Non-nucleoside Reverse Transcriptase (RT) Inhibitors Research Articles

In silico Design of Novel HIV-1 NNRTIs Based on Combined Modeling Studies of Dihydrofuro[3,4-d]pyrimidines.

A novel series of dihydrofuro[3,4-d]pyrimidine (DHPY) analogs have recently been recognized as promising HIV-1 non-nucleoside reverse transcriptase (RT) inhibitors (NNRTIs) with potent antiviral activity. To better understand the pharmacological essentiality of these DHPYs and design novel NNRTI leads, in this work, a systematic in silico study was performed on 52 DHPYs using three-dimensional quantitative structure–activity relationship (3D-QSAR), molecular docking, virtual screening, absorption-distribution-metabolism-excretion (ADME) prediction, and molecular dynamics (MD) methods. The generated 3D-QSAR models exhibited satisfactory parameters of internal validation and well-externally predictive capacity, for instance, the q2, R2, and of the optimal comparative molecular similarity indices analysis model were 0.647, 0.970, and 0.751, respectively. The docking results indicated that residues Lys101, Tyr181, Tyr188, Trp229, and Phe227 played important roles for the DHPY binding. Nine lead compounds were obtained by the virtual screening based on the docking and pharmacophore model, and three new compounds with higher docking scores and better ADME properties were subsequently designed based on the screening and 3D-QSAR results. The MD simulation studies further demonstrated that the newly designed compounds could stably bind with the HIV-1 RT. These hit compounds were supposed to be novel potential anti-HIV-1 inhibitors, and these findings could provide significant information for designing and developing novel HIV-1 NNRTIs.

Targeting the hydrophobic channel of NNIBP: discovery of novel 1,2,3-triazole-derived diarylpyrimidines as novel HIV-1 NNRTIs with high potency against wild-type and K103N mutant virus.

Enlightened by our previous efforts to modify diarylpyrimidines as HIV-1 non-nucleoside reverse transcriptase (RT) inhibitors (NNRTIs) and the reported crystallographic studies, we designed and synthesized novel 1,2,3-triazole-derived diarylpyrimidine derivatives via the CuAAC "click reaction", to make additional interactions with the hydrophobic channel in the NNRTI binding pocket. The newly synthesized compounds were evaluated for anti-HIV potency in MT-4 cells. All the compounds showed favorable activity against the wild-type HIV-1 strain with an EC50 of 0.013-5.62 μM. Interestingly, some compounds displayed remarkable potency in inhibiting K103N mutant virus, a key drug-resistant mutant to NNRTIs. Among them, meta-methylbenzoate (ZL2, EC50(IIIB) = 0.020 μM, EC50(K103N) = 0.043 μM, CC50 > 241.52 μM), para-methylbenzoate (ZL3, EC50(IIIB) = 0.013 μM, EC50 (K103N) = 0.022 μM, CC50 > 241.52 μM) and para-phenol (ZL7, EC50(IIIB) = 0.014 μM, EC50 (K103N) = 0.054 μM, CC50 = 2.1 μM) derivatives are the three most promising compounds which are superior to the first-line antiretroviral drug efavirenz (EC50(IIIB) = 0.003 μM, EC50 (K103N) = 0.11 μM, CC50 > 6.34 μM) against the K103N mutant strain. More encouragingly, ZL2 and ZL3 exhibited much lower cytotoxicity and a high selection index of >10 000 compared with all the control drugs (AZT, 3TC, NVP, EFV, and ETV). The detailed structure-activity relationship (SAR), enzymatic inhibitory activity and docking study of the representative compounds are also discussed. Furthermore, the preliminary physicochemical properties and the early metabolic stability of representative compounds were examined to evaluate their drug-like properties.

Structural and Preclinical Studies of Computationally Designed Non-Nucleoside Reverse Transcriptase Inhibitors for Treating HIV infection.

