Susan P. Colby-Germinario

In Vitro Resistance Profile of the Candidate HIV-1 Microbicide Drug Dapivirine

ABSTRACT Antiretroviral-based microbicides may offer a means to reduce the sexual transmission of HIV-1. Suboptimal use of a microbicide may, however, lead to the development of drug resistance in users that are already, or become, infected with HIV-1. In such cases, the efficacy of treatments may be compromised since the same (or similar) antiretrovirals used in treatments are being developed as microbicides. To help predict which drug resistance mutations may develop in the context of suboptimal use, HIV-1 primary isolates of different subtypes and different baseline resistance profiles were used to infect primary cells in vitro in the presence of increasing suboptimal concentrations of the two candidate microbicide antiretrovirals dapivirine (DAP) and tenofovir (TFV) alone or in combination. Infections were ongoing for 25 weeks, after which reverse transcriptase genotypes were determined and scrutinized for the presence of any clinically recognized reverse transcriptase drug resistance mutations. Results indicated that suboptimal concentrations of DAP alone facilitated the emergence of common nonnucleoside reverse transcriptase inhibitor resistance mutations, while suboptimal concentrations of DAP plus TFV gave rise to fewer mutations. Suboptimal concentrations of TFV alone did not frequently result in the development of resistance mutations. Sensitivity evaluations for stavudine (d4T), nevirapine (NVP), and lamivudine (3TC) revealed that the selection of resistance as a consequence of suboptimal concentrations of DAP may compromise the potential for NVP to be used in treatment, a finding of potential relevance in developing countries.

Effect of Mutations at Position E138 in HIV-1 Reverse Transcriptase and Their Interactions with the M184I Mutation on Defining Patterns of Resistance to Nonnucleoside Reverse Transcriptase Inhibitors Rilpivirine and Etravirine

ABSTRACT Impacts of mutations at position E138 (A/G/K/Q/R/V) alone or in combination with M184I in HIV-1 reverse transcriptase (RT) were investigated. We also determined why E138K is the most prevalent nonnucleoside reverse transcriptase inhibitor mutation in patients failing rilpivirine (RPV) therapy. Recombinant RT enzymes and viruses containing each of the above-mentioned mutations were generated, and drug susceptibility was assayed. Each of the E138A/G/K/Q/R mutations, alone or in combination with M184I, resulted in decreased susceptibility to RPV and etravirine (ETR). The maximum decrease in susceptibility to RPV was observed for E138/R/Q/G by both recombinant RT assay and cell-based assays. E138Q/R-containing enzymes and viruses also showed the most marked decrease in susceptibility to ETR by both assays. The addition of M184I to the E138 mutations did not significantly change the levels of diminution in drug susceptibility. These findings indicate that E138R caused the highest level of loss of susceptibility to both RPV and ETR, and, accordingly, E138R should be recognized as an ETR resistance-associated mutation. The E138K/Q/R mutations can compensate for M184I in regard to both enzymatic fitness and viral replication capacity. The favored emergence of E138K over other mutations at position E138, together with M184I, is not due to an advantage in either the level of drug resistance or viral replication capacity but may reflect the fact that E138R and E138Q require two distinct mutations to occur, one of which is a disfavorable G-to-C mutation, whereas E138K requires only a single favorable G-to-A hypermutation. Of course, other factors may also affect the concept of barrier to resistance.

Maraviroc and Other HIV-1 Entry Inhibitors Exhibit a Class-Specific Redistribution Effect That Results in Increased Extracellular Viral Load

ABSTRACT HIV entry inhibitors, such as maraviroc (MVC), prevent cell-free viruses from entering the cells. In clinical trials, patients who were treated with MVC often displayed viral loads that were above the limit of conventional viral load detection compared to efavirenz-based regimens. We hypothesize that viruses blocked by entry inhibitors may be redistributed to plasma, where they artificially increase viral load measurements compared to those with the use of antiretroviral drugs (ARVs) that act intracellularly. We infected PM-1 cells with CCR5-tropic HIV-1 BaL or CXCR4-tropic HIV-1 NL4-3 in the presence of inhibitory concentrations of efavirenz, raltegravir, enfuvirtide, maraviroc, and AMD3100, the latter three being entry inhibitors. Supernatant viral load, reverse transcriptase enzyme activity, and intracellular nucleic acid levels were measured at times up to 24 h postinfection. Infectivity of redistributed dual-tropic HIV-1 was assessed using TZM-bl cells. Extracellular viral load analysis revealed that entry inhibitor-treated cells had higher levels of virus in the supernatant than the cells treated with other ARVs at 8 h postinfection. By 24 h, the supernatant viral load was still higher for entry inhibitors than other ARVs. We observed a correlation between viral load and the step of entry inhibition. Dual-tropic virus infectivity was undiminished utilizing the CCR5 coreceptor following redistribution by CXCR4 entry inhibition. This in vitro model indicates that entry inhibitors exhibit a redistribution effect unseen with intracellular ARV drugs. Based on these results, the effectiveness of some entry inhibitors may be underestimated if plasma viral load is used as a sole indicator of clinical success.

