Rapid Screening of HIV Reverse Transcriptase and Integrase Inhibitors

JAIDS Journal of Acquired Immune Deficiency Syndromes

Pauly

Akram

et al. 2016

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Background Rilpivirine (RPV) is the latest non-nucleoside reverse transcriptase inhibitor (NNRTI) to be FDA-approved to combat HIV-1 infections. NNRTIs inhibit the chemical step in viral DNA synthesis by binding to an allosteric site located about 10 Å from the polymerase active site of reverse transcriptase (RT). Although NNRTIs potently inhibit the replication of WT HIV-1, the binding site is not conserved, and mutations arise in the binding pocket. Doravirine (DOR) is a new a NNRTI in phase III clinical trials. Methods Using a single round HIV-1 infection assay, we tested RPV and DOR against a broad panel of NNRTI resistant mutants to determine their respective activities. We also used molecular modeling to determine if the susceptibility profile of each compound was related to how they bind RT. Results Several mutants displayed decreased susceptibility to DOR. However, with the exception of E138K, our data suggest that the mutations that reduce the potency of DOR and RPV are non-overlapping. Thus, these two NNRTIs have the potential to be used together in combination therapy. We also show that the location at which DOR and RPV bind with the NNRTI binding pocket of RT correlates with the differences in their respective susceptibility to the panel of NNRTI resistance mutations. Conclusions This shows that (1) DOR is susceptible to a number of well-known NNRTI resistance mutations and (2) an understanding of the mutational susceptibilities and binding interactions of NNRTIs with RT could be used to develop pairs of compounds with non-overlapping mutational susceptibilities.

Section: Resultsmentioning

confidence: 99%

“…Virion production and single-round infectivity assays were used to determine antiviral activity (IC 50 values) of the compounds as described [19] and discussed in the supplemental information. Selection of NNRTI mutations in HIV-1 is described in supplemental information.…”

Section: Methodsmentioning

confidence: 99%

Rilpivirine and Doravirine Have Complementary Efficacies Against NNRTI-Resistant HIV-1 Mutants

JAIDS Journal of Acquired Immune Deficiency Syndromes

Pauly

Akram

et al. 2016

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“…The human osteosarcoma cell line HOS was obtained from Richard Schwartz (Michigan State University, East Lansing, MI) and grown in Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA) supplemented with 5% (vol/vol) fetal bovine serum, 5% newborn calf serum, and penicillin (50 U/ml) plus streptomycin (50 g/ml; Quality Biological, Gaithersburg, MD). The transfection vector pNLNgoMIVR Ϫ ΔLUC was made from pNLNgoMIVR Ϫ ΔEnv.HSA by removing the HSA reporter gene and replacing it with a luciferase reporter gene using the NotI and XhoI restriction sites (30,31).…”

mentioning

confidence: 99%

Developing and Evaluating Inhibitors against the RNase H Active Site of HIV-1 Reverse Transcriptase

Boyer

Zhao

et al. 2018

J Virol

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We tested three compounds for their ability to inhibit the RNase H (RH) and polymerase activities of HIV-1 reverse transcriptase (RT). A high-resolution crystal structure (2.2 Å) of one of the compounds showed that it chelates the two magnesium ions at the RH active site; this prevents the RH active site from interacting with, and cleaving, the RNA strand of an RNA-DNA heteroduplex. The compounds were tested using a variety of substrates: all three compounds inhibited the polymerase-independent RH activity of HIV-1 RT. Time-of-addition experiments showed that the compounds were more potent if they were bound to RT before the nucleic acid substrate was added. The compounds significantly inhibited the site-specific cleavage required to generate the polypurine tract (PPT) RNA primer that initiates the second strand of viral DNA synthesis. The compounds also reduced the polymerase activity of RT; this ability was a result of the compounds binding to the RH active site. These compounds appear to be relatively specific; they do not inhibit either RNase HI or human RNase H2. The compounds inhibit the replication of an HIV-1-based vector in a one-round assay, and their potencies were only modestly decreased by mutations that confer resistance to integrase strand transfer inhibitors (INSTIs), nucleoside analogs, or nonnucleoside RT inhibitors (NNRTIs), suggesting that their ability to block HIV replication is related to their ability to block RH cleavage. These compounds appear to be useful leads that can be used to develop more potent and specific compounds. Despite advances in HIV-1 treatment, drug resistance is still a problem. Of the four enzymatic activities found in HIV-1 proteins (protease, RT polymerase, RT RNase H, and integrase), only RNase H has no approved therapeutics directed against it. This new target could be used to design and develop new classes of inhibitors that would suppress the replication of the drug-resistant variants that have been selected by the current therapeutics.

“…Cell-based assays. WT and mutant HIV-based viral vectors were used in single-round infectivity assays to determine the antiviral activities (EC 50 s) of the compounds and the effects of the mutants on the EC 50 s, as previously described (34).…”

Section: Methodsmentioning

confidence: 99%

“…A modified version of the single-round infectivity assay was used to determine the replication capacities of the INSTI-resistant mutants. Briefly, 200 ng of a WT or INSTI-resistant mutant HIV-1-based vector was added to 96-well plates and incubated for 48 h, and luciferase activity was measured as was done previously (34). The luciferase activity of the WT virions was set to 100%, from which the infectivity of the mutant virions was measured as a percentage of the WT activity.…”

Section: Methodsmentioning

confidence: 99%

HIV-1 Integrase Inhibitors That Are Broadly Effective against Drug-Resistant Mutants

Antimicrob Agents Chemother

Zhao

Burke

et al. 2018

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Integrase strand transfer inhibitors (INSTIs) have emerged as clinically effective therapeutics that inhibit HIV-1 replication by blocking the strand transfer reaction catalyzed by HIV-1 integrase (IN). Of the three FDA-approved INSTIs, dolutegravir (DTG) is the least apt to select for resistance. However, recent salvage therapy regimens had low response rates with therapies that included DTG, suggesting that DTG resistance can be selected in patients. Using a single-round infection assay, we evaluated a collection of our best inhibitors and DTG against a broad panel of INSTI-resistant mutants. Two of the new compounds, 4c and 4d, had antiviral profiles against the mutants we tested superior to that of DTG. The susceptibility profiles of 4c and 4d suggest that the compounds are candidates for development as INSTIs. Modeling the binding of 4d to HIV-1 IN reinforced the significance of mimicking the DNA substrate in developing compounds that are broadly effective in their abilities to inhibit HIV-1 INs with mutations in the active site.