61 Comparative rates of in vitro resistance development of HIV-1 to new non-nucleoside analog RT inhibitors

ABSTRACT Using an indicator cell assay that directly quantifies viral replication, we show that human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2, respectively) exhibit similar sensitivities to 3′-azido-3′-deoxythymidine (zidovudine) as well as other nucleoside analog inhibitors of reverse transcriptase. These data support the use of nucleoside analogs for antiviral therapy of HIV-2 infection.

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Identification and Characterization of a Novel HIV-1 Nucleotide-Competing Reverse Transcriptase Inhibitor Series

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00113-13 ◽

2013 ◽

Vol 57 (6) ◽

pp. 2712-2718 ◽

Cited By ~ 17

Author(s):

D. Rajotte ◽

S. Tremblay ◽

A. Pelletier ◽

P. Salois ◽

L. Bourgon ◽

...

Keyword(s):

Reverse Transcriptase ◽

Transcriptase Inhibitor ◽

Cross Resistance ◽

Compound A ◽

New Class ◽

Measurable Activity ◽

Rt Inhibitors ◽

Reverse Transcriptase Inhibitor ◽

Hiv 1

ABSTRACTSeveral groups have recently reported on the identification of nucleotide-competing reverse transcriptase inhibitors (NcRTIs), a new class of RT inhibitors. NcRTIs reversibly inhibit binding of the incoming nucleotide to the RT active site but do not act as chain terminators, unlike the nucleos(t)ide reverse transcriptase inhibitor (NRTI) class. We identified a novel benzo[4,5]furo[3,2,d]pyrimidin-2-one NcRTI chemical series. Structure-activity relationship evaluation of this series with both RT and viral replication assays led to the identification of compound A, a new NcRTI. Compound A inhibited HIV-1 RT in a primer extension assay (50% inhibitory concentration, 2.6 nM) but had no measurable activity against human DNA polymerase γ at 10 μM. It potently inhibited HIV-1 replicationin vitro(50% effective concentration, 1.5 nM). The antiviral potency of compound A was unaffected by the presence of nonnucleotide RT inhibitor (NNRTI) mutations tested (L100I, K103N/Y181C, V106A, or Y188L). Notably, viruses encoding K65R were hypersusceptible to inhibition by compound A. Compound A also retained full activity against viruses encoding M184V.In vitroselection for resistant virus to compound A led to the selection of a single substitution within RT: W153L. A recombinant virus encoding the RT W153L was highly resistant to compound A (fold change, 160). W153 is a highly conserved residue in HIV RT and has not been previously associated with drug resistance. In summary, a novel NcRTI series with optimized antiviral activity, minimal cross-resistance to existing RT inhibitor classes, and a distinct resistance profile has been discovered. These results further establish NcRTIs as an emerging class of antiretroviral agents.

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Insights into Biophysical Methods to Study Interactions Between HIV-1 Reverse Transcriptase and Non-nucleoside Reverse Transcriptase Inhibitors

Letters in Drug Design & Discovery ◽

10.2174/1570180816666190723121845 ◽

2020 ◽

Vol 17 (6) ◽

pp. 818-825

Author(s):

Julien Dumond ◽

Jean-Marcel Julien Tronchet ◽

Serge Kirkiacharian ◽

Michel Seman ◽

Michèle Reboud-Ravaux

Keyword(s):

Reverse Transcriptase ◽

Biological Effects ◽

Tryptophan Fluorescence ◽

Plasmonic Resonance ◽

Reverse Transcriptase Inhibitors ◽

Nucleoside Reverse Transcriptase Inhibitors ◽

Surface Plasmonic Resonance ◽

Rt Inhibitors ◽

Hiv 1

Background: Reverse Transcriptase (RT) of immunodeficiency virus type-1 (HIV-1) remains an essential target for new antiretroviral therapies. Non-nucleoside reverse transcriptase inhibitors (or NNRTIs) constitute a major class of RT inhibitors whose characterization is essential. Introduction: Several biochemical, biological, and biophysical methods have been previously used to analyze the biological effects of NNRTIs. We explored here the use of surface plasmonic resonance to characterize the affinity of RT towards selected NNRTIs and compared the results with those obtained with in vitro and in cellulo assays. Methods: The solubility and stability in buffers of the tested NNRTIs were assessed by spectrophotometry and fluorescence. Surface plasmonic resonance experiments to study direct NNRTIs binding to immobilized RT and intramolecular quenching of RT tryptophan fluorescence were used to determine the KA association constants (= 1/KD) between RT and the inhibitors. The in vitro inhibition constants of RT were determined using kinetics and the effects on three other potential targets (proteasome, HIV-1 integrase, and HIV-1 protease) were analyzed. Results: The results obtained with two typical molecules belonging to our previous N-hydroxyureido acylnucleoside derivatives series using the above biophysical assays matched those obtained in in vitro and previous in cellulo assays. Conclusion: Surface plasmonic resonance provides reliable thermodynamic information on the interaction of RT with NNRTIs and appears as a useful method for understanding their inhibitory mechanism.

