2-(Arylamino)-6-(trifluoromethyl)nicotinic Acid Derivatives: New HIV-1 RT Dual Inhibitors Active on Viral Replication

The persistence of the AIDS epidemic, and the life-long treatment required, indicate the constant need of novel HIV-1 inhibitors. In this scenario the HIV-1 Reverse Transcriptase (RT)-associated ribonuclease H (RNase H) function is a promising drug target. Here we report a series of compounds, developed on the 2-amino-6-(trifluoromethyl)nicotinic acid scaffold, studied as promising RNase H dual inhibitors. Among the 44 tested compounds, 34 inhibited HIV-1 RT-associated RNase H function in the low micromolar range, and seven of them showed also to inhibit viral replication in cell-based assays with a selectivity index up to 10. The most promising compound, 21, inhibited RNase H function with an IC50 of 14 µM and HIV-1 replication in cell-based assays with a selectivity index greater than 10. Mode of action studies revealed that compound 21 is an allosteric dual-site compound inhibiting both HIV-1 RT functions, blocking the polymerase function also in presence of mutations carried by circulating variants resistant to non-nucleoside inhibitors, and the RNase H function interacting with conserved regions within the RNase H domain. Proving compound 21 as a promising lead for the design of new allosteric RNase H inhibitors active against viral replication with not significant cytotoxic effects.

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A Novel Leu92 Mutant of HIV-1 Reverse Transcriptase with a Selective Deficiency in Strand Transfer Causes a Loss of Viral Replication

Journal of Virology ◽

10.1128/jvi.00809-15 ◽

2015 ◽

Vol 89 (16) ◽

pp. 8119-8129 ◽

Cited By ~ 4

Author(s):

Eytan Herzig ◽

Nickolay Voronin ◽

Nataly Kucherenko ◽

Amnon Hizi

Keyword(s):

Life Cycle ◽

Viral Replication ◽

Reverse Transcriptase ◽

Dna Polymerase ◽

Reverse Transcription ◽

Polymerase Activity ◽

Rnase H ◽

Strand Transfer ◽

Hiv 1

ABSTRACTThe process of reverse transcription (RTN) in retroviruses is essential to the viral life cycle. This key process is catalyzed exclusively by the viral reverse transcriptase (RT) that copies the viral RNA into DNA by its DNA polymerase activity, while concomitantly removing the original RNA template by its RNase H activity. During RTN, the combination between DNA synthesis and RNA hydrolysis leads to strand transfers (or template switches) that are critical for the completion of RTN. The balance between these RT-driven activities was considered to be the sole reason for strand transfers. Nevertheless, we show here that a specific mutation in HIV-1 RT (L92P) that does not affect the DNA polymerase and RNase H activities abolishes strand transfer. There is also a good correlation between this complete loss of the RT's strand transfer to the loss of the DNA clamp activity of the RT, discovered recently by us. This finding indicates a mechanistic linkage between these two functions and that they are both direct and unique functions of the RT (apart from DNA synthesis and RNA degradation). Furthermore, when the RT's L92P mutant was introduced into an infectious HIV-1 clone, it lost viral replication, due to inefficient intracellular strand transfers during RTN, thus supporting thein vitrodata. As far as we know, this is the first report on RT mutants that specifically and directly impair RT-associated strand transfers. Therefore, targeting residue Leu92 may be helpful in selectively blocking this RT activity and consequently HIV-1 infectivity and pathogenesis.IMPORTANCEReverse transcription in retroviruses is essential for the viral life cycle. This multistep process is catalyzed by viral reverse transcriptase, which copies the viral RNA into DNA by its DNA polymerase activity (while concomitantly removing the RNA template by its RNase H activity). The combination and balance between synthesis and hydrolysis lead to strand transfers that are critical for reverse transcription completion. We show here for the first time that a single mutation in HIV-1 reverse transcriptase (L92P) selectively abolishes strand transfers without affecting the enzyme's DNA polymerase and RNase H functions. When this mutation was introduced into an infectious HIV-1 clone, viral replication was lost due to an impaired intracellular strand transfer, thus supporting thein vitrodata. Therefore, finding novel drugs that target HIV-1 reverse transcriptase Leu92 may be beneficial for developing new potent and selective inhibitors of retroviral reverse transcription that will obstruct HIV-1 infectivity.

