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Structural and pharmacological evaluation of a novel non-nucleoside reverse transcriptase inhibitor as a promising long acting nanoformulation for treating HIV.

ABSTRACT: Combination antiretroviral therapy (cART) has been proven effective in inhibiting human immunodeficiency virus type 1 (HIV-1) infection and has significantly improved the health outcomes in acquired immune deficiency syndrome (AIDS) patients. The therapeutic benefits of cART have been challenged because of the toxicity and emergence of drug-resistant HIV-1 strains along with lifelong patient compliance resulting in non-adherence. These issues also hinder the clinical benefits of non-nucleoside reverse transcriptase inhibitors (NNRTIs), which are one of the vital components of cART for the treatment of HIV-1 infection. In this study, using a computational and structural based drug design approach, we have discovered an effective HIV -1 NNRTI, compound I (Cmpd I) that is very potent in biochemical assays and which targets key residues in the allosteric binding pocket of wild-type (WT)-RT as revealed by structural studies. Furthermore, Cmpd I exhibited very potent antiviral activity in HIV-1 infected T cells, lacked cytotoxicity (therapeutic index >100,000), and no significant off-target effects were noted in pharmacological assays. To address the issue of non-adherence, we developed a long-acting nanoformulation of Cmpd I (Cmpd I-NP) using poly (lactide-coglycolide) (PLGA) particles. The pharmacokinetic studies of free and nanoformulated Cmpd I were carried out in BALB/c mice. Intraperitoneal administration of Cmpd I and Cmpd I-NP in BALB/c mice revealed prolonged serum residence time of 48?h and 30 days, respectively. The observed serum concentrations of Cmpd I in both cases were sufficient to provide >97% inhibition in HIV-1 infected T-cells. The significant antiviral activity along with favorable pharmacological and pharmacokinetic profile of Cmpd I, provide compelling and critical support for its further development as an anti-HIV therapeutic agent.

SUBMITTER: Kudalkar SN

PROVIDER: S-EPMC6554724 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

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Structural and pharmacological evaluation of a novel non-nucleoside reverse transcriptase inhibitor as a promising long acting nanoformulation for treating HIV.

Kudalkar Shalley N SN Ullah Irfan I Bertoletti Nicole N Mandl Hanna K HK Cisneros José A JA Beloor Jagadish J Chan Albert H AH Quijano Elias E Saltzman W Mark WM Jorgensen William L WL Kumar Priti P Anderson Karen S KS

Antiviral research 20190426

Combination antiretroviral therapy (cART) has been proven effective in inhibiting human immunodeficiency virus type 1 (HIV-1) infection and has significantly improved the health outcomes in acquired immune deficiency syndrome (AIDS) patients. The therapeutic benefits of cART have been challenged because of the toxicity and emergence of drug-resistant HIV-1 strains along with lifelong patient compliance resulting in non-adherence. These issues also hinder the clinical benefits of non-nucleoside r ...[more]