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Synthesis and biological evaluation of N-acetyl-β-aryl-1,2-didehydroethylamines as new HIV-1 RT inhibitors in vitro

Bioorganic & Medicinal Chemistry Letters ◽

10.1016/j.bmcl.2007.06.008 ◽

2007 ◽

Vol 17 (16) ◽

pp. 4476-4480 ◽

Cited By ~ 19

Author(s):

Pi Cheng ◽

Zhi-Yong Jiang ◽

Rui-Rui Wang ◽

Xue-Mei Zhang ◽

Qian Wang ◽

...

Keyword(s):

Biological Evaluation ◽

Rt Inhibitors ◽

Synthesis And Biological Evaluation ◽

Hiv 1

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The Nucleoside Analog BMS-986001 Shows GreaterIn VitroActivity against HIV-2 than against HIV-1

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01326-15 ◽

2015 ◽

Vol 59 (12) ◽

pp. 7437-7446 ◽

Cited By ~ 8

Author(s):

Robert A. Smith ◽

Dana N. Raugi ◽

Vincent H. Wu ◽

Sally S. Leong ◽

Kate M. Parker ◽

...

Keyword(s):

Reverse Transcriptase ◽

Treatment Options ◽

Nucleoside Analog ◽

Single Amino Acid ◽

Intrinsic Resistance ◽

Immunodeficiency Virus ◽

Treatment Naïve ◽

Hiv 1

ABSTRACTTreatment options for individuals infected with human immunodeficiency virus type 2 (HIV-2) are restricted by the intrinsic resistance of the virus to nonnucleoside reverse transcriptase inhibitors (NNRTIs) and the reduced susceptibility of HIV-2 to several protease inhibitors (PIs) used in antiretroviral therapy (ART). In an effort to identify new antiretrovirals for HIV-2 treatment, we evaluated thein vitroactivity of the investigational nucleoside analog BMS-986001 (2′,3′-didehydro-3′-deoxy-4′-ethynylthymidine; also known as censavudine, festinavir, OBP-601, 4′-ethynyl stavudine, or 4′-ethynyl-d4T). In single-cycle assays, BMS-986001 inhibited HIV-2 isolates from treatment-naive individuals, with 50% effective concentrations (EC50s) ranging from 30 to 81 nM. In contrast, EC50s for group M and O isolates of HIV-1 ranged from 450 to 890 nM. Across all isolates tested, the average EC50for HIV-2 was 9.5-fold lower than that for HIV-1 (64 ± 18 nM versus 610 ± 200 nM, respectively; mean ± standard deviation). BMS-986001 also exhibited full activity against HIV-2 variants whose genomes encoded the single amino acid changes K65R and Q151M in reverse transcriptase, whereas the M184V mutant was 15-fold more resistant to the drug than the parental HIV-2ROD9strain. Taken together, our findings show that BMS-986001 is an effective inhibitor of HIV-2 replication. To our knowledge, BMS-986001 is the first nucleoside analog that, when tested against a diverse collection of HIV-1 and HIV-2 isolates, exhibits more potent activity against HIV-2 than against HIV-1 in culture.

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Synthesis and Biological Evaluation of N-Acetyl-β-aryl-1,2-didehydroethylamines as New HIV-1 RT Inhibitors in vitro.

ChemInform ◽

10.1002/chin.200752071 ◽

2007 ◽

Vol 38 (52) ◽

Author(s):

Pi Cheng ◽

Zhi-Yong Jiang ◽

Rui-Rui Wang ◽

Xue-Mei Zhang ◽

Qian Wang ◽

...

Keyword(s):

Biological Evaluation ◽

Rt Inhibitors ◽

Synthesis And Biological Evaluation ◽

Hiv 1

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Synergistic Inhibition of HIV-1 Reverse Transcriptase and HIV-1 Replication by Combining Trovirdine with AZT, ddl and ddC in Vitro

Antiviral Chemistry and Chemotherapy ◽

10.1177/095632029600700501 ◽

1996 ◽

Vol 7 (5) ◽

pp. 221-229 ◽

Cited By ~ 3

Author(s):

H. Zhang ◽

L. Vrang ◽

C. Rydergård ◽

C. Åhgren ◽

B. Öberg

Keyword(s):