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Effects of the W153L Substitution in HIV Reverse Transcriptase on Viral Replication and Drug Resistance to Multiple Categories of Reverse Transcriptase Inhibitors

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02729-14 ◽

2014 ◽

Vol 58 (8) ◽

pp. 4515-4526 ◽

Cited By ~ 2

Author(s):

Hong-Tao Xu ◽

Susan P. Colby-Germinario ◽

Maureen Oliveira ◽

Daniel Rajotte ◽

Richard Bethell ◽

...

Keyword(s):

Viral Replication ◽

Reverse Transcriptase ◽

Rnase H ◽

Hiv Reverse Transcriptase ◽

Compound A ◽

Enzymatic Function ◽

Biochemical Assays ◽

Rt Inhibitors ◽

The Impact ◽

Hiv 1

ABSTRACTA W153L substitution in HIV-1 reverse transcriptase (RT) was recently identified by selection with a novel nucleotide-competing RT inhibitor (NcRTI) termed compound A that is a member of the benzo[4,5]furo[3,2,d]pyrimidin-2-one NcRTI family of drugs. To investigate the impact of W153L, alone or in combination with the clinically relevant RT resistance substitutions K65R (change of Lys to Arg at position 65), M184I, K101E, K103N, E138K, and Y181C, on HIV-1 phenotypic susceptibility, viral replication, and RT enzymatic function, we generated recombinant RT enzymes and viruses containing each of these substitutions or various combinations of them. We found that W153L-containing viruses were impaired in viral replicative capacity and were hypersusceptible to tenofovir (TFV) while retaining susceptibility to most nonnucleoside RT inhibitors. The nucleoside 3TC retained potency against W153L-containing viruses but not when the M184I substitution was also present. W153L was also able to reverse the effects of the K65R substitution on resistance to TFV, and K65R conferred hypersusceptibility to compound A. Biochemical assays demonstrated that W153L alone or in combination with K65R, M184I, K101E, K103N, E138K, and Y181C impaired enzyme processivity and polymerization efficiency but did not diminish RNase H activity, providing mechanistic insights into the low replicative fitness associated with these substitutions. We show that the mechanism of the TFV hypersusceptibility conferred by W153L is mainly due to increased efficiency of TFV-diphosphate incorporation. These results demonstrate that compound A and/or derivatives thereof have the potential to be important antiretroviral agents that may be combined with tenofovir to achieve synergistic results.

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Alizarine derivatives as new dual inhibitors of the HIV-1 reverse transcriptase-associated DNA polymerase and RNase H activities effective also on the RNase H activity of non-nucleoside resistant reverse transcriptases

FEBS Journal ◽

10.1111/j.1742-4658.2011.08057.x ◽

2011 ◽

Vol 278 (9) ◽

pp. 1444-1457 ◽

Cited By ~ 28

Author(s):

Francesca Esposito ◽

Tatyana Kharlamova ◽

Simona Distinto ◽

Luca Zinzula ◽

Yung-Chi Cheng ◽

...

Keyword(s):

Reverse Transcriptase ◽

Dna Polymerase ◽

Rnase H ◽

Reverse Transcriptases ◽

Dual Inhibitors ◽

Hiv 1

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The M230L Nonnucleoside Reverse Transcriptase Inhibitor Resistance Mutation in HIV-1 Reverse Transcriptase Impairs Enzymatic Function and Viral Replicative Capacity

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01795-09 ◽

2010 ◽

Vol 54 (6) ◽

pp. 2401-2408 ◽

Cited By ~ 19

Author(s):

Hong-Tao Xu ◽

Yudong Quan ◽

Susan M. Schader ◽

Maureen Oliveira ◽

Tamara Bar-Magen ◽

...

