TMC114, a newly designed human immunodeficiency virus type 1 (HIV-1) protease inhibitor, is extremely potent against both wild-type (wt) and multidrug-resistant (MDR) viruses in vitro as well as in vivo. Although chemically similar to amprenavir (APV), the potency of TMC114 is substantially greater. To examine the basis for this potency, we solved crystal structures of TMC114 complexed with wt HIV-1 protease and TMC114 and APV complexed with an MDR (L63P, V82T, and I84V) protease variant. In addition, we determined the corresponding binding thermodynamics by isothermal titration calorimetry. TMC114 binds approximately 2 orders of magnitude more tightly to the wt enzyme (K d ؍ 4.5 ؋ 10 ؊12 M) than APV (K d ؍ 3.9 ؋ 10 ؊10 M). Our X-ray data (resolution ranging from 2.2 to 1.2 Å) reveal strong interactions between the bis-tetrahydrofuranyl urethane moiety of TMC114 and main-chain atoms of D29 and D30. These interactions appear largely responsible for TMC114's very favorable binding enthalpy to the wt protease (؊12.1 kcal/mol). However, TMC114 binding to the MDR HIV-1 protease is reduced by a factor of 13.3, whereas the APV binding constant is reduced only by a factor of 5.1. However, even with the reduction in binding affinity to the MDR HIV protease, TMC114 still binds with an affinity that is more than 1.5 orders of magnitude tighter than the first-generation inhibitors. Both APV and TMC114 fit predominantly within the substrate envelope, a property that may be associated with decreased susceptibility to drug-resistant mutations relative to that of first-generation inhibitors. Overall, TMC114's potency against MDR viruses is likely a combination of its extremely high affinity and close fit within the substrate envelope.
The purpose of this study was to characterize the antiviral activity, cytotoxicity, and mechanism of action of TMC114, a novel human immunodeficiency virus type 1 (HIV-1) protease inhibitor (PI). TMC114 exhibited potent anti-HIV activity with a 50% effective concentration (EC 50 ) of 1 to 5 nM and a 90% effective concentration of 2.7 to 13 nM. TMC114 exhibited no cytotoxicity at concentrations up to 100 M (selectivity index, >20,000). All viruses in a panel of 19 recombinant clinical isolates carrying multiple protease mutations and demonstrating resistance to an average of five other PIs, were susceptible to TMC114, defined as a fold change in EC 50 of <4. TMC114 was also effective against the majority of 1,501 PI-resistant recombinant viruses derived from recent clinical samples, with EC 50 s of <10 nM for 75% of the samples. In sequential passage experiments using HIV-1 LAI, two mutations (R41T and K70E) were selected. One selected virus showed a 10-fold reduction in susceptibility to TMC114, but <10-fold reductions in susceptibility to the current PIs (atazanavir was not assessed), except saquinavir. However, when the selected mutations were introduced into a laboratory strain by site-directed mutagenesis, they had no effect on susceptibility to TMC114 or other PIs. There was no evidence of antagonism between TMC114 and any currently available PIs or reverse transcriptase inhibitors. Combinations with ritonavir, nelfinavir, and amprenavir showed some evidence of synergy. These results suggest that TMC114 is a potential candidate for the treatment of both naïve and PI-experienced patients with HIV.
The screening of known HIV-1 protease inhibitors against a panel of multi-drug-resistant viruses revealed the potent activity of TMC126 on drug-resistant mutants. In comparison to amprenavir, the improved affinity of TMC126 is largely the result of one extra hydrogen bond to the backbone of the protein in the P2 pocket. Modification of the substitution pattern on the phenylsulfonamide P2' substituent of TMC126 created an interesting SAR, with the close analogue TMC114 being found to have a similar antiviral activity against the mutant and the wild-type viruses. X-ray and thermodynamic studies on both wild-type and mutant enzymes showed an extremely high enthalpy driven affinity of TMC114 for HIV-1 protease. In vitro selection of mutants resistant to TMC114 starting from wild-type virus proved to be extremely difficult; this was not the case for other close analogues. Therefore, the extra H-bond to the backbone in the P2 pocket cannot be the only explanation for the interesting antiviral profile of TMC114. Absorption studies in animals indicated that TMC114 has pharmacokinetic properties comparable to currently approved HIV-1 protease inhibitors.
