-696,229 (pM) FIG. 3. Inhibition of HIV-1 RT activity by combinations of L-696,229 and PFA. L-696,229 dose-response curves were constructed in the absence of PFA or in the presence of the indicated concentrations of PFA: 0, 0 1LM; 0, 2 ,uM; A, 4 FxM; A, 8 ,uM; El, 16 ,uM; *, 32 ,uM. Results were calculated by the procedure of Yonetani and Theorell (22). V/Vt, control velocity in the absence or presence of PFA/velocity in the presence of L-696,229.
results were obtained when L-696,229 was added 1 day after HIV-lIIIb infection (CIC95, 0.1 ,uM). When added to MT-4 lymphoid cells after infection, L-696,229 possessed potent antiviral activity for all laboratory HIV-1 isolates examined, except rA17 (Table 1). The rA17 virus is a variant selected for resistance to the pyridinone class of specific HIV-1 RT inhibitors and was found to be resistant to TIBO R82150 and nevirapine as well (15). The degree of resistance of rA17 to L-696,229 (1,000-fold) was equivalent to that previously reported for L-697,639 (15). L-696,229 also prevented the spread of HIV-lIIIb infection in phytohemagglutinin-stimulated human peripheral blood mononuclear cells maintained in medium containing interleukin-2. In contrast to the nar-
0.15 0.10 L-696,229 (pM) FIG. 4. Inhibition of HIV-1 RT activity by combinations of L-696,229 and AZT- iP. L-696,229 dose-response curves were constructed in the absence of AZT-TP or in the presence of the indicated concentrations of AZT-TP: 0, 0 ,uM; 0, 0.04 ,uM; A, 0.08 ,uM; A, 0.16 ,uM; El, 0.32 ,uM. The remainder of the assay was carried out as described in the legend to Fig. 3. V/VN, control velocity in the absence or presence of AZT-TP/velocity in the presence of L-696,229.
L-696,229
Cell type
Median
Range
0.20 0.10 0.10 0.10 0.10 0.10 0.10 0.10 100 0.10 0.050
0.10-0.30 0.10 0.05-0.20 0.10 0.10 0.05-0.10
100
No. of
expts. 3 2 18 1 2 2 3 1 4 1 1
AZT (median)
>50 >50 0.01 0.20
0.012 1.6
a See reference 5. b For this experiment, cells were treated with inhibitor 1 day before infection. For all other experiments, cells were infected 1 day before the addition of inhibitor. c PBMC, human peripheral blood mononuclear cells.
row range of HIV-11Hb-inhibitory activities of L-696,229 with different host cell types (CIC95s, 0.050 to 0.20 ,uM), those of AZT were more varied (CIC95s, 0.010 to >50 ,uM). With viral isolates A018AIH112-2 and A018C/G910-6, which were obtained, respectively, from an HIV-1-infected person before the initiation of AZT therapy and after 6 months of AZT therapy (10), L-696,229 was found to possess antiviral activity, with similar CIC95s; no observable resistance to L-696,229 was demonstrated following chronic AZT administration (Table 1). In contrast, AZT was significantly less effective as an inhibitor of isolate A018C/G910-6 (Table
1). DISCUSSION
L-696,229 has biological properties equivalent to those of the aminomethyl linker pyridinone RT inhibitors L-697,639 and L-697,661. These similarities include the templateprimer dependence of activity, noncompetitive kinetics with respect to deoxynucleoside triphosphates, the lack of inhibitory activity for other polymerases, and potent antiviral activity against all HIV-1 isolates tested, except rA17, which is resistant to all members of this pharmacological class (15). Like TIBO R82913 (21), L-696,229 and related compounds were potent inhibitors of HIV-1 RT activity when rRNA-15mer was used as a template-primer. In contrast to the complete (100%) inhibition of HIV-1 RNase H activity by Erie violet, L-696,229 caused only partial inhibition of HIV-1 RNase H activity (maximum, 36%). Similar partial RNase H inhibition results have been reported with nevirapine (13), which appears to act by the same mechanism as the pyridinones (5). Recently, other naphthalenesulfonic acid derivatives related to Erie violet were shown to possess antiviral activity against HIV-1 in cell culture (14). Because of (i) the ability of PFA to displace pyridinones from the RT-substrate complex (5) and (ii) the synergistic antiviral activity of combinations of specific HIV-1 RT inhibitors and nucleoside analogs (5, 17), dual inhibitor studies were performed. The results demonstrated that, under these conditions, L-696,229 produced mutually exclusive enzyme inhibition patterns with respect to either PFA or
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GOLDMAN ET AL.
