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Tenofovir alafenamide (TAF) is a new drug from the nucleotide reverse transcriptase inhibitor group approved for the treatment of chronic Hepatitis B in 2016. With this study, we aimed to test whether possible cellular toxicity can be reduced by controlled drug release as a result of loading with chitosan nanoparticles (CHS). We investigated the genotoxic and mitotoxic effects of 45 µM TAF-loaded CHS and TAF-only on HepG2 cells by micronucleus (MN), comet assay, determination of mtDNA quantification, mitochondrial membrane potential (ΔΨm), and ROS levels. Additionally, we compared the samples by RNAseq analyses to reveal the transcriptional responses to each regimen. In terms of genotoxic tests, although MN and comet were found higher in all experimental treatment conditions, the encapsulation of CHS reduced the genotoxicity of TAF. MtDNA level was found to be lower in the TAF treatment, whereas it was higher in CHS and CHS-TAF treatments. The TAF-loaded CHS and TAF treatments had an impaired ΔΨm value. Cellular ROS levels were higher in all treatment conditions. According to the analyses of gene expression patterns; CHS-only changed the expression of relatively few genes (187 genes), while TAF changed the expression of the 1974 genes and TAF-loaded CHS changed the expression of 734 genes. Considering the gene expression numbers, CHS encapsulation of TAF significantly reduced the number of genes that were differentially expressed by TAF-only. Overall, we observed that TAF has genotoxic and mitotoxic effects on HepG2 cells, and upon encapsulation with CHS, its genotoxic and mitotoxic effects were decreased.

This study synthesized and evaluated a series of benzotriazole derivatives denoted 3(a-j) and 6(a-j) for their anti-HIV-1 RT activities compared to the standard drug efavirenz. Notably, compound 3 h, followed closely by 6 h, exhibited significant anti-HIV-1 RT efficacy relative to the standard drug. In vivo oral toxicity studies were conducted for the most active compound 3 h, confirming its nontoxic nature to ascertain the safety profile. By employing molecular docking techniques, we explored the potential interactions between the synthesized compounds (ligands) and a target biomolecule (protein)(PDB ID 1RT2) at the molecular level. We undertook the molecular dynamics study of 3 h, the most active compound, within the active binding pocket of the cocrystallized structure of HIV-1 RT (PDB ID 1RT2). We aimed to learn more about how biomolecular systems behave, interact, and change at the atomic or molecular level over time. Finally, the DFT-derived HOMO and LUMO orbitals, as well as analysis of the molecular electrostatic potential map, aid in discerning the reactivity characteristics of our molecule., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Objective: This study aimed to determine the HIV-1 subtype distribution and HIV drug resistance (HIVDR) in patients with ART failure from 2014 to 2020 in Hainan, China., Methods: A 7-year cross-sectional study was conducted among HIV/AIDS patients with ART failure in Hainan. We used online subtyping tools and the maximum likelihood phylogenetic tree to confirm the HIV subtypes with pol sequences. Drug resistance mutations (DRMs) were analyzed using the Stanford University HIV Drug Resistance Database., Results: A total of 307 HIV-infected patients with ART failure were included, and 241 available pol sequences were obtained. Among 241 patients, CRF01_AE accounted for 68.88%, followed by CRF07_BC (17.00%) and eight other subtypes (14.12%). The overall prevalence of HIVDR was 61.41%, and the HIVDR against non-nucleoside reverse transcriptase inhibitors (NNRTIs), nucleotide reverse transcriptase inhibitors (NRTIs), and protease inhibitors (PIs) were 59.75%, 45.64%, and 2.49%, respectively. Unemployed patients, hypoimmunity or opportunistic infections in individuals, and samples from 2017 to 2020 increased the odd ratios of HIVDR. Also, HIVDR was less likely to affect female patients. The common DRMs to NNRTIs were K103N (21.99%) and Y181C (20.33%), and M184V (28.21%) and K65R (19.09%) were the main DRMs against NRTIs., Conclusion: The present study highlights the HIV-1 subtype diversity in Hainan and the importance of HIVDR surveillance over a long period., (Copyright © 2023 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.)

In the past, one of the most widely used non-nucleoside reverse transcriptase inhibitors (NNRTI) in first-line antiretroviral therapy (ART) of HIV infection was efavirenz (EFV), which is already used as a cost-effective treatment in developing countries due to its efficacy, tolerability, and availability. However, EFV also demonstrates several adverse effects, like hepatotoxicity, altered lipid profile, neuropsychological symptoms, and behavioral effects in children after in utero exposure. In 2018, another NNRTI, doravirine (DOR), was approved due to its similar efficacy but better safety profile. Preclinical safety studies demonstrated that DOR is not genotoxic and exhibits no developmental toxicity or effects on fertility in rats. Zebrafish ( Danio rerio ) embryos have been widely accepted as a vertebrate model for pharmacological and developmental studies. We used zebrafish embryos as an in vivo model to investigate the developmental toxicity of DOR compared to EFV. After exposure of the embryos to the drugs from the gastrula stage up to different developmental stages (30 embryos for each arm, in three independent experiments), we assessed their survival, morphology, hatching rate, apoptosis in the developing head, locomotion behavior, vasculature development, and neutral lipid distribution. Overall, DOR showed a better safety profile than EFV in our model. Therapeutic and supra-therapeutic doses of DOR induced very low mortality [survival rates: 92, 90, 88, 88, and 81% at 1, 5, 10, 25, and 50 μM, respectively, at 24 h post fecundation (hpf), and 88, 85, 88, 89, and 75% at the same doses, respectively, at 48 hpf] and mild morphological alterations compared to EFV exposure also in the sub-therapeutic ranges (survival rates: 80, 77, 69, 63, and 44% at 1, 5, 10, 25, and 50 μM, respectively, at 24 hpf and 72, 70, 63, 52, and 0% at the same doses, respectively, at 48 hpf). Further, DOR only slightly affected the hatching rate at supra-therapeutic doses (97, 98, 96, 87, and 83% at 1, 5, 10, 25, and 50 μM, respectively, at 72 hpf), while EFV already strongly reduced hatching at sub-therapeutic doses (83, 49, 11, 0, and 0% at 1, 5, 10, 25, and 50 μM, respectively, at the same time endpoint). Both DOR at therapeutic doses and most severely EFV at sub-therapeutic doses enhanced apoptosis in the developing head during crucial phases of embryo neurodevelopment and perturbed the locomotor behavior. Furthermore, EFV strongly affected angiogenesis and disturbed neutral lipid homeostasis even at sub-therapeutic doses compared to DOR at therapeutic concentrations. Our findings in zebrafish embryos add further data confirming the higher safety of DOR with respect to EFV regarding embryo development, neurogenesis, angiogenesis, and lipid metabolism. Further studies are needed to explore the molecular mechanisms underlying the better pharmacological safety profile of DOR, and further human studies are required to confirm these results in the zebrafish animal model.