Keyword(s):

Viral Replication ◽

Reverse Transcriptase ◽

First Generation ◽

Resistance Mutation ◽

Transcriptase Inhibitor ◽

Rnase H ◽

Replication Capacity ◽

Dna And Rna ◽

Enzymatic Function ◽

Hiv 1

ABSTRACT The M230L mutation in HIV-1 reverse transcriptase (RT) is associated with resistance to first-generation nonnucleoside reverse transcriptase inhibitors (NNRTIs). The present study was designed to determine the effects of M230L on enzyme function, viral replication capacity (RC), and the extent to which M230L might confer resistance to the second-generation NNRTI etravirine (ETR) as well as to the first-generation NNRTIs efavirenz (EFV) and nevirapine (NVP). Phenotyping assays with TZM-bl cells confirmed that M230L conferred various degrees of resistance to each of the NNRTIs tested. Recombinant viruses containing M230L displayed an 8-fold decrease in RC compared to that of the parental wild-type (WT) virus. Recombinant HIV-1 WT and M230L mutant RT enzymes were purified; and both biochemical and cell-based phenotypic assays confirmed that M230L conferred resistance to each of EFV, NVP, and ETR. RT that contained M230L was also deficient in regard to each of minus-strand DNA synthesis, both DNA- and RNA-dependent polymerase activities, processivity, and RNase H activity, suggesting that this mutation contributes to diminished viral replication kinetics.

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Design, synthesis and biological evaluation of 3-hydroxyquinazoline-2,4(1H,3H)-diones as dual inhibitors of HIV-1 reverse transcriptase-associated RNase H and integrase

Bioorganic & Medicinal Chemistry ◽

10.1016/j.bmc.2019.07.011 ◽

2019 ◽

Vol 27 (17) ◽

pp. 3836-3845 ◽

Cited By ~ 2

Author(s):

Ping Gao ◽

Xiqiang Cheng ◽

Lin Sun ◽

Shu Song ◽

Mar Álvarez ◽

...

Keyword(s):

Reverse Transcriptase ◽

Biological Evaluation ◽

Rnase H ◽

Design Synthesis ◽

Dual Inhibitors ◽

Synthesis And Biological Evaluation ◽

Hiv 1

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Activity-based selection of HIV-1 reverse transcriptase variants with decreased polymerization fidelity

Biological Chemistry ◽

10.1515/bc.2010.067 ◽

2010 ◽

Vol 391 (6) ◽

Cited By ~ 3

Author(s):

Sascha N. Stumpp ◽

Bianca Heyn ◽

Susanne Brakmann

Keyword(s):

Viral Replication ◽

Reverse Transcriptase ◽

Error Rate ◽

Antiviral Treatment ◽

Rnase H ◽

Single Amino Acid ◽

Selection Scheme ◽

Single Amino Acid Residue ◽

Hiv 1

AbstractHIV-1 reverse transcriptase (HIV-1 RT) copies the RNA genome of HIV-1 into DNA, thereby committing errors at an exceptionally high frequency. Viral offspring evolve rapidly and consequently are capable of evading the immune response as well as antiviral treatment. However, error-prone viral replication could drive HIV close to extinction owing to an intolerable load of deleterious mutations. We applied a genetic selection scheme to identify variants of HIV-1 RT with a further increased error rate to study the relationship between error rate and viral replication. Using this approach, we identified 16 mutator candidates, two of which were purified and further studiedin vitro. One of these variant enzymes showed a generally increased mutation frequency as compared with the reference enzyme. A single amino acid residue, R448, is probably responsible for the observed effect. Mutation of this residue, which is located within the RNase H domain of HIV-1 RT, seems to perturb the interaction with template RNA and consequently affects polymerase activity and fidelity.

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Madurahydroxylactone Derivatives as Dual Inhibitors of Human Immunodeficiency Virus Type 1 Integrase and RNase H

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00883-07 ◽

2007 ◽

Vol 52 (1) ◽

pp. 361-364 ◽

Cited By ~ 22

Author(s):

Christophe Marchand ◽

John A. Beutler ◽

Antony Wamiru ◽

Scott Budihas ◽

Ute Möllmann ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Rnase H ◽

Immunodeficiency Virus ◽

Dual Inhibitors ◽

Dual Inhibition ◽

Pharmacophore Models ◽

Hiv 1

ABSTRACT A series of 29 madurahydroxylactone derivatives was evaluated for dual inhibition of human immunodeficiency virus type 1 (HIV-1) integrase and RNase H. While most of the compounds exhibited similar potencies for both enzymes, two of the derivatives showed 10- to 100-fold-higher selectivity for each enzyme, suggesting that distinct pharmacophore models could be generated. This study exemplifies the common and divergent structural requirements for the inhibition of two structurally related HIV-1 enzymes and demonstrates the importance of systematically screening for both integrase and RNase H when developing novel inhibitors.

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