AZT-TP, indicating that the pairs of compounds cannot bind to RT at the same time. These data suggest, therefore, that the binding sites on HIV-1 RT for the pyridinones, PFA, and AZT-TP kinetically overlap. Since PFA can displace pyridinones from the RT-substrate complex (5), it is logical that PFA and AZT-TP would be mutually exclusive. HIV-1 RT that is resistant to the pyridinones (as well as to TIBO R82150 and nevirapine), however, is susceptible to PFA (15). In addition, the interaction between L-696,229 and AZT-TP was also mutually exclusive, yet specific HIV-1 RT inhibitors and nucleoside analogs interacted synergistically to prevent the spread of HIV-1 infection in cell culture (5, 17). If these compounds inhibit HIV-1 infection by interacting solely with RT, it could be expected that they may bind simultaneously to RT to cause the observed synergistic inhibition. Taken together, therefore, these inconsistencies lend support to the hypothesis that the interactions between the specific HIV-1 RT inhibitors and HIV-1 RT are quite complex (4, 5, 9, 13) and will require a great deal of biochemical and structural (X-ray crystallography) investigation before their mechanisms are understood. In addition to the in vitro experiments, L-696,229 was studied in animals by use of several ancillary pharmacology models to determine whether it produces untoward effects (19). At intravenous doses of 1 to 5 mg/kg in dogs, this compound did not cause significant changes in cardiovascular parameters, responses to autonomic nervous system interventions, renal function, respiratory parameters, or basal or gastrin-stimulated gastric acid secretion. Following oral doses of 200 mg/kg in rhesus monkeys, no central nervous system effects were noted. These results suggest that L-696,229 is well tolerated in vivo. In summary, these results demonstrate that L-696,229 possesses biochemical and antiviral properties similar to those of L-697,639 and L-697,661. Although the effect of protein binding on in vivo antiviral activity is unknown, L-696,229, which attains a significantly higher free concentration in plasma, may possess enhanced antiviral activity in vivo compared with the aminomethyl compounds. In addition, L-696,229 is metabolized differently from the aminomethyl compounds, a fact which may be important in the event that toxicities occur with long-term administration of L-697,639 or L-697,661. Finally, the eventual clinical usefulness of L-696,229 will largely depend on the rate of resistant virus emergence in treated individuals. We have demonstrated by using cell cultures that virus resistant to L-697,639 and L-697,661 is equivalently resistant to L-696,229. It remains to be determined whether reduced protein binding, yielding higher free plasma L-696,229 levels, would result in a decreased incidence or a delayed appearance of resistant virus. ACKNOWLEDGMENTS HIV-1 isolates A018A/H112-2 and A018C/G910-6 were supplied by D. Richman through the AIDS Research and Reference Reagent Program of the NIH. ADDENDUM IN PROOF
Recent studies from our institution (S. Carroll, D. Olsen, and L. Kuo, unpublished data) have demonstrated synergistic inhibition of purified HIV-1 RT between L-697,661 and AZT-TP at high inhibitor concentrations (>90% inhibitory concentrations). This result implies that under steady-state conditions and at high inhibitor concentrations, L-697,661
ANTimICROB. AGENTS CHEMOTHER.
(and presumably L-696,229 as well) and AZT-TP can bind simultaneously to HIV-1 RT. REFERENCES 1. Balani, S. K., S. M. Pitzenberger, L. R. Kauffnan, B. H. Arison, H. G. Ramjit, M. E. Goldman, J. A. O'Brien, J. M. Hoffman, and A. D. Theoharides. 1992. Metabolism of a new HIV-1 reverse transcriptase inhibitor, 3-[2-(benzoxazol-2-yl)ethyl]5ethyl-6-methylpyridin-2(1H)-one, in rat and liver slices. Program Abstr. Fed. Am. Soc. Exp. Biol. J. 6:abstr. 3672. 2. Balani, S. K., and A. D. Theoharides (Merck Research Laboratories). 1991. Personal communication. 3. Davey, R., Jr., 0. Laskin, M. Decker, D. O'Neill, S. Haneiwich, J. Metcalf, M. Polis, J. Kovacs, S. Davis, M. Mauer, C. Yoder, P. Patterson, S. Justice, K. C. Yeh, E. Woolf, T. Au, and H. C. Lane. 1991. L-697,639 and L-697,661, novel agents for treatment of human immunodeficiency virus type 1 infection. Program Abstr. 31st Intersci. Conf. Antimicrob. Agents Chemother., abstr. 697. 4. Debyser, Z., R. Pauwels, K. Andries, J. Desmyter, Y. Engelborghs, P. A. J. Janssen, and E. De Clercq. 1991. Allosteric properties of heterodimeric HIV-1 reverse transcriptase. Program Abstr. 7th Int. Conf. AIDS, abstr M. A. 1144. 