HIV gp120 H375 Is Unique to HIV-1 Subtype CRF01_AE and Confers Strong Resistance to the Entry Inhibitor BMS-599793, a Candidate Microbicide Drug

ABSTRACT BMS-599793 is a small molecule entry inhibitor that binds to human immunodeficiency virus type 1 (HIV-1) gp120, resulting in the inhibition of CD4-dependent entry into cells. Since BMS-599793 is currently considered a candidate microbicide drug, we evaluated its efficacy against a number of primary patient HIV isolates from different subtypes and circulating recombinant forms (CRFs) and showed that activity varied between ∼3 ρM and 7 μM at 50% effective concentrations (EC50s). Interestingly, CRF01_AE HIV-1 isolates consistently demonstrated natural resistance against this compound. Genotypic analysis of >1,600 sequences (Los Alamos HIV sequence database) indicated that a single amino acid polymorphism in Env, H375, may account for the observed BMS-599793 resistance in CRF01_AE HIV-1. Results of site-directed mutagenesis experiments confirmed this hypothesis, and in silico drug docking simulations identified a drug resistance mechanism at the molecular level. In addition, CRF01_AE viruses were shown to be resistant to multiple broadly neutralizing monoclonal antibodies. Thus, our results not only provide insight into how Env polymorphisms may contribute to entry inhibitor resistance but also may help to elucidate how HIV can evade some broadly neutralizing antibodies. Furthermore, the high frequency of H375 in CRF01_AE HIV-1, and its apparent nonoccurrence in other subtypes, could serve as a means for rapid identification of CRF01_AE infections.

Purification of Zika virus RNA-dependent RNA polymerase and its use to identify small-molecule Zika inhibitors

Background: The viral RNA-dependent RNA polymerase (RdRp) enzymes of the Flaviviridae family are essential for viral replication and are logically important targets for development of antiviral therapeutic agents. Zika virus (ZIKV) is a rapidly re-emerging human pathogen for which no vaccine or antiviral agent is currently available. Methods: To facilitate development of ZIKV RdRp inhibitors, we have established an RdRp assay using purified recombinant ZIKV NS5 polymerase. Results: We have shown that both the hepatitis C virus (HCV) nucleoside inhibitor sofosbuvir triphosphate and a pyridoxine-derived non-nucleoside small-molecule inhibitor, DMB213, can act against ZIKV RdRp activity at IC50s of 7.3 and 5.2 &mgr;M, respectively, in RNA synthesis reactions catalysed by recombinant ZIKV NS5 polymerase. Cell-based assays confirmed the anti-ZIKV activity of sofosbuvir and DMB213 with 50% effective concentrations (EC50s) of 8.3 and 4.6 &mgr;M, respectively. Control studies showed that DMB213 did not inhibit recombinant HIV-1 reverse transcriptase and showed only very weak inhibition of HIV-1 integrase strand-transfer activity. The S604T substitution in motif B of the ZIKV RdRp, which corresponds to the S282T substitution in motif B of HCV RdRp, which confers resistance to nucleotide inhibitors, also conferred resistance to sofosbuvir triphosphate, but not to DMB213. Enzyme assays showed that DMB213 appears to be competitive with natural nucleoside triphosphate (NTP) substrates. Conclusions: Recombinant ZIKV RdRp assays can be useful tools for the screening of both nucleos(t)ide compounds and non-nucleotide metal ion-chelating agents that interfere with ZIKV replication.

Identification of a Pyridoxine-Derived Small-Molecule Inhibitor Targeting Dengue Virus RNA-Dependent RNA Polymerase

ABSTRACT The viral RNA-dependent RNA polymerase (RdRp) activity of the dengue virus (DENV) NS5 protein is an attractive target for drug design. Here, we report the identification of a novel class of inhibitor (i.e., an active-site metal ion chelator) that acts against DENV RdRp activity. DENV RdRp utilizes a two-metal-ion mechanism of catalysis; therefore, we constructed a small library of compounds, through mechanism-based drug design, aimed at chelating divalent metal ions in the catalytic site of DENV RdRp. We now describe a pyridoxine-derived small-molecule inhibitor that targets DENV RdRp and show that 5-benzenesulfonylmethyl-3-hydroxy-4-hydroxymethyl-pyridine-2-carboxylic acid hydroxyamide (termed DMB220) inhibited the RdRp activity of DENV serotypes 1 to 4 at low micromolar 50% inhibitory concentrations (IC50s of 5 to 6.7 μM) in an enzymatic assay. The antiviral activity of DMB220 against DENV infection was also verified in a cell-based assay and showed a 50% effective concentration (EC50) of <3 μM. Enzyme assays proved that DMB220 was competitive with nucleotide incorporation. DMB220 did not inhibit the enzymatic activity of recombinant HIV-1 reverse transcriptase and showed only weak inhibition of HIV-1 integrase strand transfer activity, indicating high specificity for DENV RdRp. S600T substitution in the DENV RdRp, which was previously shown to confer resistance to nucleoside analogue inhibitors (NI), conferred 3-fold hypersusceptibility to DMB220, and enzymatic analyses showed that this hypersusceptibility may arise from the decreased binding/incorporation efficiency of the natural NTP substrate without significantly impacting inhibitor binding. Thus, metal ion chelation at the active site of DENV RdRp represents a viable anti-DENV strategy, and DMB220 is the first of a new class of DENV inhibitor.