Reverse Transcriptase ◽

Synergistic Effects ◽

Inhibitory Effect ◽

Molar Ratio ◽

Immunodeficiency Virus ◽

A Cell ◽

Infected Cells ◽

Rt Inhibitors ◽

Hiv 1

Trovirdine (LY300046·HCI) is a potent and selective non-nucleoside human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) inhibitor (Åhgren et al., Antimicrob Ag Chemother 39: 1329, 1995). Combinations of trovirdine with other RT inhibitors, AZT, ddC., ddl and their triphosphates, were studied as well as the pyrophosphate analogue PFA in both cell-free HIV-1 polymerase assays and HIV-1-infected MT-4 cell cultures. Synergistic effects and weak synergism were observed both using RT and HIV-1 - infected cells and using different HIV-1 RT mutants and HIV-1 drug-resistant variants known to be resistant to the inhibitory effects of trovirdine. The best combination with substantial synergism was ddC-TP and trovirdine at a 20:1 molar ratio combination in a cell-free enzyme assay. This combination showed the weak synergy in MT-4 cells. Synergism was judged by the median-effect method. The inhibitory effect of trovirdine was independent of increased concentrations of AZT triphosphate and ddC triphosphate implying that trovirdine acts in a mutually exclusive manner with AZT-TP and ddC-TP as determined by the Dixon plot. The combination effects were expressed by the combination index (Cl) using end points of 50%, 70% and 90% inhibition of HIV-1 RT activity and HIV-1 replication in MT-4 cells.

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1448. Forgiveness of BIC/FTC/TAF: In Vitro Simulations of Intermittent Poor Adherence Find Limited HIV-1 Breakthrough and High Barrier to Resistance

Open Forum Infectious Diseases ◽

10.1093/ofid/ofaa439.1629 ◽

2020 ◽

Vol 7 (Supplement_1) ◽

pp. S726-S727

Author(s):

Andrew Mulato ◽

Rima K Acosta ◽

Stephen R Yant ◽

Tomas Cihlar ◽

Kirsten L White

Keyword(s):

Virologic Failure ◽

Resistance Development ◽

Drug Concentrations ◽

Suboptimal Adherence ◽

Trough Concentrations ◽

Tenofovir Alafenamide ◽

Emergence Of Resistance ◽

Perfect Adherence ◽

Hiv 1

Abstract Background Short lapses in adherence to ARVs can lead to virologic failure and emergence of resistance. Previous in vitro studies of regimen “forgiveness” simulated drug exposures of perfect adherence or short-term suboptimal adherence with bictegravir+emtricitabine+tenofovir alafenamide (BIC+FTC+TAF) and with dolutegravir and lamivudine (DTG+3TC). Here, viral breakthrough (VB) and resistance development were evaluated under alternating high and low drug exposures simulating variable adherence levels. Methods Wild-type HIV-1 (IIIb)-infected MT-2 cells were exposed to drug combinations and monitored for VB. Experiments alternated between high and low drug concentrations of either BIC+FTC+TAF or DTG+3TC (Table 1). Drug concentrations for each regimen were determined using human plasma-free adjusted clinical trough concentrations (Cmin), at simulated Cmin after missing 2 or 4 consecutive doses (Cmin-2 and Cmin-4) based on drug half-lives. Emergent HIV-1 were genotyped by deep sequencing and a 2% threshold. Results In these experiments, constant drug concentrations corresponding to full adherence (Cmin) did not lead to VB. Using Cmin concentrations for one week followed by constant Cmin-2 exposures for 4 weeks, DTG+3TC had VB and emergence of M184V/I in reverse transcriptase (RT) but there was no VB for BIC+FTC+TAF. Using alternating drug exposures of Cmin (weeks 1 and 3) and Cmin-2 or Cmin -4 (weeks 2, 4, and 5), VB was not observed with BIC+FTC+TAF, and VB was decreased or delayed with DTG+3TC compared to DTG+3TC held at Cmin-2 or Cmin-4. Resistance development was observed in some cultures with VB: 1 culture with BIC+FTC+TAF had G163R in IN and 19 cultures with DTG+3TC had INSTI and RT resistance including 10 with M184V/I. Table 1. Summary of Breakthrough Frequency and Resistance Development Conclusion BIC+FTC+TAF has high in vitro forgiveness and consistent protection against emergence of drug resistance during simulations of short lapses in adherence. Higher DTG+3TC exposure, whether constant or intermittent, was better at preventing or delaying VB than lower DTG+3TC exposures, but DTG+3TC was less forgiving than BIC+FTC+TAF. Prevention of viral replication and resistance development is necessary to maintain lifelong viral suppression, particularly in the real world where drug adherence is often imperfect. Disclosures Andrew Mulato, BS, MBA, Gilead Sciences, Inc. (Employee, Shareholder) Rima K. Acosta, BS, Gilead Sciences, Inc. (Employee, Shareholder) Stephen R. Yant, PhD, Gilead Sciences, Inc. (Employee, Shareholder) Tomas Cihlar, PhD, Gilead Sciences, Inc. (Employee, Shareholder) Kirsten L. White, PhD, Gilead Sciences, Inc. (Employee, Shareholder)