The hepatitis C virus (HCV) NS3/4A serine protease has been explored as a target for the inhibition of viral replication in preclinical models and in HCV-infected patients. TMC435350 is a highly specific and potent inhibitor of NS3/4A protease selected from a series of novel macrocyclic inhibitors. In biochemical assays using NS3/4A proteases of genotypes 1a and 1b, inhibition constants of 0.5 and 0.4 nM, respectively, were determined. TMC435350 inhibited HCV replication in a cellular assay (subgenomic 1b replicon) with a half-maximal effective concentration (EC 50 ) of 8 nM and a selectivity index of 5,875. The compound was synergistic with alpha interferon and an NS5B inhibitor in the replicon model and additive with ribavirin. In rats, TMC435350 was extensively distributed to the liver and intestinal tract (tissue/plasma area under the concentration-time curve ratios of >35), and the absolute bioavailability was 44% after a single oral administration. Compound concentrations detected in both plasma and liver at 8 h postdosing were above the EC 99 value measured in the replicon. In conclusion, given the selective and potent in vitro anti-HCV activity, the potential for combination with other anti-HCV agents, and the favorable pharmacokinetic profile, TMC435350 has been selected for clinical development.
We have discovered a novel class of human immunodeficiency virus (HIV) reverse transcriptase (RT) inhibitors that block the polymerization reaction in a mode distinct from those of the nucleoside or nucleotide RT inhibitors (NRTIs) and nonnucleoside RT inhibitors (NNRTIs). For this class of indolopyridone compounds, steady-state kinetics revealed competitive inhibition with respect to the nucleotide substrate. Despite substantial structural differences with classical chain terminators or natural nucleotides, these data suggest that the nucleotide binding site of HIV RT may accommodate this novel class of RT inhibitors. To test this hypothesis, we have studied the mechanism of action of the prototype compound indolopyridone-1 (INDOPY-1) using a variety of complementary biochemical tools. Time course experiments with heteropolymeric templates showed "hot spots" for inhibition following the incorporation of pyrimidines (T>C). Moreover, binding studies and site-specific footprinting experiments revealed that INDOPY-1 traps the complex in the posttranslocational state, preventing binding and incorporation of the next complementary nucleotide. The novel mode of action translates into a unique resistance profile. While INDOPY-1 susceptibility is unaffected by mutations associated with NNRTI or multidrug NRTI resistance, mutations M184V and Y115F are associated with decreased susceptibility, and mutation K65R confers hypersusceptibility to INDOPY-1. This resistance profile provides additional evidence for active site binding. In conclusion, this class of indolopyridones can occupy the nucleotide binding site of HIV RT by forming a stable ternary complex whose stability is mainly dependent on the nature of the primer 3 end.The reverse transcriptase (RT) enzyme of human immunodeficiency virus type 1 (HIV-1) remains a major target in antiretroviral therapy, with the current standard of care being the use of two nucleoside or nucleotide analogue reverse transcriptase inhibitors (NRTIs), combined with one nonnucleoside reverse transcriptase inhibitor (NNRTI) or protease inhibitor (32).Upon entry into the cell, the virus is uncoated, and the viral RT enzyme converts its single-stranded RNA genome into double-stranded proviral DNA. Inhibition of this crucial event in the early viral life cycle ultimately precludes the virus from proliferating. Following the intracellular phosphorylation of NRTIs, NRTI-triphosphates compete with natural deoxyribonucleoside triphosphate (dNTP) pools and bind to the RT active site. They act as chain terminators due to the lack of a 3Ј-hydroxyl group (31). In contrast, NNRTIs represent a chemically diverse class of compounds that bind to a pocket in the vicinity of the catalytic site (25,29,30). Binding of these inhibitors is noncompetitive with respect to both dNTPs and template/primer (16).Despite the potency of combinations of NRTIs and NNRTIs, the emergence of mutations conferring resistance remains a major cause for treatment failure. The advent of novel RT inhibitors with a different mechani...
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