PMID: 31034849

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Project description:Background & aimsWhile certain nucleos(t)ide reverse transcriptase inhibitors (NRTIs) are efficacious in treating HBV infection, their effects are yet to be optimized and the emergence of NRTI-resistant HBV variants is an issue because of the requirement for lifelong treatment. The development of agents that more profoundly suppress wild-type and drug-resistant HBVs, and that have a long-acting effect, are crucial to improve patient outcomes.MethodsHerein, we synthesized a novel long-acting 4'-modified NRTI termed E-CFCP. We tested its anti-HBV activity in vitro, before evaluating its anti-HBV activity in HBV-infected human-liver-chimeric mice (PXB-mice). E-CFCP's long-acting features and E-CFCP-triphosphate's interactions with the HBV reverse transcriptase (HBV-RT) were examined.ResultsE-CFCP potently blocked HBVWTD1 production (IC50qPCR_cell=1.8 nM) in HepG2.2.15 cells and HBVWTC2 (IC50SB_cell=0.7 nM), entecavir (ETV)-resistant HBVETV-RL180M/S202G/M204V (IC50SB_cell=77.5 nM), and adefovir-resistant HBVADV-RA181T/N236T production (IC50SB_cell=14.1 nM) in Huh7 cells. E-CFCP profoundly inhibited intracellular HBV DNA production to below the detection limit, but ETV and tenofovir alafenamide (TAF) failed to do so. E-CFCP also showed less toxicity than ETV and TAF. E-CFCP better penetrated hepatocytes and was better tri-phosphorylated; E-CFCP-triphosphate persisted intracellularly for longer than ETV-triphosphate. Once-daily peroral E-CFCP administration over 2 weeks (0.02~0.2 mg/kg/day) reduced HBVWTC2-viremia by 2-3 logs in PXB-mice without significant toxicities and the reduction persisted over 1-3 weeks following treatment cessation, suggesting once-weekly dosing capabilities. E-CFCP also reduced HBVETV-RL180M/S202G/M204V-viremia by 2 logs over 2 weeks, while ETV completely failed to reduce HBVETV-RL180M/S202G/M204V-viremia. E-CFCP's 4'-cyano and fluorine interact with both HBVWT-RT and HBVETV-RL180M/S202G-M204 -RT via Van der Waals and polar forces, being important for E-CFCP-triphosphate's interactions and anti-HBV potency.ConclusionE-CFCP represents the first reported potential long-acting NRTI with potent activity against wild-type and treatment-resistant HBV.Lay summaryAlthough there are currently effective treatment options for HBV, treatment-resistant variants and the need for lifelong therapy pose a significant challenge. Therefore, the development of new treatment options is crucial to improve outcomes and quality of life. Herein, we report preclinical evidence showing that the anti-HBV agent, E-CFCP, has potent activity against wild-type and treatment-resistant variants. In addition, once-weekly oral dosing may be possible, which is preferrable to the current daily dosing regimens.

Project description:Equilibrative nucleoside transporters (ENTs) transport nucleosides across the blood-testis barrier (BTB). ENTs are of interest to study the disposition of nucleoside reverse-transcriptase inhibitors (NRTIs) in the human male genital tract because of their similarity in structure to nucleosides. HeLa S3 cells express ENT1 and ENT2 and were used to compare relative interactions of these transporters with selected NRTIs. Inhibition of [3H]uridine uptake by NBMPR was biphasic, with IC50 values of 11.3 nM for ENT1 and 9.6 μM for ENT2. Uptake measured with 100 nM NBMPR represented ENT2-mediated transport; subtracting that from total uptake represented ENT1-mediated transport. The kinetics of ENT1- and ENT2-mediated [3H]uridine uptake revealed no difference in Jmax (16.53 and 30.40 pmol cm-2 min-1) and an eightfold difference in Kt (13.6 and 108.9 μM). The resulting fivefold difference in intrinsic clearance (Jmax/Kt) for ENT1- and ENT2 transport accounted for observed inhibition of [3H]uridine uptake by 100 nM NBMPR. Millimolar concentrations of the NRTIs emtricitabine, didanosine, lamivudine, stavudine, tenofovir disoproxil, and zalcitabine had no effect on ENT transport activity, whereas abacavir, entecavir, and zidovudine inhibited both transporters with IC50 values of ∼200 µM, 2.5 mM, and 2 mM, respectively. Using liquid chromatography-tandem mass spectrometry and [3H] compounds, the data suggest that entecavir is an ENT substrate, abacavir is an ENT inhibitor, and zidovudine uptake is carrier-mediated, although not an ENT substrate. These data show that HeLa S3 cells can be used to explore complex transporter selectivity and are an adequate model for studying ENTs present at the BTB. SIGNIFICANCE STATEMENT: This study characterizes an in vitro model using S-[(4-nitrophenyl)methyl]-6-thioinosine to differentiate between equilibrative nucleoside transporter (ENT) 1- and ENT2-mediated uridine transport in HeLa cells. This provides a method to assess the influence of nucleoside reverse-transcriptase inhibitors on natively expressed transporter function. Determining substrate selectivity of the ENTs in HeLa cells can be effectively translated into the activity of these transporters in Sertoli cells that comprise the blood-testis barrier, thereby assisting targeted drug development of compounds capable of circumventing the blood-testis barrier.