The clinical benefits of HIV-1 non-nucleoside reverse transcriptase (RT) inhibitors (NNRTIs) are hindered by their unsatisfactory pharmacokinetic (PK) properties along with the rapid development of drug-resistant variants. However, the clinical efficacy of these inhibitors can be improved by developing compounds with enhanced pharmacological profiles and heightened antiviral activity. We used computational and structure-guided design to develop two next-generation NNRTI drug candidates, compounds I and II, which are members of a class of catechol diethers. We evaluated the preclinical potential of these compounds in BALB/c mice because of their high solubility (510 µg/ml for compound I and 82.9 µg/ml for compound II), low cytotoxicity, and enhanced antiviral activity against wild-type (WT) HIV-1 RT and resistant variants. Additionally, crystal structures of compounds I and II with WT RT suggested an optimal binding to the NNRTI binding pocket favoring the high anti-viral potency. A single intraperitoneal dose of compounds I and II exhibited a prolonged serum residence time of 48 hours and concentration maximum (Cmax) of 4000- to 15,000-fold higher than their therapeutic/effective concentrations. These Cmax values were 4- to 15-fold lower than their cytotoxic concentrations observed in MT-2 cells. Compound II showed an enhanced area under the curve (0-last) and decreased plasma clearance over compound I and efavirenz, the standard of care NNRTI. Hence, the overall (PK) profile of compound II was excellent compared with that of compound I and efavirenz. Furthermore, both compounds were very well tolerated in BALB/c mice without any detectable acute toxicity. Taken together, these data suggest that compounds I and II possess improved anti-HIV-1 potency, remarkable in vivo safety, and prolonged in vivo circulation time, suggesting strong potential for further development as new NNRTIs for the potential treatment of HIV infection.

Commonly Transmitted HIV-1 Drug Resistance Mutations in Reverse-Transcriptase and Protease in Antiretroviral Treatment-Naive Patients and Response to Regimens Containing Tenofovir Disoproxil Fumarate or Tenofovir Alafenamide.

The presence of transmitted drug resistance mutations (TDRMs) in antiretroviral treatment (ART)-naive patients can adversely affect the outcome of ART. Resistance testing was conducted in 6704 ART-naive subjects predominantly from the United States and Europe in 9 clinical studies conducted by Gilead Sciences from 2000 to 2013. The presence of TDRMs increased during this period (from 5.2% to 11.4%), primarily driven by an increase in nonnucleoside reverse-transcriptase (RT) inhibitor (NNRTI) resistance mutations (from 0.3% to 7.1%), particularly K103N/S (increase from 0.3% to 5.3%). Nucleoside/nucleotide RT inhibitor mutations were found in 3.1% of patients. Only 1 patient had K65R (0.01%) and 7 had M184V/I (0.1%), despite high use of tenofovir disoproxil fumarate (TDF), emtricitabine, and lamivudine and potential transmission of resistance to these drugs. At least 1 thymidine-analogue mutations was present in 2.7% of patients with 0.07% harboring T215Y/F and 2.7% harboring T215 revertant mutations (T215rev). Patients with the combination of M41L + L210W + T215rev showed full human immunodeficiency virus RNA suppression while receiving a TDF- or tenofovir alafenamide-containing regimen. There was an overall increase of TDRMs among patients enrolling in clinical trials from 2000 through 2013, driven primarily by an increase in NNRTI resistance. However, the presence of common TDRMs, including thymidine-analogue mutations/T215rev, showed no impact on response to TDF- or tenofovir alafenamide-containing regimens.

Different Effects of Nonnucleoside Reverse Transcriptase Inhibitor Resistance Mutations on Cytotoxic T Lymphocyte Recognition between HIV-1 Subtype B and Subtype A/E Infections.