Abstract: The two non-nucleoside reverse transcriptase inhibitors (NNRTIs), efavirenz (EFV) and nevirapine (NVP), are currently the core antiretroviral drugs for treatment of HIV in sub-Saharan Africa including Botswana. The drugs are metabolized by Cytochrome P450 2B6 (CYP2B6) liver enzyme. The CYP2B6 gene that encodes for metabolism of these drugs is known to be highly polymorphic. One of the polymorphism in the CYP2B6 gene, 516G>T, particularly the 516T allele, is known to confer poor metabolism of EFV and NVP. This may lead to high levels of plasma drug concentrations and development of treatment toxicities, like central nervous system toxicities, and cutaneous and hepatic toxicities, for EFV and NVP, respectively. The CYP2B6 516G allele on the other hand is associated with an extensive metabolism of the two NNRTIs drugs. We sought to establish association between possible developments of NNRTIs toxicities with CYP2B6 516G>T variation in Botswana.A total of 316 peripheral blood mononuclear cells samples were used in a retrospective view. All the samples were from participants on EFV/NVP-containing regimen with known toxicity output. TaqMan Real-Time PCR approach was applied for assessing CYP2B6 516 allele variation in cases with treatment toxicity and those without. Analysis was performed by chi-square statistics and logistic regression analysis.The rate of poor metabolizers among participants with toxicity and those without toxicity was 18.4% and 15.1%, respectively. The CYP2B6 516 genotype distribution comparisons between the participants with toxicity and those without were not statistically different (chi-square = .326; P = .568).CYP2B6 516 variation was not associated with NNRTI toxicity. No other factors were associated with toxicity when considering age, baseline body mass index, baseline CD4, baseline HIV viral load and adherence. The results were discussed in the context of all the studies done in Botswana to date., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2022 the Author(s). Published by Wolters Kluwer Health, Inc.)

Efavirenz (EFV) is a non-nucleoside reverse transcriptase inhibitor (NNRTI), which is widely used for anti-HIV-1. Evidences revealed that several central nervous system side effects could be observed in mice and patients with administration of EFV. However, the detailed mechanisms are still unknown. In this study, we investigated the effects of long-term EFV treatment on cognitive functions and the potential underlying mechanisms in mice. We maintained C57BL/6 mice aged 2 months with treatment containing 40 or 80 mg/kg/day EFV for 5 months, while control group treated with saline. The cognitive functions were evaluated by novel object recognition test, Barnes maze test and Morris water maze. The results showed significant short-term memory impairment in 40 and 80 mg/kg groups, and notable spatial learning and memory impairments in 80 mg/kg group, without any spontaneous activity alteration. Moreover, EFV induced impairments in dendritic integrity and synaptic plasticity in hippocampus. Furthermore, Significant increases were observed in the expression levels of pro-IL-1β, a similar tendency of TNF-α and phosphorylation of p65 of the 80 mg/kg group compared with control group. These results imply that long-term EFV treatment causes synaptic dysfunction resulting in cognitive deficits, which might be induced by the enhanced pro-inflammatory cytokines IL-1β and TNF-α via activating NF-κB pathway., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Inc. All rights reserved.)

In the present study, we newly synthesized four types of novel fullerene derivatives: pyridinium/ethyl ester-type derivatives 3b-3l, pyridinium/carboxylic acid-type derivatives 4a, 4e, 4f, pyridinium/amide-type derivative 5a, and pyridinium/2-morpholinone-type derivative 6a. Among the assessed compounds, cis-3c, cis-3d, trans-3e, trans-3h, cis-3l, cis-4e, cis-4f, trans-4f, and cis-5a were found to inhibit HIV-1 reverse transcriptase (HIV-RT), HIV-1 protease (HIV-PR), and HCV NS5B polymerase (HCV NS5B), with IC 50 values observed in the micromolar range. Cellular uptake of pyridinium/ethyl ester-type derivatives was higher than that of corresponding pyridinium/carboxylic acid-type derivatives and pyridinium/amide-type derivatives. This result might indicate that pyridinium/ethyl ester-type derivatives are expected to be lead compounds for multitargeting drugs to treat HIV/HCV coinfection., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Enlightened by the available structural biology information, a novel series of dihydrothiopyrano[4,3- d ]pyrimidine derivatives were rationally designed via scaffold hopping and molecular hybridization strategies. Notably, compound 20a yielded exceptionally potent antiviral activities (EC 50 = 4.44-54.5 nM) against various HIV-1 strains and improved resistance profiles (RF = 0.5-5.6) compared to etravirine and rilpivirine. Meanwhile, 20a exhibited reduced cytotoxicity (CC 50 = 284 μM) and higher SI values (SI = 5210-63992). Molecular dynamics simulations were performed to rationalize the distinct resistance profiles. Besides, 20a displayed better solubility (sol. = 12.8 μg/mL) and no significant inhibition of the main CYP enzymes. Furthermore, 20a was characterized for prominent metabolic stability and in vivo safety properties. Most importantly, the hERG inhibition profile of 20a (IC 50 = 19.84 μM) was a remarkable improvement. Overall, 20a possesses huge potential to serve as a promising drug candidate due to its excellent potency, low toxicity, and favorable drug-like properties.