5. Goldman, M. E., J. H. Nunberg, J. A. O'Brien, J. C. Quintero, W. A. Schleif, K. F. Freund, S. L. Gaul, W. S. Saarl, J. S. Wai, J. M. Hoffman, P. S. Anderson, D. J. Hupe, E. A. Emini, and A. M. Stern. 1991. Pyridinone derivatives: specific human immunodeficiency virus type 1 reverse transcriptase inhibitors with antiviral activity. Proc. Natl. Acad. Sci. USA 88:68636867. 6. Goldman, M. E., G. S. Salituro, J. A. Bowen, J. M. Williamson, D. L. Zink, W. A. Schleif, and E. A. Emini. 1990. Inhibition of human immunodeficiency virus-1 reverse transcriptase activity by rubromycins: competitive interaction at the template-primer site. Mol. Pharmacol. 38:20-25. 7. Halpin, R. A. (Merck Research Laboratories). 1991. Personal communication. 8. Halpin, R. A., L. A. Geer, D. W. Wyatt, K. P. Vyas, and M. Hichens. 1992. The disposition of L-696,229, a potent and specific inhibitor of human immunodeficiency virus reverse transcriptase in rats and rhesus monkeys. Program Abstr. Fed. Am. Soc. Exp. Biol. J. 6:abstr. 332. 9. Kopp, E. B., J. J. Miglietta, A. G. Shrutkowski, C.-K. Shih, P. M. Grob, and M. T. Skoog. 1991. Steady state kinetics and inhibition of HIV-1 reverse transcriptase by a non-nucleoside dipyridodiazepinone, BI-RG-587, using a heteropolymeric template. Nucleic Acids Res. 19:3035-3039. 10. Larder, B. A., G. Darby, and D. D. Richman. 1989. HIV with reduced sensitivity to zidovudine (AZT) isolated during prolonged therapy. Science (Washington, D.C.) 243:1731-1734. 11. Laskin, 0. L., A. G. Dupont, A. Buntinx, D. Schoors, M. De Pre, A. Van Hecken, K. C. Yeh, E. Woolf, R. Eisenhandler, M. De Smet, P. Patterson, I. De Lepeleire, and P. J. De Schepper. 1991. Human pharmacokinetics and tolerability of L-697,661 and L-697,639: non-nucleoside HIV-1 reverse transcriptase inhibitors. Program Abstr. 31st Intersci. Conf. Antimicrob. Agents Chemother., abstr. 698. 12. Lineweaver, H., and D. Burk. 1934. The determination of enzyme dissociation constants. J. Am. Chem. Soc. 56:658666. 13. Merluzzi, V. J., K. D. Hargrave, M. Labadia, K. Grozinger, M. Skoog, J. C. Wu, C.-K. Shih, K. Eckner, S. Hattox, J. Adams, A. S. Rosenthal, R. Faanes, R. J. Eckner, R. A. Koup, and J. L. Sullivan. 1990. Inhibition of HIV-1 replication by a nonnucleoside reverse transcriptase inhibitor. Science (Washington, D.C.) 250:1411-1413. 14. Mohan, P., A. J. Hopfilnger, and M. Baba. 1991. Naphthalenesulphonic acid derivatives as potential anti-HIV-1 agents. Chemistry, biology and molecular modelling of their inhibition of reverse transcriptase. Antiviral Chem. Chemother. 2:215222. 15. Nunberg, J. H., W. A. Schleif, E. J. Boots, J. A. O'Brien, J. C. Quintero, J. M. Hoffman, E. A. Emini, and M. E. Goldman.
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1991. Viral resistance to human immunodeficiency virus type 1-specific pyridinone reverse transcriptase inhibitors. J. Virol. 65:4887-4892. 16. Pauwels, R, K. Andries, J. Desmyter, D. Schols, M. J. Kukla, H. J. Breslin, A. Raeymaeckers, J. Van Gelder, R. Woestenborghs, J. Heykants, K. Schellenkens, M. A. C. Janssen, E. De Clercq, and P. A. J. Janssen. 1990. Potent and selective inhibition of HIV-1 replication by a novel series of TIBO derivatives. Nature (London) 343:470-474. 17. Richman, D., A. S. Rosenthal, M. Skoog, R. J. Eckner, T.-C. Chou, R. P. Sabo, and V. J. Merluzzi. 1991. BI-RG-587 is active against zidovudine-resistant human immunodeficiency virus type 1 and synergistic with zidovudine. Antimicrob. Agents Chemother. 35:305-308. 18. Saari, W. S., J. M. Hoffman, J. S. Wai, T. E. Fisher, C. S. Rooney, A. M. Smith, C. M. Thomas, M. E. Goldman, J. A. O'Brien, J. H. Nunberg, J. C. Quintero, W. A. Schleif, E. A.
19. 20. 21.
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Emini, and P. S. Anderson. 1991. 2-Pyridinone derivatives: a new class of nonnucleoside HIV-1 specific reverse transcriptase inhibitors. J. Med. Chem. 34:2922-2925. Siegl, P. K. S. (Merck Research Laboratories). 1991. Personal communication. Starnes, M. C., and Y.-C. Cheng. 1989. Human immunodeficiency virus reverse transcriptase-associated RNase H activity. J. Biol. Chem. 264:7073-7077. White, E. L., R. W. Buckheit, Jr., L. J. Ross, J. M. Germany, K. Andries, R. Pauwels, P. A. J. Janssen, W. M. Shannon, and M. A. Chingos. 1991. A TIBO derivative, R82913, is a potent inhibitor of HIV-1 reverse transcriptase with heteropolymeric templates. Antiviral Res. 16:257-266. Yonetani, Y., and H. Theorell. 1964. Studies on liver alcohol dehydrogenase complexes. III. Multiple inhibition kinetics in the presence of two competitive inhibitors. Arch. Biochem. Biophys. 106:243-251.