Project description:ObjectivesTwo nucleos(t)ide reverse-transcriptase inhibitors (NRTIs) plus 1 non-NRTI (nNRTI) remain the preferred or alternative combination antiretroviral therapy (cART) for antiretroviral-naive HIV-positive patients in Taiwan. The three most commonly used nNRTIs are nevirapine (NVP), efavirenz (EFV) and rilpivirine (RPV). This study aimed to determine the incidences of hepatotoxicity and skin rashes within 4 weeks of initiation of cART containing 1 nNRTI plus 2 NRTIs.MethodsBetween June, 2012 and November, 2015, all antiretroviral-naive HIV-positive adult patients initiating nNRTI-containing cART at 8 designated hospitals for HIV care were included in this retrospective observational study. According to the national HIV treatment guidelines, patients were assessed at baseline, 2 and 4 weeks of cART initiation, and subsequently every 8 to 12 weeks. Plasma HIV RNA load, CD4 cell count and aminotransferases were determined. The toxicity grading scale of the Division of AIDS (DAIDS) 2014 was used for reporting clinical and laboratory adverse events.ResultsDuring the 3.5-year study period, 2,341 patients initiated nNRTI-containing cART: NVP in 629 patients, EFV 1,363 patients, and RPV 349 patients. Rash of any grade occurred in 14.1% (n = 331) of the patients. In multiple logistic regression analysis, baseline CD4 cell counts (per 100-cell/μl increase, adjusted odds ratio [AOR], 1.125; 95% confidence interval [95% CI], 1.031-1.228) and use of NVP (AOR, 2.443; 95% CI, 1.816-3.286) (compared with efavirenz) were independently associated with the development of skin rashes. Among the 1,455 patients (62.2%) with aminotransferase data both at baseline and week 4, 72 (4.9%) developed grade 2 or greater hepatotoxicity. In multiple logistic regression analysis, presence of antibody for hepatitis C virus (HCV) (AOR, 2.865; 95% CI, 1.439-5.704) or hepatitis B surface antigen (AOR, 2.397; 95% CI, 1.150-4.997), and development of skin rashes (AOR, 2.811; 95% CI, 1.051-7.521) were independently associated with the development of hepatotoxicity.ConclusionsThe baseline CD4 cell counts and use of NVP were associated with increased risk of skin rashes, while hepatotoxicity was independently associated with HCV or hepatitis B virus coinfection, and development of skin rashes in antiretroviral-naïve HIV-positive Taiwanese patients within 4 weeks of initiation of nNRTI-containing regimens.

Project description:Reverse transcriptase (RT) is the target for the majority of anti-HIV-1 drugs. As with all anti-AIDS treatments, continued success of RT inhibitors is persistently disrupted by the occurrence of resistance mutations. To explore latent resistance mechanisms potentially accessible to therapeutically challenged HIV-1 viruses, we examined RT from the related feline immunodeficiency virus (FIV). FIV closely parallels HIV-1 in its replication and pathogenicity, however, is resistant to all non-nucleoside inhibitors (NNRTI). The intrinsic resistance of FIV RT is particularly interesting since FIV harbors the Y181 and Y188 sensitivity residues absent in both HIV-2 and SIV. Unlike RT from HIV-2 or SIV, previous efforts have failed to make FIV RT susceptible to NNRTIs concluding that the structure or flexibility of the feline enzyme must be profoundly different. We report the first crystal structure of FIV RT and, being the first structure of an RT from a non-primate lentivirus, enrich the structural and species repertoires available for RT. The structure demonstrates that while the NNRTI binding pocket is conserved, minor subtleties at the entryway can render the FIV RT pocket more restricted and unfavorable for effective NNRTI binding. Measuring NNRTI binding affinity to FIV RT shows that the "closed" pocket configuration inhibits NNRTI binding. Mutating the loop residues rimming the entryway of FIV RT pocket allows for NNRTI binding, however, it does not confer sensitivity to these inhibitors. This reveals a further layer of resistance caused by inherent FIV RT variances that could have enhanced the dissociation of bound inhibitors, or, perhaps, modulated protein plasticity to overcome inhibitory effects of bound NNRTIs. The more "closed" conformation of FIV RT pocket can provide a template for the development of innovative drugs that could unlock the constrained pocket, and the resilient mutant version of the enzyme can offer a fresh model for the study of NNRTI-resistance mechanisms overlooked in HIV-1.

Dataset Information

Structural and pharmacological evaluation of a novel non-nucleoside reverse transcriptase inhibitor as a promising long acting nanoformulation for treating HIV.

Publications

Structural and pharmacological evaluation of a novel non-nucleoside reverse transcriptase inhibitor as a promising long acting nanoformulation for treating HIV.

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