Introduction: Combination antiretroviral therapy is currently the main component of treatment for human immunodeficiency virus (HIV) infected patients. At the same time, the high mutational potential of the virus and the frequency of side effects of existing drugs dictate the need for the development and preclinical study of new, more effective and safer compounds.The aim of the study is to evaluate the specific types of toxicity of a new non-nucleoside inhibitor of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RNA-dependent DNA revertase) (NNRTI) based on the substance 1-[2-(2-benzoylphenoxy)ethyl]-6-methyluracil, a benzophenone derivative., Material and Methods: The study investigated reproductive toxicity, embryotoxicity, immunotoxicity, genotoxic (in micronucleus test in and comet assay) and allergenic properties of the test itemcompound. It was tested on three species of animals in two doses: the estimated therapeutic dose (1 TD) and its tenfold equivalent (10 TD). Taking into account the metabolic coefficients, the doses for rats (Rattus) were 9 and 90 mg/kg, for mice (Mus musculus), 21 and 210 mg/kg, and for guinea pigs (Cavia porcellus), 8 and 80 mg/kg, respectively., Results and Discussion: According to the obtained results, a favorable safety profile of the tested compound was established. Negative effects on the immune system, reproductive function, the body of pregnant animals and the fetus were not observed, as well as the compound did not have genotoxic and allergenic properties., Conclusion: These data allows to consider the studied compound as a promising therapeutic candidate for the treatment of HIV-1 infection.

Highly active antiretroviral therapy (HAART) works effectively in inhibiting HIV replication in patients. However, the use of nucleoside reverse transcriptase inhibitors (NRTIs) often causes side effects of neuropathic pain, and its mechanism remains to be elucidated. Therefore, we aim to explore the mechanism of NRTIs-induced neuropathic pain at the transcriptome level. C57BL/6 J mice were given intraperitoneal injection of zalcitabine (ddC) or saline (control) for 2 weeks, during which the mechanical pain threshold of the mice was detected by von Frey test. Then the L3~L5 spinal segments of the mice were isolated and subsequently used for RNA sequencing (RNA-seq) on the last day of treatment. The mechanical pain threshold of mice given ddC decreased significantly. Compared with the control group, ddC caused significant changes in the expression of 135 genes, of which 66 upregulated and 69 downregulated. Enrichment analysis showed that the functions of these genes are mainly enriched in regulation of transcription, multicellular organism development, and cell differentiation, and the pathway is mainly enriched in the cGMP-PKG signaling pathway and AMPK signaling pathway. Furthermore, key genes such as Gabrd, Kcnd3, Npcd, Insr, Lypd6, Scd2, and Mef2d were also identified. These may serve as drug targets for the prevention or treatment of NRTI-induced neuropathic pain.

Nucleoside reverse transcriptase inhibitors (NRTIs) were the first drugs used to treat human immunodeficiency virus infection, and their use can cause mitochondrial toxicity, including mitochondrial DNA (mtDNA) depletion in several cases. The first-generation NRTIs, including 2',3'-dideoxycytidine (ddC), were originally and are still pursued as anticancer agents. NRTI-sensitive DNA polymerases localizing to mitochondria allow for the opportunity to poison proliferating cancer cell mtDNA replication as certain cancers rely heavily on mitochondrial functions. However, mtDNA replication is independent of the cell cycle creating a significant concern that toxicants such as ddC impair mtDNA maintenance in both proliferating and nonproliferating cells. To examine this possibility, we tested the utility of the HepaRG cell line to study ddC-induced toxicity in isogenic proliferating (undifferentiated) and nonproliferating (differentiated) cells. Following ddC exposures, we measured cell viability, mtDNA copy number, and mitochondrial bioenergetics utilizing trypan blue, Southern blotting, and extracellular flux analysis, respectively. After 13 days of 1 μM ddC exposure, proliferating and differentiated HepaRG harbored mtDNA levels of 0.9% and 17.9% compared with control cells, respectively. Cells exposed to 12 μM ddC contained even less mtDNA. By day 13, differentiated cell viability was maintained but declined for proliferating cells. Proliferating HepaRG bioenergetic parameters were severely impaired by day 8, with 1 and 12 μM ddC, whereas differentiated cells displayed defects of spare and maximal respiratory capacities (day 8) and proton-leak linked respiration (day 14) with 12 μM ddC. These results indicate HepaRG is a useful model to study proliferating and differentiated cell mitochondrial toxicant exposures., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Ocular lesions in patients with human immunodeficiency virus (HIV) are commonly due to underlying opportunistic infections. With highly active antiretroviral therapy (HAART), infectious lesions have reduced and noninfectious ocular manifestations including drug-related side effects have been noted. While retinal toxicity has been noted with few other HAART drugs, there are not many on the same with Efavirenz usage. We report a series of five patients with possible efavirenz-related retinal toxicity, visual function abnormalities, and its management. Efavirenz was replaced with alternate anti-retroviral drug. Reversal of ocular side effects were noted subjectively in the form of symptom amelioration of the patients. Objectively, it could be documented with electroretinogram changes and other visual function tests reverting back to normal after change in HAART regime. Early identification of this uncommon side effect in select patients can prevent irreversible vision loss due to efavirenz-associated retinal toxicity.