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Copyright X 1992, American Society for Microbiology
L-696,229 Specifically Inhibits Human Immunodeficiency Virus Type 1 Reverse Transcriptase and Possesses Antiviral Activity In Vitro MARK E. GOLDMAN,l* JULIE A. O'BRIEN,1 THOMAS L. RUFFING,1 JACK H. NUNBERG,2 WILLIAM A. SCHLEIF,2 JULIO C. QUINTERO,2 PETER K. S. SIEGL,3 JACOB M. HOFFMAN,4 ANTHONY M. SMITH,4 AND EMILIO A. EMINI2 Departments of New Lead Pharmacology, 1 Virus and Cell Biology,2 Pharmacology, 3 and Medicinal Chemistry, Merck Research Laboratories, West Point, Pennsylvania 19486-0004 Received 11 December 1991/Accepted 11 February 1992
L-696,229 {3-[2-(benzoxazol-2-yl)ethylj-5-ethyl-6-methyl-pyridin-2(1H)-one} is a specific inhibitor of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) activity that possesses antiviral activity in cell culture (W. S. Saari, J. M. Hoffman, J. S. Wai, T. E. Fisher, C. S. Rooney, A. M. Smith, C. M. Thomas, M. E. Goldman, J. A. O'Brien, J. H. Nunberg, J. C. Quintero, W. A. Schleif, E. A. Emini, and P. S. Anderson, J. Med. Chem. 34:2922-2925, 1991). In the present study, the RT-inhibitory activity and antiviral properties were characterized in detail. The inhibition of RT activity was template-primer dependent with 50% inhibitory concentrations of 0.018 to 0.50 pM and was noncompetitive with respect to deoxynucleoside triphosphates. L-696,229 inhibited RT activity in a mutually exclusive manner with respect to either phosphonoformate or azidothymidine triphosphate and was a weak partial inhibitor of the RNase H activity associated with HIV-1 RT. The compound did not significantly inhibit other retroviral or cellular polymerases at 300 ,uM. L-696,229 inhibited the spread of HIV-1 infection in cell cultures with all cell types and viral isolates tested, including human peripheral blood mononuclear cells and a virus isolate resistant to azidothymidine. The aminomethyl linker pyridinone derivatives L-697,639 and L-697,661 (Fig. 1) inhibit by at least 95% the progression of human immunodeficiency virus type 1 (HIV-1) infections in cell culture at concentrations of 0.012 to 0.20 ,uM, depending on the host cell type and viral isolate (5). The mechanism of antiviral activity is inhibition of the virally encoded polymerase reverse transcriptase (RT). These compounds inhibit both RNA- and DNA-directed DNA syntheses at 50% inhibitory concentrations of 0.020 to 0.80 p.M. The inhibition is reversible and noncompetitive with respect to all deoxynucleoside triphosphates. It is magnesium dependent and highly specific for HIV-1 RT. Binding (5) and viral cross-resistance (15) studies have demonstrated that these compounds probably inhibit HIV-1 RT activity by the same mechanism as tetrahydroimidazobenzodiazepinone (TIBO) R82150 (16) and nevirapine (13). While phosphonoformate (PFA) can displace [3HJL-697,639 from the enzymesubstrate complex, its mechanism of HIV-1 RT inhibition is different from that of the pyridinones, nevirapine, and TIBO R82150 (5, 15). Both L-697,639 and L-697,661 have entered clinical trials (3, 11). These compounds, however, are highly bound to human plasma and are extensively metabolized (2, 7). L-696,229 is related structurally to L-697,639 and L-697,661 but contains an ethylene linker instead of an aminomethyl linker and is devoid of nuclear substituents on the benzoxazole ring (Fig. 1) (18). This compound prevents the spread of HIV-1 infection in cell culture and inhibits HIV-1 RT activity at concentrations similar to those of L-697,639 and L-697,661 (18). L-696,229, however, binds to human plasma 10- to 20-fold less than do L-697,639 and L-697,661 (8). Unlike L-697,639 and L-697,661, the metabolic cleavage reaction at the ethylene linker is not observed in L-696,229 (1). Given these potential advantages, *
L-696,229 is presently undergoing safety and pharmacokinetic evaluation in humans. The detailed characterization of the RT-inhibitory and antiviral properties of L-696,229 is the subject of this communication. MATERIALS AND METHODS
HIV-1 RT assays. HIV-1 RT assays were performed at 37°C with purified, recombinant HIV-1 RT (NY5 isolate) and appropriate substrates as described previously (5, 6). The globin mRNA-dT assay mixture contained 55 mM Tris-HCI (pH 8.2); 70 mM KCI; 1 mM MgCl2; 1 mM dithiothreitol (DTT); 1 mg of bovine serum albumin (BSA) per ml; 1 ,ug of globin mRNA (Bethesda Research Laboratories, Gaithersburg, Md.) per ml; 1 ,ug of p(dT)12,18 (Pharmacia, Piscataway, N.J.) per ml; 50 ,uM ethylene glycol-bis(,-aminoethyl ether)-N,N,N',N'-tetraacetic acid, 150 ,uM each dATP, dCTP, and dGTP; 0.5 ,uM [3H]TTP (New England Nuclear, Boston, Mass.); 0.0025 U of RNase Block II (Stratagene, La Jolla, Calif.) per ml; 0.01% (vol/vol) Triton X-100; and 0.63
N
R
0
H FIG. 1. Structures of pyridinone HIV-1 RT inhibitors. X and R are CH2 and H, respectively, in L-696,229, NH and CH3, respectively, in L-697,639, and NH and Cl, respectively, in L-697,661.
Corresponding author. 1019
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ANTIMICROB. AGENTS CHEMOTHER.