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Imidazo[1,5-b]Pyridazine-D4T Conjugates: Synthesis and Anti-Human Immunodeficiency Virus Evaluation

Antiviral Chemistry and Chemotherapy ◽

10.1177/095632029800900302 ◽

1998 ◽

Vol 9 (3) ◽

pp. 205-223 ◽

Cited By ~ 12

Author(s):

M Renoud-Grappin ◽

C Fossey ◽

G Fontaine ◽

D Ladurée ◽

AM Aubertin ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Clear Trend ◽

The Third ◽

Immunodeficiency Virus ◽

Infected Cells ◽

Rt Inhibitors ◽

Anti Hiv ◽

Hiv 1

In an attempt to combine the human immunodeficiency virus type 1 (HIV-D-inhibitory capacity of 2′,3 -dideoxy-2,3 -didehydronucleoside analogues [nucleoside reverse transcriptase (RT) inhibitors; NRTI] and non-nucleoside RT inhibitors (NNRTI), we have designed, synthesized and evaluated for their anti-HIV activity several heterodimers of the general formula [d4T]-NH-(CH2)n-NH-[imidazo[1,5–b]pyridazine]. The synthesis of these heterodimers was conducted in three parts. The first part focused on the synthesis of the NRTI. The second part was devoted to the NNRTI and the NNRTI linked to appropriate spacers; [NNRTI]-NH-(CH2)n-NH2. In the third part, the condensation between the NRTI and the [NNRTI]-NH-(CH2)n-NH2 was performed. The in vitro inhibitory activities against HIV-1 of the [d4T]-NH-(CH2)n-NH-[imidazo[1,5–b]pyridazine] heterodimers were found to be comparable to that of d4T (stavudine) in HIV-infected cells. Moreover, the heterodimers were endowed with anti-HlV-2 activity and with anti-nevirapine-resistant HIV-1 activity. None of the heterodimers proved markedly cytotoxic to CEM-SS or MT-4 cells. There was not a clear trend toward antiviral potency on lengthening the methylene spacer in the [d4T]-NH-(CH2)n-NH-[imidazo[1,5–b]pyridazine] heterodimers.

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Combination of mutations in human immunodeficiency virus type 1 reverse transcriptase required for resistance to the carbocyclic nucleoside 1592U89.

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.41.5.1094 ◽

1997 ◽

Vol 41 (5) ◽

pp. 1094-1098 ◽

Cited By ~ 144

Author(s):

M Tisdale ◽

T Alnadaf ◽

D Cousens

Keyword(s):

Human Immunodeficiency Virus ◽

Reverse Transcriptase ◽

In Vitro Selection ◽

Fold Reduction ◽

Immunodeficiency Virus ◽

Rt Inhibitors ◽

Carbocyclic Nucleoside ◽

Hiv 1

The carbocyclic nucleoside 1592U89 is a selective inhibitor of the human immunodeficiency virus (HIV), targeting the reverse transcriptase (RT). In vitro selection studies were undertaken to generate resistant variants with both HIV type 1 (HIV-1) wild-type strain HIV-1(HXB2) and 3'-azido-3'-deoxythymidine (AZT)-resistant strain HIV-1(RTMC). At least two or three mutations in RT were required to produce a 10-fold reduction in susceptibility. The first RT mutation selected was at codon 184, methionine (M) to valine (V), for HIV-1(HXB2) and HIV-1(RTMC), conferring two- and fivefold resistance, respectively. Two additional mutations were selected with HIV-1(HXB2), either leucine (L) 74 to V and lysine (K) 65 to arginine (R) (first-passage series) or L74 to V and tyrosine (Y) 115 to phenylalanine (F) (second-passage series). Cloned variants, obtained from the 1592U89 selection, were either double RT mutants 65R/184V and 74V/184V or triple RT mutant 74V/115Y/184V. Molecular clones were constructed with single, double, and triple combinations of these mutations for resistance analysis with different RT inhibitors. Each individual mutation conferred only low-level resistance (two- to fourfold) to 1592U89 in the HXB2 background. Double mutants containing the 184V mutation and triple mutants showed slightly greater levels of resistance to 1592U89 (7- to 11-fold). Some of the 1592U89-resistant variants were cross-resistant with 2',3'-dideoxycytidine, 2',3'-dideoxyinosine, and (-)-2'-deoxy-3'-thiacytidine, but none were resistant to 2',3'-didehydro-3'-deoxythymidine or AZT.

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