The effect of antiretroviral drug resistance mutations on cytotoxic T lymphocyte (CTL) recognition has been analyzed in HIV-1 subtype B infections, but it remains unclear in infections by other HIV-1 subtypes that are epidemic in countries where antiretroviral drugs are not effectively used. We investigated the effect of nonnucleoside reverse transcriptase (RT) inhibitor (NNRTI)-resistance mutations (Y181C, Y181I, and Y181V) on epitope recognition by CTLs specific for 3 different HIV-1 epitopes (HLA-A*02:01-restricted IV10, HLA-B*35:01-restricted NY9, and HLA-C*12:02-restricted KY9) in subtype B and subtype A/E infections and the accumulation of these mutations in treatment-naive Japanese and Vietnamese. These NNRTI-resistance mutations critically affected NY9-specific and KY9-specific T cell responses in the subtype B infections, whereas they showed a different effect on IV10-specific T cell responses among the subtype B-infected individuals. These mutations affected IV10-specific T cell responses but weakly affected NY9-specific T cell responses in the subtype A/E infections. The substitution at position 3 of NY9 epitope which was found in the subtype A/E virus differently influenced the peptide binding to HLA-B*35:01, suggesting that the differences in peptide binding may result in the differences in T cell recognition between the subtype B virus and A/E virus infections. The Y181C mutation was found to be accumulating in treatment-naive Vietnamese infected with the subtype A/E virus. The present study demonstrated different effects of NNRTI-resistance RT181 mutations on CTL responses between the 2 subtype infections. The Y181C mutation may influence HIV-1 control by the CTLs in Vietnam, since this mutation has been accumulating in treatment-naive Vietnamese. Antiretroviral therapy leads to the emergence of drug-resistant HIV-1, resulting in virological and clinical failures. Though HIV-1-specific CTLs play a critical role in HIV-1 infection, some of drug resistance mutations located in CTL epitopes are known to affect HIV-1-specific CTL responses. Nonnucleoside reverse transcriptase inhibitor (NNRTI)-resistance RT181 mutations are frequently observed in patients treated with NNRTIs. Such drug resistance mutations may have an influence on immune control by HIV-1-specific CTLs, especially in countries where antiretroviral drugs are not effectively used. We here investigated the effect of three NNRTI-resistance RT181 mutations on immune responses by HIV-1-specific CTLs and the recent accumulation of these mutations in treatment-naive Vietnamese infected with HIV-1 subtype A/E virus. RT181 mutations affected CTL recognition in both subtype A/E and B infections, while the RT Y181C mutation has been accumulating in treatment-naive Vietnamese. The results suggest that the Y181C mutation may influence HIV-1 control by CTLs in Vietnam.

Fused heterocycles bearing bridgehead nitrogen as potent HIV-1 NNRTIs. Part 3: Optimization of [1,2,4]triazolo[1,5-a]pyrimidine core via structure-based and physicochemical property-driven approaches

In our arduous efforts to develop new potent HIV-1 non-nucleoside reverse transcriptase (RT) inhibitors (NNRTIs), novel piperidine-linked [1,2,4]triazolo[1,5-a]pyrimidine derivatives were designed, synthesized and evaluated for their antiviral activities in MT-4 cell cultures. Biological results showed that all of the title compounds displayed moderate to excellent activities against wild-type (wt) HIV-1 strain (IIIB) with EC50 values ranging from 8.1 nM to 2284 nM in a cell-based assay. Among them, the most promising analog 7d possessed an EC50 value of 8.1 nM against wt HIV-1, which was much more potent than the reference drugs DDI, 3 TC, NVP and DLV. Additionally, 7d demonstrated weak activity against the double mutant HIV-1 strain (K103N + Y181C), and was more efficient than NVP in a RT inhibition assay. Besides, some measured and calculated physicochemical properties of 7d, like log P and water solubility, as well as the structure–activity relationships (SARs) analysis have been discussed in detail. Furthermore, the binding mode of the active compound 7d was rationalized by molecular simulation studies.

Mechanism of allosteric inhibition of HIV-1 reverse transcriptase revealed by single-molecule and ensemble fluorescence.

Non-nucleoside reverse transcriptase (RT) inhibitors (NNRTIs) are routinely used to treat HIV-1 infection, yet their mechanism of action remains unclear despite intensive investigation. In this study, we developed complementary single-molecule fluorescence and ensemble fluorescence anisotropy approaches to discover how NNRTIs modulate the intra-molecular conformational changes and inter-molecular dynamics of RT-template/primer (T/P) and RT–T/P–dNTP complexes. We found that NNRTI binding to RT induces opening of the fingers and thumb subdomains, which increases the dynamic sliding motion of the enzyme on the T/P and reduces dNTP binding affinity. Further, efavirenz promotes formation of the E138-K101 salt bridge between the p51 and p66 subunits of RT, which contributes to opening of the thumb/fingers subdomains. Engineering a more polar salt bridge between p51 and p66 resulted in even greater increases in the thumb/fingers opening, RT sliding, dNTP binding disruption and in vitro and in vivo RT inhibition than were observed with wild-type RT. We also observed that K103N, a clinically relevant NNRTI resistance mutation, does not prevent binding between efavirenz and RT-T/P but instead allows formation of a stable and productive RT–T/P–dNTP complex, possibly through disruption of the E138-K101 salt bridge. Collectively, these data describe unique structure–activity–resistance relationships that could be exploited for drug development.