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nM recombinant HIV-1 RT. The rRNA-15-mer assay mixture contained 55 mM Tris-HCl (pH 8.2); 50 mM KCI; 10 mM MgCl2; 1 mM DTT; 1 mg of BSA per ml; 3 jig of 16S rRNA (Bethesda Research Laboratories) per ml annealed to 5'-TAACCrTGCGGCCGT-3' (1:1 molar ratio; 21); 100 ,uM each dATP, dCTP, and dGTP; 0.5 PM [3H]TTP; 0.025 U of RNase Block II per ml; 0.01% (vol/vol) Triton X-100; and 3.15 nM recombinant HIV-1 RT. The dC-dG assay mixture contained 55 mM Tris-HCl (pH 8.2), 30 mM MgCl2, 1 mg of BSA per ml, 1.38 ,ug of dC-dG (Pharmacia) per ml, 30 ,uM [3H]dGTP, 0.01% (vol/vol) Triton X-100, and 0.63 nM recombinant HIV-1 RT. HIV-1 RNase H assay. A modification of the procedure of Starnes and Cheng (20) was used to prepare the [3H]rG-dC substrate and to perform the nuclease assay. In brief, the hybrid was prepared with Eschenichia coli RNA polymerase (100 U/500 ,u; Pharmacia), poly(dC) (1 A260 unit; Pharmacia), and 0.5 mM [3H]GTP in a buffer composed of 40 mM Tris-HCI (pH 8.0), 100 mM KCI, 8 mM MgCl2, and 2 mM DTT. Incubation was performed at 37°C, and hybrid formation was monitored by 7% perchloric acid precipitation on glass fiber filters and was typically complete in 60 to 90 min. The HIV-1 RNase H assay was optimized to yield assay component concentrations of 50 mM Tris-HCl (pH 8.0), 30 mM KCI, 2 mM MgCl2, 2 mM DTT, 0.001% (vo/vol) Triton X-100, 1 mg of BSA per ml, 20,000 cpm of ['H]rG-dC, and purified HIV-1 RT (0.1 nM) in a volume of 50 ,ul. Following incubation at 37°C for 45 min in Skatron tube strips, racks were transferred to an ice-water bath. Ten microliters of a 10-mg/ml solution of bakers' yeast RNA type III (Sigma Chemical Co., St. Louis, Mo.) was added, and then 300 ,u of 7% perchloric acid was added. The tube strips were vortexed and incubated for 30 min. The racks were centrifuged at 1,100 x g for 15 min, and 200 pl of supernatant was transferred to scintillation vials with the aid of a Tecan robotic sample processor. Two milliliters of scintillation cocktail was added to each vial, and radioactivity was determined. Antiviral assays. The antiviral properties of L-696,229 in cell culture were determined as described previously (5, 15). The cell culture 95% inhibitory concentration (CIC95) was defined as the concentration of compound that prevented the spread of HIV-1 infection in the culture by at least 95%, as measured by p24 core antigen production (enzyme-linked immunosorbent assay; Coulter Immunology, Hialeah, Fla.). Cytotoxicity was assessed by microscopic evaluation of cell viability. Compounds. L-696,229 was synthesized as described previously (18). Erie violet {5,5'[(1,1'-biphenyl)-4,4'diylbis(azo)I bis(6-amino-4-hydroxy-2-naphthalenesulfonic acid)} was purchased from Allied Chemical and Dye Corp. (New York, N.Y.). Azidothymidine (AZT) triphosphate (AZT-TP) was custom synthesized by Sierra Bioresearch (Tucson, Ariz.). Data analysis. Enzyme inhibition assays were performed in duplicate, and results were expressed as the mean + standard error of at least three experiments. In some experiments, the results were analyzed by the procedures of Lineweaver and Burk (12) or Yonetani and Theorell (22). RESULTS Inhibition of HIV-1 RT activity. L-696,229 inhibited wildtype HIV-1 RT activity in a concentration-dependent manner, and its 50% inhibitory concentration was a function of the template-primer substrate. With rC-dG, dA-dT, rA-dT, dC-dG, globin mRNA-dT, and rRNA-15-mer, the 50% in-
c
0
cJ
50-
a1)
2a)
a.
25-
9
8 7 6 5 4 Concentration (-Log M)
3
FIG. 2. Inhibition of HIV-1 RNase H activity by L-696,229 (0) or Erie violet (A). Although Erie violet inhibited HIV-1 RNase H activity, it did not inhibit E. coli RNase H activity (at up to 300 ,uM). In the spread assay, Erie violet had a CIC95 of 25 p.M.
hibitory concentrations were 0.018 + 0.002, 0.02 + 0.006, 0.50 ± 0.11, 0.10 t 0.03, 0.37 ± 0.20, and 0.030 ± 0.01 ,uM, respectively. As noted previously with other members of this class, L-696,229 inhibited HIV-1 RT activity most effectively with the homogeneous templates-primers rC-dG and dA-dT and least effectively with rA-dT. The effectiveness of the compound with rRNA-15-mer in the assay was similar to those with rC-dG and dA-dT. Effectiveness was intermediate with the other templates-primers tested, including dC-dG and globin mRNA-dT. In addition, LineweaverBurk analysis as a function of dGTP (with rC-dG) at various L-696,229 concentrations demonstrated that the inhibition was noncompetitive with respect to dGTP (results not shown). Specificity. While L-696,229 was a potent, submicromolar inhibitor of HIV-1 RT activity, it did not significantly inhibit, at 300 ,uM, other polymerases of retroviral, bacterial, or mammalian origin (results not shown). L-696,229 was a partial inhibitor of the RNase H activity associated with HIV-1 RT (Fig. 2). Unlike the inhibition of RT activity, the inhibition of RNase H activity yielded a very shallow doseresponse curve. In contrast, Erie violet caused 100% inhibition of HIV-1 RNase H activity (Fig. 2). Mutual exclusivity. V/Vi was plotted against various concentrations of L-696,229 at fixed concentrations of PFA (Fig. 3) or AZT-TP (Fig. 4). The slope of L-696,229 inhibition of HIV-1 RT activity at each PFA or AZT-TP concentration was independent of that of the second inhibitor (PFA or AZT-TP), providing evidence for mutually exclusive inhibition between pairs of compounds. Since the concentration of L-696,229 was varied by the same factor as that of the second compound, the Yagi-Ozawa plot (broken lines in Fig. 3 and 4) (22) was determined and found to be linear, providing further evidence for mutually exclusive interactions between L-696,229 and PFA or AZT-TP. Antiviral activity. L-696,229 prevented the spread of HIV-1 infection in cell culture (Table 1); at concentrations of up to 200 ,uM, however, cytotoxicity was not evident. When L-696,229 was added to H9 human T-lymphoid cells before HIV-lIIIb infection, the CIC95 value was 0.20 ,uM. Similar