Despite the availability of modern antiretroviral therapy (ART), neurocognitive impairment persists among some persons with HIV (PWH). We investigated the role of exposure to four major classes of ARTs in neurocognitive impairment in PWH. A single-site cohort of 343 PWH was recruited. Lifetime ART medication history was obtained from medical health records. We evaluated the role of ART exposure as a predictor of neurocognitive impairment using univariate analyses and machine learning, while accounting for potential effects of demographic, clinical, and comorbidity-related risk factors. Out of a total of 26 tested variables, two random forest analyses identified the most important characteristics of a neurocognitively impaired group (N = 59): Compared with a neurocognitively high-performing group (N = 132; F 1 -score = 0.79), we uncovered 13 important risk factors; compared with an intermediate-performing group (N = 152; F 1 -score = 0.75), 16 risk factors emerged. Longer lifetime ART exposure, especially to integrase inhibitors, was one of the most important predictors of neurocognitive impairment in both analyses (rank 2 of 13 and rank 4 of 16, respectively), superseding effects of age (rank 11/13, rank 15/16) and HIV duration (rank 13/13, rank 16/16). Concerning specific integrase inhibitors, the impaired group had significantly longer dolutegravir exposure (p = 0.011) compared with the high-performing group (p = 0.012; trend compared with the intermediate group p = 0.063). A longer duration to integrase inhibitor intake was negatively related to cognition in this cohort. Our findings suggest that possible cognitive complications of long-term exposure to integrase inhibitors, in particular dolutegravir, should be closely monitored in PWH.

Efavirenz (EFV) is used for antiretroviral treatment of HIV infection, and successfully inhibits viral replication and mother-to-child transmission of HIV during pregnancy and childbirth. Unfortunately, the drug induces neuropsychiatric symptoms such as anxiety and depressed mood and potentially affects cognitive performance. EFV acts on, among others, the serotonin transporter and serotonin receptors that are expressed in the developing brain. Yet, how perinatal EFV exposure affects brain cytoarchitecture remains unclear. Here, we exposed pregnant and lactating rats to EFV, and examined in the medial prefrontal cortex (mPFC) of their adult offspring the effects of the maternal EFV exposure on cortical architecture. We observed a significant decrease in the number of cells, mainly mature neurons, in the infra/prelimbic and cingulate cortices of adult offspring. Next, we found an altered cortical cytoarchitecture characterized by a significant reduction in deep- and superficial-layer cells. This was accompanied by a sharp increase in programmed cell death, as we identified a significantly higher number of cleaved Caspase-3-positive cells. Finally, the serotonergic and dopaminergic innervation of the mPFC subdomains was increased. Thus, the perinatal exposure to EFV provoked in the mPFC of adult offspring cell death, significant changes in cytoarchitecture, and disturbances in serotonergic and dopaminergic innervation. Our results are important in the light of EFV treatment of HIV-positive pregnant women, and its effect on brain development and cognitive behavior., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Highly active antiretroviral therapy (HAART) regimens are based on the use of nucleoside reverse transcriptase inhibitors (NRTIs), which are the main drugs used by patients infected with the human immunodeficiency virus (HIV). The use of NRTIs combinations has afforded clear clinical benefits to patients undergoing HAART. However, the combination of two NRTIs may increase the risk of genomic instability in comparison with the drugs administered individually. We analyzed the ability of zidovudine (AZT) and lamivudine (3TC), and the combination AZT +3TC to induce complex genomic alterations using the cytokinesis-block micronucleus (CBMN) assay in Chinese hamster ovary (CHO)-K1 cells. The 24-h cell treatment with individual NRTIs showed that AZT increased micronucleus frequencies and nucleoplasmic bridges (NPBs). No significant differences were observed for any parameters investigated after exposure of CHO-K1 cells to 3TC. The combination AZT +3TC significantly increased micronucleus frequencies. Analysis of interaction between these drugs suggested that antagonism occurs in all AZT +3TC concentrations. These results highlight the importance to investigate the genotoxic profile of NRTIs to develop safer intervention strategies in antiretroviral treatment protocols.

Efavirenz (EFV) is a well-known, effective anti-retroviral drug long used in first-line treatment for children and adults with HIV and HIV/AIDS. Due to its narrow window of effective concentrations, between 1 and 4 μg/mL, and neurological side effects at supratherapeutic levels, several investigations into the pharmacokinetics of the drug and its genetic underpinnings have been carried out, primarily with adult samples. A number of studies, however, have examined the genetic influences on the metabolism of EFV in children. Their primary goal has been to shed light on issues of appropriate pediatric dosing, as well as the manifestation of neurotoxic effects of EFV in some children. Although EFV is currently being phased out of use for the treatment of both adults and children, we share this line of research to highlight an important aspect of medical treatment that is relevant to understanding the development of children diagnosed with HIV., (© 2020 Wiley Periodicals, Inc.)

Our previous efforts have led to the development of two potent NNRTIs, K-5a2 and 25a, exhibiting effective anti-HIV-1 potency and resistance profiles compared with etravirine. However, both inhibitors suffered from potent hERG inhibition and short half-life. In this article, with K-5a2 and etravirine as leads, series of novel fluorine-substituted diarylpyrimidine derivatives were designed via molecular hybridization and bioisosterism strategies. The results indicated 24b was the most active inhibitor, exhibiting broad-spectrum activity (EC 50 = 3.60-21.5 nM) against resistant strains, significantly lower cytotoxicity (CC 50 = 155 μM), and reduced hERG inhibition (IC 50 > 30 μM). Crystallographic studies confirmed the binding of 24b and the role of the fluorine atom, as well as optimal contacts of a nitrile group with the main-chain carbonyl group of H235. Furthermore, 24b showed longer half-life and favorable safety properties. All the results demonstrated that 24b has significant promise in circumventing drug resistance as an anti-HIV-1 candidate.