The Connection Domain Mutation N348I in HIV-1 Reverse Transcriptase Enhances Resistance to Etravirine and Rilpivirine but Restricts the Emergence of the E138K Resistance Mutation by Diminishing Viral Replication Capacity

ABSTRACT Clinical resistance to rilpivirine (RPV), a novel nonnucleoside reverse transcriptase (RT) inhibitor (NNRTI), is associated an E-to-K mutation at position 138 (E138K) in RT together with an M184I/V mutation that confers resistance against emtricitabine (FTC), a nucleoside RT inhibitor (NRTI) that is given together with RPV in therapy. These two mutations can compensate for each other in regard to fitness deficits conferred by each mutation alone, raising the question of why E138K did not arise spontaneously in the clinic following lamivudine (3TC) use, which also selects for the M184I/V mutations. In this context, we have investigated the role of a N348I connection domain mutation that is prevalent in treatment-experienced patients. N348I confers resistance to both the NRTI zidovudine (ZDV) and the NNRTI nevirapine (NVP) and was also found to be associated with M184V and to compensate for deficits associated with the latter mutation. Now, we show that both N348I alone and N348I/M184V can prevent or delay the emergence of E138K under pressure with RPV or a related NNRTI, termed etravirine (ETR). N348I also enhanced levels of resistance conferred by E138K against RPV and ETR by 2.2- and 2.3-fold, respectively. The presence of the N348I or M184V/N348I mutation decreased the replication capacity of E138K virus, and biochemical assays confirmed that N348I, in a background of E138K, impaired RT catalytic efficiency and RNase H activity. These findings help to explain the low viral replication capacity of viruses containing the E138K/N348I mutations and how N348I delayed or prevented the emergence of E138K in patients with M184V-containing viruses.

Complete inactivation of HIV-1 using photo-labeled non-nucleoside reverse transcriptase inhibitors

We demonstrate that a photo-labeled derivative of the non-nucleoside reverse transcriptase inhibitor (NNRTI) dapivirine termed DAPY, when used together with exposure to ultraviolet light, was able to completely and irreversibly inactivate both HIV-1 RT activity as well as infectiousness in each of a T cell line and peripheral blood mononuclear cells. Control experiments using various concentrations of DAPY revealed that a combination of exposure to ultraviolet light together with use of the specific, high affinity photo-labeled compound was necessary for complete inactivation to occur. This method of HIV RT inactivation may have applicability toward preservation of an intact viral structure and warrants further investigation in regard to the potential of this approach to elicit a durable, broad protective immune response.

Role of the K101E Substitution in HIV-1 Reverse Transcriptase in Resistance to Rilpivirine and Other Nonnucleoside Reverse Transcriptase Inhibitors

ABSTRACT Resistance to the recently approved nonnucleoside reverse transcriptase inhibitor (NNRTI) rilpivirine (RPV) commonly involves substitutions at positions E138K and K101E in HIV-1 reverse transcriptase (RT), together with an M184I substitution that is associated with resistance to coutilized emtricitabine (FTC). Previous biochemical and virological studies have shown that compensatory interactions between substitutions E138K and M184I can restore enzyme processivity and the viral replication capacity. Structural modeling studies have also shown that disruption of the salt bridge between K101 and E138 can affect RPV binding. The current study was designed to investigate the impact of K101E, alone or in combination with E138K and/or M184I, on drug susceptibility, viral replication capacity, and enzyme function. We show here that K101E can be selected in cell culture by the NNRTIs etravirine (ETR), efavirenz (EFV), and dapivirine (DPV) as well as by RPV. Recombinant RT enzymes and viruses containing K101E, but not E138K, were highly resistant to nevirapine (NVP) and delavirdine (DLV) as well as ETR and RPV, but not EFV. The addition of K101E to E138K slightly enhanced ETR and RPV resistance compared to that obtained with E138K alone but restored susceptibility to NVP and DLV. The K101E substitution can compensate for deficits in viral replication capacity and enzyme processivity associated with M184I, while M184I can compensate for the diminished efficiency of DNA polymerization associated with K101E. The coexistence of K101E and E138K does not impair either viral replication or enzyme fitness. We conclude that K101E can play a significant role in resistance to RPV.

Identification of a dibenzocyclooctadiene lignan as a HIV-1 non-nucleoside reverse transcriptase inhibitor:

Background Due to resistance to all classes of anti-HIV drugs and drug toxicity, there is a need for the discovery and development of new anti-HIV drugs. Methods HIV-1 inhibitors were identified and biologically characterized for mechanism of action. Results We identified a dibenzocyclooctadiene lignan, termed HDS2 that possessed anti-HIV activity against a wide variety of viral strains with EC50 values in the 1–3 µM range. HDS2 was shown to act as an NNRTI by qPCR and in vitro enzyme assays. Conclusions This compound provides a new scaffold for further optimization of activity through structure-guided design.