INHIBITION OF HIV-1 RT AND REPLICATION BY L-696,229
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TABLE 1. Comparison of antiviral properties of L-696,229 in cell cultures with those of AZT CIC95 (piM) of:
4.0-
3.0-
HIV-1
isolatea
IIIbb
H9 H9 MT4 IlIb PBMCC IIIb MT4 MN MT4 RF MT4 WMJ-2 MT4 RUTZ MT4 rA17 A018A/H112-2 CEM A018C/G910-6 CEM IIIb
0
0.2
0.4
0.6
0.8
1.0
L-696,229 (pM) FIG. 3. Inhibition of HIV-1 RT activity by combinations of L-696,229 and PFA. L-696,229 dose-response curves were constructed in the absence of PFA or in the presence of the indicated concentrations of PFA: 0, 0 1LM; 0, 2 ,uM; A, 4 FxM; A, 8 ,uM; El, 16 ,uM; *, 32 ,uM. Results were calculated by the procedure of Yonetani and Theorell (22). V/Vt, control velocity in the absence or presence of PFA/velocity in the presence of L-696,229.
results were obtained when L-696,229 was added 1 day after HIV-lIIIb infection (CIC95, 0.1 ,uM). When added to MT-4 lymphoid cells after infection, L-696,229 possessed potent antiviral activity for all laboratory HIV-1 isolates examined, except rA17 (Table 1). The rA17 virus is a variant selected for resistance to the pyridinone class of specific HIV-1 RT inhibitors and was found to be resistant to TIBO R82150 and nevirapine as well (15). The degree of resistance of rA17 to L-696,229 (1,000-fold) was equivalent to that previously reported for L-697,639 (15). L-696,229 also prevented the spread of HIV-lIIIb infection in phytohemagglutinin-stimulated human peripheral blood mononuclear cells maintained in medium containing interleukin-2. In contrast to the nar-
0.15 0.10 L-696,229 (pM) FIG. 4. Inhibition of HIV-1 RT activity by combinations of L-696,229 and AZT- iP. L-696,229 dose-response curves were constructed in the absence of AZT-TP or in the presence of the indicated concentrations of AZT-TP: 0, 0 ,uM; 0, 0.04 ,uM; A, 0.08 ,uM; A, 0.16 ,uM; El, 0.32 ,uM. The remainder of the assay was carried out as described in the legend to Fig. 3. V/VN, control velocity in the absence or presence of AZT-TP/velocity in the presence of L-696,229.
L-696,229
Cell type
Median
Range
0.20 0.10 0.10 0.10 0.10 0.10 0.10 0.10 100 0.10 0.050
0.10-0.30 0.10 0.05-0.20 0.10 0.10 0.05-0.10
100
No. of
expts. 3 2 18 1 2 2 3 1 4 1 1
AZT (median)
>50 >50 0.01 0.20
0.012 1.6
a See reference 5. b For this experiment, cells were treated with inhibitor 1 day before infection. For all other experiments, cells were infected 1 day before the addition of inhibitor. c PBMC, human peripheral blood mononuclear cells.
row range of HIV-11Hb-inhibitory activities of L-696,229 with different host cell types (CIC95s, 0.050 to 0.20 ,uM), those of AZT were more varied (CIC95s, 0.010 to >50 ,uM). With viral isolates A018AIH112-2 and A018C/G910-6, which were obtained, respectively, from an HIV-1-infected person before the initiation of AZT therapy and after 6 months of AZT therapy (10), L-696,229 was found to possess antiviral activity, with similar CIC95s; no observable resistance to L-696,229 was demonstrated following chronic AZT administration (Table 1). In contrast, AZT was significantly less effective as an inhibitor of isolate A018C/G910-6 (Table
1). DISCUSSION
L-696,229 has biological properties equivalent to those of the aminomethyl linker pyridinone RT inhibitors L-697,639 and L-697,661. These similarities include the templateprimer dependence of activity, noncompetitive kinetics with respect to deoxynucleoside triphosphates, the lack of inhibitory activity for other polymerases, and potent antiviral activity against all HIV-1 isolates tested, except rA17, which is resistant to all members of this pharmacological class (15). Like TIBO R82913 (21), L-696,229 and related compounds were potent inhibitors of HIV-1 RT activity when rRNA-15mer was used as a template-primer. In contrast to the complete (100%) inhibition of HIV-1 RNase H activity by Erie violet, L-696,229 caused only partial inhibition of HIV-1 RNase H activity (maximum, 36%). Similar partial RNase H inhibition results have been reported with nevirapine (13), which appears to act by the same mechanism as the pyridinones (5). Recently, other naphthalenesulfonic acid derivatives related to Erie violet were shown to possess antiviral activity against HIV-1 in cell culture (14). Because of (i) the ability of PFA to displace pyridinones from the RT-substrate complex (5) and (ii) the synergistic antiviral activity of combinations of specific HIV-1 RT inhibitors and nucleoside analogs (5, 17), dual inhibitor studies were performed. The results demonstrated that, under these conditions, L-696,229 produced mutually exclusive enzyme inhibition patterns with respect to either PFA or
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GOLDMAN ET AL.