Combination antiretroviral therapy (cART) has improved the life expectancy of HIV patients, thus increasing the number of people living with HIV (PLWH). However, cardiovascular diseases (CVD) are now one of the most prevalent causes of death among PLWH. Nucleoside reverse transcriptase inhibitors (NRTIs) are the backbone of cART, and the emtricitabine (FTC) and tenofovir disoproxil fumarate (TDF) coformulation is commonly used. In prior studies, acute NRTI treatment-induced endothelial dysfunction, increased reactive oxygen species production, and mitophagic activity, suggesting that mitochondrial dysfunction may be critical to NRTI-induced endothelial dysfunction. Mitochondrial dysfunction plays a causal role in endothelial senescence, whereas premature endothelial senescence can promote the development of CVD. We hypothesize that for chronic NRTI treatment, a disruption in mitochondrial homeostasis leads to premature endothelial senescence and predisposes PLWH to CVD. We used human aortic endothelial cells (HAEC) and HIV-1 transgenic (Tg26) mice to test the interrelationship between mitochondrial and vascular dysfunction after chronic NRTI treatment in vitro and in vivo. Mitochondrial DNA copy number was decreased in late-passage HAEC treated with NRTIs, and senescence-associated β-galactosidase accumulation was elevated. In late-passage HAEC, NRTIs decreased the activity of Parkin-mediated mitophagy. In Tg26 mice treated with FTC, plasma nitrite levels were decreased. Endothelium-dependent vasodilation in NRTI-treated Tg26 mice was also reduced. Our work suggests that long-term use of NRTI may disrupt mitochondrial homeostasis, induce premature endothelial senescence, and impair vascular function., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Human immunodeficiency virus (HIV)-infected patients undergoing antiretroviral therapy are afforded an increased lifespan but also exhibit an elevated incidence of cardiovascular disease. HIV therapy uses a combination drug approach, and nucleoside reverse transcriptase inhibitors (NRTI) are a backbone of this therapy. Endothelial dysfunction is an initiating event in cardiovascular disease etiology, and in our prior studies, NRTIs induced an endothelial dysfunction that was dependent upon mitochondrial oxidative stress. Moreover, short-term NRTI administration induced a mitophagy-associated endothelial toxicity and increased reactive oxygen species (ROS) production that was rescued by coenzyme Q10 (Q10) or overexpression of a mitochondrial antioxidant enzyme. Thus, our objective was to examine mitochondrial toxicity in endothelial cells after chronic NRTI treatment and evaluate Q10 as a potential adjunct therapy for preventing NRTI-induced mitochondrial toxicity. Human aortic endothelial cells (HAEC) were exposed to chronic NRTI treatment, with or without Q10. ROS production, cell proliferation rate, levels of senescence, and mitochondrial bioenergetic function were determined. Chronic NRTI increased ROS production but decreased population doubling. In addition, NRTI increased the accumulation of β-galactosidase, indicative of an accelerated rate of senescence. Moreover, ATP-linked respiration was diminished. Co-treatment with Q10 delayed the onset of NRTI-induced senescence, decreased ROS production and rescued the cells' mitochondrial respiration rate. Thus, our findings may suggest antioxidant enrichment approaches for reducing the cardiovascular side effects of NRTI therapy.

Efavirenz (more specifically the S- enantiomer) is a cornerstone antiretroviral therapy for treatment of HIV infection. The major primary metabolite is S -8-hydroxyefavirenz, which does not have antiretroviral activity but is neurotoxic. Cytochrome P450 2B6 (CYP2B6) is the major enzyme catalyzing S -8-hydroxyefavirenz formation. CYP2B6 genetics and drug interactions are major determinants of clinical efavirenz disposition and dose adjustment. In addition, as a prototypic CYP2B6 substrate, S- efavirenz and analogs can inform on the structure, activity, catalytic mechanisms, and stereoselectivity of CYP2B6. Metabolism of R- efavirenz by CYP2B6 remains unexplored. This investigation assessed S- efavirenz metabolism by clinically relevant CYP2B6 genetic variants. This investigation also evaluated R- efavirenz hydroxylation by wild-type CYP2B6.1 and CYP2B6 variants. S -Efavirenz 8-hydroxylation by wild-type CYP2B6.1 and variants exhibited positive cooperativity and apparent cooperative substrate inhibition. On the basis of Cl max values, relative activities for S -efavirenz 8-hydroxylation were in the order CYP2B6.4 > CYP2B6.1 ≈ CYP2B6.5 ≈ CYP2B6.17 > CYP2B6.6 ≈ CYP2B6.7 ≈ CYP2B6.9 ≈ CYP2B6.19 ≈ CYP2B6.26; CYP2B6.16 and CYP2B6.18 showed minimal activity. Rates of R -efavirenz metabolism were approximately 1/10 those of S -efavirenz for wild-type CYP2B6.1 and variants. On the basis of Cl max values, there was 14-fold enantioselectivity ( S > R -efavirenz) for wild-type CYP2B6.1, and 5- to 22-fold differences for other CYP2B6 variants. These results show that both CYP2B6 516G > T ( CYP2B6*6 and CYP2B6*9 ) and 983T > C ( CYP2B6*16 and CYP2B6*18 ) polymorphisms cause canonical diminishment or loss-of-function variants for S -efavirenz 8-hydroxylation, provide a mechanistic basis for known clinical pharmacogenetic differences in efavirenz disposition, and may predict additional clinically important variant alleles. Efavirenz is the most stereoselective CYP2B6 drug substrate yet identified and may be a useful probe for the CYP2B6 active site and catalytic mechanisms. SIGNIFICANCE STATEMENT: Clinical disposition of the antiretroviral S -efavirenz is affected by CYP2B6 polymorphisms. Expressed CYP2B6 with 516G>T (CYP2B6*6 and CYP2B6*9), and 983T>C (CYP2B6*16 and CYP2B6*18) polymorphisms had a diminishment or loss of function for efavirenz 8-hydroxylation. This provides a mechanistic basis for efavirenz clinical pharmacogenetics and may predict additional clinically important variant alleles. Efavirenz metabolism showed both cooperativity and cooperative substrate inhibition. With greater than 10-fold enantioselectivity ( S - vs. R - metabolism), efavirenz is the most stereoselective CYP2B6 drug substrate yet identified. These findings may provide mechanistic insights., (Copyright © 2019 by The American Society for Pharmacology and Experimental Therapeutics.)