Fifteen Years Later: Hard and Soft Selection Sweeps Confirm a Large Population Number for HIV In Vivo

Fifteen Years Later: Hard and Soft Selection Sweeps Confirm a Large Population Number for HIV In Vivo

Competitive Fitness Assays Indicate that the E138A Substitution in HIV-1 Reverse Transcriptase Decreases In Vitro Susceptibility to Emtricitabine

We characterized the relative fitness of multiple nonnucleoside reverse transcriptase (RT) inhibitor (NNRTI)-resistant HIV-1 variants in the presence of etravirine (ETV), rilpivirine (RPV), and/or the nucleoside RT inhibitor emtricitabine (FTC) by simultaneous competitive culture and 454 deep sequencing. The E138A substitution, typically associated with decreased virologic responses to ETV- and RPV-containing regimens, confers a clear fitness advantage to the virus in the presence of FTC and decreases FTC susceptibility 4.7-fold.

Biochemical Mechanism of HIV-1 Resistance to Rilpivirine

Rilpivirine (RPV) is a second generation nonnucleoside reverse transcriptase (RT) inhibitor (NNRTI) that efficiently inhibits HIV-1 resistant to first generation NNRTIs. Virological failure during therapy with RPV and emtricitabine is associated with the appearance of E138K and M184I mutations in RT. Here we investigate the biochemical mechanism of RT inhibition and resistance to RPV. We used two transient kinetics approaches (quench-flow and stopped-flow) to determine how subunit-specific mutations in RT p66 or p51 affect association and dissociation of RPV to RT as well as their impact on binding of dNTP and DNA and the catalytic incorporation of nucleotide. We compared WT with four subunit-specific RT mutants, p66(M184I)/p51(WT), p66(E138K)/p51(E138K), p66(E138K/M184I)/p51(E138K), and p66(M184I)/p51(E138K). Ile-184 in p66 (p66(184I)) decreased the catalytic efficiency of RT (k(pol)/K(d)(.dNTP)), primarily through a decrease in dNTP binding (K(d)(.dNTP)). Lys-138 either in both subunits or in p51 alone abrogated the negative effect of p66(184I) by restoring dNTP binding. Furthermore, p51(138K) reduced RPV susceptibility by altering the ratio of RPV dissociation to RPV association, resulting in a net reduction in RPV equilibrium binding affinity (K(d)(.RPV) = k(off.RPV)/k(on.RPV)). Quantum mechanics/molecular mechanics hybrid molecular modeling revealed that p51(E138K) affects access to the RPV binding site by disrupting the salt bridge between p51(E138) and p66(K101). p66(184I) caused repositioning of the Tyr-183 active site residue and decreased the efficiency of RT, whereas the addition of p51(138K) restored Tyr-183 to a WT-like conformation, thus abrogating the Ile-184-induced functional defects.

Anti-human immunodeficiency virus type 1 activity of novel 6-substituted 1-benzyl-3-(3,5-dimethylbenzyl)uracil derivatives.

Nonnucleoside reverse transcriptase (RT) inhibitors (NNRTIs) are important components of current combination therapies for human immunodeficiency virus type 1 (HIV-1) infection. In screening of chemical libraries, we found 6-azido-1-benzyl-3-(3,5-dimethylbenzyl)uracil (AzBBU) and 6-amino-1-benzyl-3-(3,5-dimethylbenzyl)uracil (AmBBU) to be highly active and selective inhibitors of HIV-1 replication in vitro. To determine the resistance profiles of these compounds, we conducted a long-term culture of HIV-1-infected MT-4 cells with escalating concentrations of each compound. After serial passages of the infected cells, escape viruses were obtained, and they were more than 500-fold resistant to the uracil derivatives compared to the wild type. Sequence analysis was conducted for RT of the escape viruses at passages 12 and 24. The amino acid mutation Y181C in the polymerase domain of RT was detected for all escape viruses. Docking studies using the crystal structure of RT showed that AmBBU requires the amino acid residues Leu100, Val106, Tyr181, and Trp229 for exerting its inhibitory effect on HIV-1. Four additional amino acid changes (K451R, R461K, T468P, and D471N) were identified in the RNase H domain of RT; however, their precise role in the acquisition of resistance is still unclear. In conclusion, the initial mutation Y181C seems sufficient for the acquisition of resistance to the uracil derivatives AzBBU and AmBBU. Further studies are required to determine the precise role of each mutation in the acquisition of HIV-1 resistance.