AZT-TP, indicating that the pairs of compounds cannot bind to RT at the same time. These data suggest, therefore, that the binding sites on HIV-1 RT for the pyridinones, PFA, and AZT-TP kinetically overlap. Since PFA can displace pyridinones from the RT-substrate complex (5), it is logical that PFA and AZT-TP would be mutually exclusive. HIV-1 RT that is resistant to the pyridinones (as well as to TIBO R82150 and nevirapine), however, is susceptible to PFA (15). In addition, the interaction between L-696,229 and AZT-TP was also mutually exclusive, yet specific HIV-1 RT inhibitors and nucleoside analogs interacted synergistically to prevent the spread of HIV-1 infection in cell culture (5, 17). If these compounds inhibit HIV-1 infection by interacting solely with RT, it could be expected that they may bind simultaneously to RT to cause the observed synergistic inhibition. Taken together, therefore, these inconsistencies lend support to the hypothesis that the interactions between the specific HIV-1 RT inhibitors and HIV-1 RT are quite complex (4, 5, 9, 13) and will require a great deal of biochemical and structural (X-ray crystallography) investigation before their mechanisms are understood. In addition to the in vitro experiments, L-696,229 was studied in animals by use of several ancillary pharmacology models to determine whether it produces untoward effects (19). At intravenous doses of 1 to 5 mg/kg in dogs, this compound did not cause significant changes in cardiovascular parameters, responses to autonomic nervous system interventions, renal function, respiratory parameters, or basal or gastrin-stimulated gastric acid secretion. Following oral doses of 200 mg/kg in rhesus monkeys, no central nervous system effects were noted. These results suggest that L-696,229 is well tolerated in vivo. In summary, these results demonstrate that L-696,229 possesses biochemical and antiviral properties similar to those of L-697,639 and L-697,661. Although the effect of protein binding on in vivo antiviral activity is unknown, L-696,229, which attains a significantly higher free concentration in plasma, may possess enhanced antiviral activity in vivo compared with the aminomethyl compounds. In addition, L-696,229 is metabolized differently from the aminomethyl compounds, a fact which may be important in the event that toxicities occur with long-term administration of L-697,639 or L-697,661. Finally, the eventual clinical usefulness of L-696,229 will largely depend on the rate of resistant virus emergence in treated individuals. We have demonstrated by using cell cultures that virus resistant to L-697,639 and L-697,661 is equivalently resistant to L-696,229. It remains to be determined whether reduced protein binding, yielding higher free plasma L-696,229 levels, would result in a decreased incidence or a delayed appearance of resistant virus. ACKNOWLEDGMENTS HIV-1 isolates A018A/H112-2 and A018C/G910-6 were supplied by D. Richman through the AIDS Research and Reference Reagent Program of the NIH. ADDENDUM IN PROOF
Recent studies from our institution (S. Carroll, D. Olsen, and L. Kuo, unpublished data) have demonstrated synergistic inhibition of purified HIV-1 RT between L-697,661 and AZT-TP at high inhibitor concentrations (>90% inhibitory concentrations). This result implies that under steady-state conditions and at high inhibitor concentrations, L-697,661
ANTimICROB. AGENTS CHEMOTHER.
(and presumably L-696,229 as well) and AZT-TP can bind simultaneously to HIV-1 RT. REFERENCES 1. Balani, S. K., S. M. Pitzenberger, L. R. Kauffnan, B. H. Arison, H. G. Ramjit, M. E. Goldman, J. A. O'Brien, J. M. Hoffman, and A. D. Theoharides. 1992. Metabolism of a new HIV-1 reverse transcriptase inhibitor, 3-[2-(benzoxazol-2-yl)ethyl]5ethyl-6-methylpyridin-2(1H)-one, in rat and liver slices. Program Abstr. Fed. Am. Soc. Exp. Biol. J. 6:abstr. 3672. 2. Balani, S. K., and A. D. Theoharides (Merck Research Laboratories). 1991. Personal communication. 3. Davey, R., Jr., 0. Laskin, M. Decker, D. O'Neill, S. Haneiwich, J. Metcalf, M. Polis, J. Kovacs, S. Davis, M. Mauer, C. Yoder, P. Patterson, S. Justice, K. C. Yeh, E. Woolf, T. Au, and H. C. Lane. 1991. L-697,639 and L-697,661, novel agents for treatment of human immunodeficiency virus type 1 infection. Program Abstr. 31st Intersci. Conf. Antimicrob. Agents Chemother., abstr. 697. 4. Debyser, Z., R. Pauwels, K. Andries, J. Desmyter, Y. Engelborghs, P. A. J. Janssen, and E. De Clercq. 1991. Allosteric properties of heterodimeric HIV-1 reverse transcriptase. Program Abstr. 7th Int. Conf. AIDS, abstr M. A. 1144. 