A novel series of dihydroquinazolin-2-amine derivatives were synthesized and evaluated for their anti-HIV-1 activity in MT-4 cell cultures. All of the molecules were active against wild-type HIV-1 with EC 50 values ranging from 0.61 μM to 0.84 nM. The most potent inhibitor, compound 4b, had an EC 50 value of 0.84 nM against HIV-1 strain IIIB, and thus was more active than the reference drugs efavirenz and etravirine. Moreover, most of the compounds maintained high activity (low-micromolar EC 50 values) against strains bearing the reverse transcriptase (RT) E138K mutation. Compound 4b had EC 50 values of 3.5 nM and 66 nM against non-nucleoside reverse transcriptase inhibitor-resistant strains bearing the RT E138K and RES056 mutations. In enzyme activity assays, compound 4b exhibited an IC 50 value of 10 nM against HIV-1 RT. Preliminary SARs and molecular docking studies provide valuable insights for further optimization., (Copyright © 2019 Elsevier Masson SAS. All rights reserved.)

Nucleoside reverse transcriptase inhibitors (NRTIs) form the backbone in combination antiretroviral therapy (cARVs). They halt chain elongation of the viral cDNA by acting as false substrates in counterfeit incorporation mechanism to viral RNA-dependent DNA polymerase. In the process genomic DNA polymerase as well as mitochondrial DNA (mtDNA) polymerase-γ (which has a much higher affinity for these drugs at therapeutic doses) are also impaired. This leads to mitochondrial toxicity that manifests clinically as mitochondrial myopathy, peripheral neuropathy, hyperlactatemia or lactic acidosis and lipoatrophy. This has led to the revision of clinical guidelines by World Health Organization to remove stavudine from first-line listing in the treatment of HIV infections. Recent reports have implicated oxidative stress besides mtDNA polymerase-γ hypothesis in NRTI-induced metabolic complications. Reduced plasma antioxidant concentrations have been reported in HIV positive patients on cARVs but clinical intervention with antioxidant supplements have not been successful either due to low efficacy or poor experimental designs. Citrus fruit-derived naringenin has previously been demonstrated to possess antioxidant and free radical scavenging properties which could prevent mitochondrial toxicity associated with these drugs. This review revisits the controversy surrounding mtDNA polymerase-γ hypothesis and evaluates the potential benefits of naringenin as a potent anti-oxidant and free radical scavenger which as a nutritional supplement or therapeutic adjunct could mitigate the development of mitochondrial toxicity associated with these drugs., (Copyright © 2019 Elsevier B.V. All rights reserved.)

All nucleoside analogues for treating HIV infection, due to their capacity to integrate into and alter human DNA, are experimentally genotoxic to some extent. The long-term oncogenic risk after in utero exposure remains to be determined. Cancer incidence in uninfected children exposed to nucleos(t)ide reverse transcriptase inhibitors (NRTIs) was evaluated, by cross-checking against the National Cancer Registry, in the French perinatal study of children born to HIV + mothers. Twenty-one cancers were identified in 15,163 children (median age: 9.9 years [interquartile range (IQR): 5.8-14.2]) exposed to at least one NRTI in utero between 1990 and 2014. Five of these children were exposed to zidovudine monotherapy, and 15 to various combinations, seven of which included didanosine. Overall, the total number of cases was not significantly different from that expected for the general population (SIR = 0.8[0.47-1.24]), but the number of cases after didanosine exposure was twice that expected (SIR = 2.5 [1.01-5.19]). Didanosine accounted for only 10% of prescriptions but was associated with one-third of cancers. In multivariate analysis, didanosine exposure was significantly associated with higher risk (HR = 3.0 [0.9-9.8]). This risk was specifically linked to first-trimester exposure (HR = 5.5 [2.1-14.4]). Three cases of pineoblastoma, a very rare cancer, were observed, whereas 0.03 were expected. Two were associated with didanosine exposure. Despite reassuring data overall, there is strong evidence to suggest that didanosine displays transplacental oncogenicity. These findings cannot be extrapolated to other NRTIs, but they highlight the need for comprehensive evaluations of the transplacental genotoxicity of this antiretroviral class. Environ. Mol. Mutagen., 60:404-409, 2019. © 2017 Wiley Periodicals, Inc., (© 2017 Wiley Periodicals, Inc.)

Diarylpyrimidine derivatives (DAPYs) exhibit robust anti-HIV-1 potency, although they have been compromised by E138K variant and severe side-effects and been suffering from poor water solubility. In the present work, hydrophilic morpholine or methylsulfonyl and sulfamide-substituted piperazine/piperidines were introduced into the right wing of DAPYs targeting the solvent-exposed tolerant region I. The anti-HIV-1 activities of 11c (EC 50(WT) = 0.0035 μM, EC 50(E138K) = 0.0075 μM) were the same as and 2-fold better than that of the lead etravirine against the wild-type and E138K mutant HIV-1, respectively, with a relative low cytotoxicity (CC 50 ≥ 173 μM). Further test showed a significant improvement in the water solubility of 11c. Besides, 11c displayed no significant inhibition on main cytochrome P450 enzymes and exhibited no acute/subacute toxicities at doses of 2000 mg·kg -1 /50 mg·kg -1 in mice. Taken together, we consider that 11c is a promising lead for further structural optimization.

Nucleoside reverse transcriptase inhibitors (NRTI), such as zidovudine (AZT), are constituents of HIV-1 therapy and are used for the prevention of mother-to-child transmission. Prolonged thymidine analogue exposure has been associated with mitochondrial toxicities to heart, liver, and skeletal muscle. We hypothesized that the thymidine analogue AZT might interfere with autophagy in myocytes, a lysosomal degradation pathway implicated in the regulation of mitochondrial recycling, cell survival, and the pathogenesis of myodegenerative diseases. The impact of AZT and lamivudine (3TC) on C2C12 myocyte autophagy was studied using various methods based on LC3-green fluorescent protein overexpression or LC3 staining in combination with Western blotting, flow cytometry, and confocal and electron microscopy. Lysosomal and mitochondrial functions were studied using appropriate staining for lysosomal mass, acidity, cathepsin activity, as well as mitochondrial mass and membrane potential in combination with flow cytometry and confocal microscopy. AZT, but not 3TC, exerted a significant dose- and time-dependent inhibitory effect on late stages of autophagosome maturation, which was reversible upon mTOR inhibition. Inhibition of late autophagy at therapeutic drug concentrations led to dysfunctional mitochondrial accumulation with membrane hyperpolarization and increased reactive oxygen species (ROS) generation and, ultimately, compromised cell viability. These AZT effects could be readily replicated by pharmacological and genetic inhibition of myocyte autophagy and, most importantly, could be rescued by pharmacological stimulation of autophagolysosomal biogenesis. Our data suggest that the thymidine analogue AZT inhibits autophagy in myocytes, which in turn leads to the accumulation of dysfunctional mitochondria with increased ROS generation and compromised cell viability. This novel mechanism could contribute to our understanding of the long-term side effects of antiviral agents., (Copyright © 2018 American Society for Microbiology.)