The HIV-1 Reverse Transcriptase M184I Mutation Enhances the E138K-Associated Resistance to Rilpivirine and Decreases Viral Fitness

The registrational phase III clinical trials of the nonnucleoside reverse transcriptase (RT) inhibitor (NNRTI) rilpivirine (RPV) in combination with two nucleoside/nucleotide RT inhibitors (NRTIs) found a unique genotypic resistance pattern involving the NNRTI mutation E138K with the NRTI mutation M184I. Eighty percent of subjects used emtricitabine (FTC) and tenofovir disoproxil fumarate (TDF); a single tablet regimen of FTC/RPV/TDF is in development. HIV-1 with E138K and/or M184V or I mutations were constructed and phenotyped in MT-2 cells and the PhenoSense and Antivirogram assays. Viral fitness was determined using growth competitions. Molecular models of the mutants were constructed from the RT-RPV crystal structure. The E138K mutant showed low-level reduced susceptibility to RPV (2.4-fold), but full susceptibility to FTC and tenofovir (TFV). Viruses with M184V or M184I showed high-level resistance to FTC and full susceptibility to RPV and TFV. Addition of M184I, but not M184V, to E138K, further decreased susceptibility to RPV and maintained FTC resistance. The E138K and M184V or I single and double mutants showed decreased replication fitness compared with wild type. M184V outcompeted M184I when compared directly and in the background of E138K. E138K + M184I was less fit than either E138K or M184I alone. Removing a salt bridge between E138/K101 is implicated in resistance to RPV. The higher frequency of E138K and M184I among RPV + FTC/TDF virologic failures is due to reduced susceptibility of the single mutants to RPV and FTC and the enhanced resistance to RPV for the double mutant at the cost of decreased viral fitness.

Viral Decay Kinetics in the Highly Active Antiretroviral Therapy-Treated Rhesus Macaque Model of AIDS

To prevent progression to AIDS, persons infected with human immunodeficiency virus type 1 (HIV-1) must remain on highly active antiretroviral therapy (HAART) indefinitely since this modality does not eradicate the virus. The mechanisms involved in viral persistence during HAART are poorly understood, but an animal model of HAART could help elucidate these mechanisms and enable studies of HIV-1 eradication strategies. Due to the specificity of non-nucleoside reverse transcriptase (RT) inhibitors (NNRTIs) for HIV-1, we have used RT-SHIV, a chimeric virus of simian immunodeficiency virus with RT from HIV-1. This virus is susceptible to NNRTIs and causes an AIDS-like disease in rhesus macaques. In this study, two groups of HAART-treated, RT-SHIV-infected macaques were analyzed to determine viral decay kinetics. In the first group, viral loads were monitored with a standard TaqMan RT-PCR assay with a limit of detection of 50 viral RNA copies per mL. Upon initiation of HAART, viremia decayed in a bi-phasic manner with half-lives of 1.7 and 8.5 days, respectively. A third phase was observed with little further decay. In the second group, the macaques were followed longitudinally with a more sensitive assay utilizing ultracentrifugation to concentrate virus from plasma. Bi-phasic decay of viral RNA was also observed in these animals with half-lives of 1.8 and 5.8 days. Viral loads in these animals during a third phase ranged from 2–58 RNA copies/mL, with little decay over time. The viral decay kinetics observed in these macaques are similar to those reported for HIV-1 infected humans. These results demonstrate that low-level viremia persists in RT-SHIV-infected macaques despite a HAART regimen commonly used in humans.

Synthesis and Anti-HIV-1 Activity of a Novel Series of Aminoimidazole Analogs.

There is still an urgent need to develop nonnucleoside reverse transcriptase (RT) inhibitors (NNRTI) with a high-genetic barrier to resistance that facilitate patient adherence and allow durable suppression of HIV-1 replication. In this study, we describe the synthesis of a novel series of N-aminoimidazole (NAIM) analogs. Each of the NAIM analogs display potent activity against wild-type recombinant purified HIV-1 RT as well as RTs containing the K103N or Y181C resistance mutations. The analogs, however, do not exhibit significant antiviral activity in cell culture and were, in general, cytotoxic. Nevertheless, these data suggest that the NAIM backbone may provide a suitable scaffold from which inhibitors active against NNRTI-resistant HIV-1 could be developed.