5. Goldman, M. E., J. H. Nunberg, J. A. O'Brien, J. C. Quintero, W. A. Schleif, K. F. Freund, S. L. Gaul, W. S. Saarl, J. S. Wai, J. M. Hoffman, P. S. Anderson, D. J. Hupe, E. A. Emini, and A. M. Stern. 1991. Pyridinone derivatives: specific human immunodeficiency virus type 1 reverse transcriptase inhibitors with antiviral activity. Proc. Natl. Acad. Sci. USA 88:68636867. 6. Goldman, M. E., G. S. Salituro, J. A. Bowen, J. M. Williamson, D. L. Zink, W. A. Schleif, and E. A. Emini. 1990. Inhibition of human immunodeficiency virus-1 reverse transcriptase activity by rubromycins: competitive interaction at the template-primer site. Mol. Pharmacol. 38:20-25. 7. Halpin, R. A. (Merck Research Laboratories). 1991. Personal communication. 8. Halpin, R. A., L. A. Geer, D. W. Wyatt, K. P. Vyas, and M. Hichens. 1992. The disposition of L-696,229, a potent and specific inhibitor of human immunodeficiency virus reverse transcriptase in rats and rhesus monkeys. Program Abstr. Fed. Am. Soc. Exp. Biol. J. 6:abstr. 332. 9. Kopp, E. B., J. J. Miglietta, A. G. Shrutkowski, C.-K. Shih, P. M. Grob, and M. T. Skoog. 1991. Steady state kinetics and inhibition of HIV-1 reverse transcriptase by a non-nucleoside dipyridodiazepinone, BI-RG-587, using a heteropolymeric template. Nucleic Acids Res. 19:3035-3039. 10. Larder, B. A., G. Darby, and D. D. Richman. 1989. HIV with reduced sensitivity to zidovudine (AZT) isolated during prolonged therapy. Science (Washington, D.C.) 243:1731-1734. 11. Laskin, 0. L., A. G. Dupont, A. Buntinx, D. Schoors, M. De Pre, A. Van Hecken, K. C. Yeh, E. Woolf, R. Eisenhandler, M. De Smet, P. Patterson, I. De Lepeleire, and P. J. De Schepper. 1991. Human pharmacokinetics and tolerability of L-697,661 and L-697,639: non-nucleoside HIV-1 reverse transcriptase inhibitors. Program Abstr. 31st Intersci. Conf. Antimicrob. Agents Chemother., abstr. 698. 12. Lineweaver, H., and D. Burk. 1934. The determination of enzyme dissociation constants. J. Am. Chem. Soc. 56:658666. 13. Merluzzi, V. J., K. D. Hargrave, M. Labadia, K. Grozinger, M. Skoog, J. C. Wu, C.-K. Shih, K. Eckner, S. Hattox, J. Adams, A. S. Rosenthal, R. Faanes, R. J. Eckner, R. A. Koup, and J. L. Sullivan. 1990. Inhibition of HIV-1 replication by a nonnucleoside reverse transcriptase inhibitor. Science (Washington, D.C.) 250:1411-1413. 14. Mohan, P., A. J. Hopfilnger, and M. Baba. 1991. Naphthalenesulphonic acid derivatives as potential anti-HIV-1 agents. Chemistry, biology and molecular modelling of their inhibition of reverse transcriptase. Antiviral Chem. Chemother. 2:215222. 15. Nunberg, J. H., W. A. Schleif, E. J. Boots, J. A. O'Brien, J. C. Quintero, J. M. Hoffman, E. A. Emini, and M. E. Goldman.
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1991. Viral resistance to human immunodeficiency virus type 1-specific pyridinone reverse transcriptase inhibitors. J. Virol. 65:4887-4892. 16. Pauwels, R, K. Andries, J. Desmyter, D. Schols, M. J. Kukla, H. J. Breslin, A. Raeymaeckers, J. Van Gelder, R. Woestenborghs, J. Heykants, K. Schellenkens, M. A. C. Janssen, E. De Clercq, and P. A. J. Janssen. 1990. Potent and selective inhibition of HIV-1 replication by a novel series of TIBO derivatives. Nature (London) 343:470-474. 17. Richman, D., A. S. Rosenthal, M. Skoog, R. J. Eckner, T.-C. Chou, R. P. Sabo, and V. J. Merluzzi. 1991. BI-RG-587 is active against zidovudine-resistant human immunodeficiency virus type 1 and synergistic with zidovudine. Antimicrob. Agents Chemother. 35:305-308. 18. Saari, W. S., J. M. Hoffman, J. S. Wai, T. E. Fisher, C. S. Rooney, A. M. Smith, C. M. Thomas, M. E. Goldman, J. A. O'Brien, J. H. Nunberg, J. C. Quintero, W. A. Schleif, E. A.
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Emini, and P. S. Anderson. 1991. 2-Pyridinone derivatives: a new class of nonnucleoside HIV-1 specific reverse transcriptase inhibitors. J. Med. Chem. 34:2922-2925. Siegl, P. K. S. (Merck Research Laboratories). 1991. Personal communication. Starnes, M. C., and Y.-C. Cheng. 1989. Human immunodeficiency virus reverse transcriptase-associated RNase H activity. J. Biol. Chem. 264:7073-7077. White, E. L., R. W. Buckheit, Jr., L. J. Ross, J. M. Germany, K. Andries, R. Pauwels, P. A. J. Janssen, W. M. Shannon, and M. A. Chingos. 1991. A TIBO derivative, R82913, is a potent inhibitor of HIV-1 reverse transcriptase with heteropolymeric templates. Antiviral Res. 16:257-266. Yonetani, Y., and H. Theorell. 1964. Studies on liver alcohol dehydrogenase complexes. III. Multiple inhibition kinetics in the presence of two competitive inhibitors. Arch. Biochem. Biophys. 106:243-251.