The nucleoside reverse transcriptase inhibitor zidovudine (AZT), used in HIV infection treatment, induces mitochondrial DNA (mtDNA) depletion. A cause-effect relationship between mtDNA status alterations and autophagy has been reported. Both events are common in several liver diseases, including hepatocellular carcinoma. Here, we have studied autophagy activation in rat liver with mtDNA depletion induced by AZT administration in drinking water for 35 days. AZT at a concentration of 1 mg/ml, but not 0.5 mg/ml in the drinking water, decreased mtDNA levels in rat liver and extrahepatic tissues. In liver, mtDNA-encoded cytochrome c oxidase 1 protein levels were decreased. Although serum biomarkers of liver and kidney toxicity remained unaltered, β-hydroxybutyrate levels were increased in liver of AZT-treated rats. Moreover, autophagy was dysregulated at two levels: (i) decreased induction signalling of this process as indicated by increases in autophagy inhibitors activity (AKT/mTOR), and absence of changes (Beclin-1, Atg5, Atg7) or decreases (AMPK/ULK1) in the expression/activity of pro-autophagy proteins; and (ii) reduced autophagosome degradation as indicated by decreases in the lysosome abundance (LAMP2 marker) and the transcription factor TFEB controlling lysosome biogenesis. This resulted in increased autophagosome abundance (LC3-II marker) and accumulation of the protein selectively degraded by autophagy p62, and the transcription factor Nrf2 in liver of AZT-treated rats. Nrf2 was activated as indicated by the up-regulation of antioxidant target genes Nqo1 and Hmox-1. In conclusion, rat liver with AZT-induced mtDNA depletion presented dysregulations in autophagosome formation and degradation balance, which results in accumulation of these structures in parenchymal liver cells, favouring hepatocarcinogenesis.

Genome-wide association studies indicate that several idiosyncratic adverse drug reactions are highly associated with specific human leukocyte antigen (HLA) alleles. For instance, abacavir, a human immunodeficiency virus reverse transcriptase inhibitor, induces multiorgan toxicity exclusively in patients carrying the HLA-B*57:01 allele. However, the underlying mechanism is unclear due to a lack of appropriate animal models. Previously, we developed HLA-B*57:01 transgenic mice and found that topical application of abacavir to the ears induced proliferation of CD8+ lymphocytes in local lymph nodes. Here, we attempted to reproduce abacavir-induced liver injury in these mice. However, oral administration of abacavir alone to HLA-B*57:01 transgenic mice did not increase levels of the liver injury marker alanine aminotransferase. Considering the importance of innate immune activation in mouse liver, we treated mice with CpG oligodeoxynucleotide, a toll-like receptor 9 agonist, plus abacavir. This resulted in a marked increase in alanine aminotransferase, pathological changes in liver, increased numbers of activated CD8+ T cells, and tissue infiltration by immune cells exclusively in HLA-B*57:01 transgenic mice. These results indicate that CpG oligodeoxynucleotide-induced inflammatory reactions and/or innate immune activation are necessary for abacavir-induced HLA-mediated liver injury characterized by infiltration of CD8+ T cells. Thus, we developed the first mouse model of HLA-mediated abacavir-induced idiosyncratic liver injury. Further investigation will show that the proposed HLA-mediated liver injury model can be applied to other combinations of drugs and HLA types, thereby improving drug development and contributing to the development of personalized medicine.

A high proportion of HIV-positive patients in South Africa receive concomitant efavirenz (EFV) and isoniazid (INH) therapy. EFV is metabolised in the liver via CYP2B6, and genetic polymorphism of CYP2B6 is known to result in slowed metabolism of the drug. INH is also metabolised in the liver, causing inhibition of a pathway that plays an important role in slow EFV metabolisers. Concomitant INH use therefore affects plasma levels of EFV. EFV is well known to cause neuropsychiatric side-effects on initiation, and a recent adult case series described late-onset neurotoxicity in the form of subacute ataxia and encephalopathy in patients treated with EFV for a median of 2 years, in association with toxic plasma levels of the drug. We have seen an increase in cases of EFV toxicity presenting to our neurology referral unit. All cases have been in the context of recent initiation of concomitant INH. We therefore conducted a retrospective case record audit to describe these seven cases with the additional advantage of tertiary-level assessment. We outline the clinical features and investigation results, as well as outcomes after EFV was stopped. Our main objectives are to highlight the probable role of concomitant INH use in the development of this syndrome, and to suggest that only limited work-up may be warranted in suspected cases.

A new series of diarylpyrimidines (DAPYs) were designed, synthesized and evaluated as novel HIV-1 NNRTIs to further explore the chemical space surrounding the "hydrophobic channel" of the NNRTI binding pocket (NNIBP), guided by the comprehensive analysis of X-ray structural biology data of HIV-1 RT/NNRTI complexes and molecular modeling. Encouragingly, most of the synthesized DAPYs were found to be active against the HIV-1 wild-type (WT) strain with EC 50 values ranging from 3 nM to 63 nM, and displayed significantly reduced cytotoxicity compared with etravirine (ETV) and rilpivirine (RPV). Among them, two most promising compounds Z10 (EC 50 = 3 nM) and Z13 (EC 50 = 3 nM) showed equivalent potency against the HIV-1 WT strain to the reference drugs efavirenz (EFV, EC 50 = 3 nM) and ETV (EC 50 = 3 nM). Notably, Z13 also showed the most potent activity against HIV-1 mutant strains including K103N (EC 50 = 10 nM), E138K (EC 50 = 22 nM) and RES056 (EC 50 = 0.935 μM). Against mutant strains Y181C, Y188L and F227L + V106A, Z17 showed double-digit nanomolar inhibitory activity with EC 50 values 27 nM, 98 nM and 30 nM, respectively. The structure-activity relationships (SARs) and molecular docking studies provided important clues for further molecular elaboration. Collectively, this study provides useful information to guide lead optimization and drug discovery via the exploration of this seldom investigated region.