Prioritizing Hits with Appropriate Trade-Offs Between HIV-1 Reverse Transcriptase Inhibitory Efficacy and MT4 Blood Cells Toxicity Through Desirability-Based Multiobjective Optimization and Ranking.

Nonnucleoside reverse transcriptase (RT) inhibitors (NNRTIs) constitute a promising therapeutic option for AIDS. However, the emergence of virus-NNRTIs resistance was found to be a major problem in the field. Toward that goal, a "knock-out" strategy stands out between the several options to circumvent the problem. However the high drug or drug-drug concentrations often used generate additional safety concerns. The need for approaches able to early integrate drug- or lead-likeness, toxicity and bioavailability criteria in the drug discovery phase is an emergent issue. Given that, we propose a combined strategy based on desirability-based multiobjective optimization (MOOP) and ranking for the prioritization of HIV-1 NNRTIs hits with appropriate trade-offs between inhibitory efficacy over the HIV-1 RT and toxic effects over MT4 blood cells. Through the MOOP process, the theoretical levels of the predictive variables required to reach a desirable RT inhibitor candidate with the best possible compromise between efficacy and safety were found. This information is used as a pattern to rank the library of compounds according to a similarity-based structural criterion, providing a ranking quality of 64 %/71 %/73 % in training/validation/test set. A comparative study between the sequential, parallel and multiobjective virtual screening revealed that the multiobjective approach can outperform the other approaches. These results suggest that the identification of NNRTIs hits with appropriate trade-offs between potency and safety, rather than fully optimized hits solely based on potency, can facilitate the hit to lead transition and increase the likelihood of the candidate to evolve into a successful antiretroviral drug.

Combination of V106I and V179D Polymorphic Mutations in Human Immunodeficiency Virus Type 1 Reverse Transcriptase Confers Resistance to Efavirenz and Nevirapine but Not Etravirine

Etravirine (ETV) is a second-generation nonnucleoside reverse transcriptase (RT) inhibitor (NNRTI) introduced recently for salvage antiretroviral treatment after the emergence of NNRTI-resistant human immunodeficiency virus type 1 (HIV-1). Following its introduction, two naturally occurring mutations in HIV-1 RT, V106I and V179D, were listed as ETV resistance-associated mutations. However, the effect of these mutations on the development of NNRTI resistance has not been analyzed yet. To select highly NNRTI-resistant HIV-1 in vitro, monoclonal HIV-1 strains harboring V106I and V179D (HIV-1(V106I) and HIV-1(V179D)) were propagated in the presence of increasing concentrations of efavirenz (EFV). Interestingly, V179D emerged in one of three selection experiments from HIV-1(V106I) and V106I emerged in two of three experiments from HIV-1(V179D). Analysis of recombinant HIV-1 clones showed that the combination of V106I and V179D conferred significant resistance to EFV and nevirapine (NVP) but not to ETV. Structural analysis indicated that ETV can overcome the repulsive interactions caused by the combination of V106I and V179D through fine-tuning of its binding module to RT facilitated by its plastic structure, whereas EFV and NVP cannot because of their rigid structures. Analysis of clinical isolates showed comparable drug susceptibilities, and the same combination of mutations was found in some database patients who experienced virologic NNRTI-based treatment failure. The combination of V106I and V179D is a newly identified NNRTI resistance pattern of mutations. The combination of polymorphic and minor resistance-associated mutations should be interpreted carefully.

Human Immunodeficiency Virus Type 1 Recombinant Reverse Transcriptase Enzymes Containing the G190A and Y181C Resistance Mutations Remain Sensitive to Etravirine

Etravirine (ETR) is a second-generation nonnucleoside reverse transcriptase (RT) inhibitor (NNRTI) active against common human immunodeficiency virus type 1 (HIV-1) drug-resistant strains. This study was designed to determine the extent to which each of the Y181C or G190A mutations in RT might confer resistance to ETR and other members of the NNRTI family of drugs. Recombinant HIV-1 RT enzymes containing either the Y181C or the G190A mutation, or both mutations in tandem, were purified. Both RNA- and DNA-dependent DNA polymerase assays were performed in order to determine the extent to which each of these mutations might confer resistance in cell-free biochemical assays against each of ETR, efavirenz, and nevirapine. Both the biochemical and the cell-based phenotypic assays confirmed the susceptibility of G190A-containing enzymes and viruses to ETR. The results of this study indicate that the G190A mutation is not associated with resistance to ETR.

Non-nucleoside HIV-1 reverse transcriptase inhibitors di-halo-indolyl aryl sulfones achieve tight binding to drug-resistant mutants by targeting the enzyme–substrate complex

Indolyl aryl sulfone (IAS) non-nucleoside reverse transcriptase (RT) inhibitors (NNRTIs) have been previously shown to effectively inhibit wild-type (wt) and drug-resistant human immunodeficiency virus type 1 (HIV-1) replication. IASs proved to act through different mechanisms of action, depending on the nature and position of their chemical substituents. Here we describe selected novel IAS derivatives (di-halo-IASs). Our results show that these compounds are selective for the enzyme-substrate complex. The molecular basis for this selectivity was a different dissociation rate of the drug to a particular enzymatic form along the reaction pathway. By comparing the activities of the different compounds against wild-type RT and the resistant enzymes carrying the single mutations Lys103Asn, Leu100Ile, and Tyr181Ile (K103N, L100I, and Y181I), we found that one compound (RS1914) dissociated from the mutated enzymes almost 10-fold slower than from the wild type RT. These results demonstrate that IASs are very flexible molecules, interacting dynamically with the viral RT, and that this property can be successfully exploited to design inhibitors endowed with an enhanced binding to common NNRTI-resistant mutants.

Mechanisms of inhibition of HIV replication by non-nucleoside reverse transcriptase inhibitors

The non-nucleoside reverse transcriptase (RT) inhibitors (NNRTIs) are a therapeutic class of compounds that are routinely used, in combination with other antiretroviral drugs, to treat HIV-1 infection. NNRTIs primarily block HIV-1 replication by preventing RT from completing reverse transcription of the viral single-stranded RNA genome into DNA. However, some NNRTIs, such as efavirenz, have been shown to inhibit the late stages of HIV-1 replication by interfering with HIV-1 Gag-Pol polyprotein processing, while others, such as the pyrimidinediones, have been shown to inhibit both HIV-1 RT-mediated reverse transcription and HIV-1/HIV-2 viral entry. Accordingly, in this review we describe the multiple mechanisms by which NNRTIs inhibit HIV-1 reverse transcription (and in some cases HIV-2 reverse transcription) and other key steps involved in HIV-1/HIV-2 replication.

Impact of Residues in the Nonnucleoside Reverse Transcriptase Inhibitor Binding Pocket on HIV-1 Reverse Transcriptase Heterodimer Stability

Previous studies have demonstrated that nonnucleoside reverse transcriptase (RT) inhibitors (NNRTIs) act as chemical enhancers of human immunodeficiency virus type 1 (HIV-1) RT dimerization. In the current study, we sought to define the role of key residues (101, 103, 108, 181, 188, 190, 225 and 318) in the NNRTI-binding pocket on HIV-1 RT heterodimer stability. Thirteen mutant RTs were constructed and evaluated for p66/p51 RT heterodimer formation using the well-established yeast two-hybrid assay. We found that the mutations K101A, P225H, Y318F and Y318W decreased RT heterodimer stability whereas K103N, V108I, V108W, Y181C, Y188L, G190A, G190E, G190W and P225W increased RT heterodimer stability. While these results demonstrate that residues that comprise the NNRTI-binding pocket contribute to the stability of p66/p51 HIV-1 RT, they did not suggest any obvious correlation between RT dimer stability and the extent of NNRTI resistance. Remarkably, mutations at residue G190 (A, E, W) in the p66 RT subunit were found to dramatically increase heterodimer stability. Notably, the G190W mutation increased RT dimer stability almost to the same extent as did 5 microM efavirenz. In light of these findings, we characterized the in vitro activity of recombinant RT expressing mutations at G190 in the p66 subunit only and compared them with a wild-type enzyme complexed with efavirenz. We found that while mutations at G190 had a significant effect on both the DNA polymerase and ribonuclease H activity of the enzyme, their phenotypic effects did not mirror those induced by efavirenz-binding to RT.