Non-nucleoside reverse transcriptase inhibitors (NNRTIs) are recommended components of preferred combination antiretroviral therapies used for the treatment of human immunodeficiency virus (HIV) infection. These regimens are extremely effective in suppressing virus replication. Recently, our research group identified some N 1 -aryl-2-arylthioacetamido-benzimidazoles as a novel class of NNRTIs. In this research work we report the design, the synthesis and the structure-activity relationship studies of new compounds (20-34) in which some structural modifications have been introduced in order to investigate their effects on reverse transcriptase (RT) inhibition and to better define the features needed to increase the antiviral activity. Most of the new compounds proved to be highly effective in inhibiting both RT enzyme at nanomolar concentrations and HIV-1 replication in MT4 cells with minimal cytotoxicity. Among them, the most promising N 1 -aryl-2-arylthioacetamido-benzimidazoles and N 1 -aryl-2-aryloxyacetamido-benzimidazoles were also tested toward a panel of single- and double-mutants strain responsible for resistance to NNRTIs, showing in vitro antiviral activity toward single mutants L100I, K103N, Y181C, Y188L and E138K. The best results were observed for derivatives 29 and 33 active also against the double mutants F227L and V106A. Computational approaches were applied in order to rationalize the potency of the new synthesized inhibitors., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Background and Purpose: SQSTM1/p62 is a multifunctional, stress-induced, scaffold protein involved in multiple cellular processes including autophagic clearance, regulation of inflammatory responses and redox homeostasis. Its altered function has been associated with different human pathologies, such as neurodegenerative, metabolic and bone diseases (down-regulation), and cancerogenesis (up-regulation). However, its role in the off-target effects of clinically used drugs is still not understood., Experimental Approach: We evaluated the expression of p62 in cultured Hep3B cells and their derived ρ° cells (lacking mitochondria), along with markers of autophagy and mitochondrial dysfunction. The effects of efavirenz were compared with those of known pharmacological stressors, rotenone, thapsigargin and CCCP, and we also used transient silencing with siRNA and p62 overexpression. Western blotting, quantRT-PCR and fluorescence microscopy were used to assay these effects and their underlying mechanisms., Key Results: In Hep3B cells, efavirenz augmented p62 protein content, an effect not observed in the corresponding ρ° cells. p62 up-regulation followed enhanced SQSTM1 expression mediated through the transcription factor CHOP/DDIT3, while other well-known regulators (NF-kB and Nrf2) were not involved. Inhibition of autophagy with 3MA or with transient silencing of Atg5 did not affect SQSTM1 expression in efavirenz-treated cells while p62 overexpression ameliorated the deleterious effect of efavirenz on cell viability., Conclusion and Implications: In our model, p62 exerted a specific, autophagy-independent role and protected against efavirenz-induced mitochondrial ROS generation and activation of the NLRP3 inflammasome. These findings add to the multifunctional nature of p62 and may help to understand the off-target effects of clinically useful drugs., (© 2017 The British Pharmacological Society.)

Although antiretroviral (ARV) therapy has reduced the incidence of severe dementia associated with HIV infection, there has been a rise in milder neurocognitive complaints. Data from HIV patients taking ARVs have shown measurable neurocognitive improvements during drug cessation, suggesting a neurotoxic role of the therapy itself. Mechanisms underlying potential ARV neurotoxicity have not been thoroughly investigated, however pathologic oxidative stress and mitochondrial dysfunction have been suspected. Using DIV 16 primary rat cortical neuron culture, we tested eight ARVs from the three most commonly prescribed ARV classes: nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs/NtRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs), and protease inhibitors (PIs) for effects on neuron viability and morphology after 24 h of drug exposure. Of the tested NRTIs, only stavudine at nearly 100 times the target plasma concentration affected neuron viability with no appreciable change in morphology. Dideoxyinosine induced dendritic simplification at 100 times target plasma concentrations, but did not adversely affect viability. The sole NtRTI, tenofovir, induced dendritic simplification at approximately 3-4 times target plasma concentration, but did not affect viability. Of the tested PIs, only amprenavir decreased neuron viability at nearly 100 times the target plasma concentration. The non-nucleoside reverse transcriptase inhibitor, efavirenz, consistently reduced viability (at 50 µM) and induced dendritic simplification (at 20 µM) nearest the target plasma concentration. Probing mitochondrial energetics of DIV16 cortical neurons after exposure to 20 µM efavirenz showed rapid diminution of mitochondrial-dependent oxygen consumption. Further, 20 µM efavirenz decreased excitability in ex vivo slice culture whereas 2 µM had no effect.

We designed and synthesized a series of chiral indolyarylsulfones (IASs) as new HIV-1 NNRTIs. The new IASs 8-37 showed potent inhibition of the HIV-1 WT NL4-3 strain and of the mutant K103N, Y181C, Y188L, and K103N-Y181C HIV-1 strains. Six racemic mixtures, 8, 23-25, 31, and 33, were separated at semipreparative level into their pure enantiomers. The (R)-8 enantiomer bearing the chiral (α-methylbenzyl) was superior to the (S)-counterpart. IAS derivatives bearing the (S) alanine unit, (S)-23, (S,R)-25, (S)-31, and (S)-33, were remarkably more potent than the corresponding (R)-enantiomers. Compound 23 protected hippocampal neuronal cells from the excitotoxic insult, while efavirenz (EFV) did not contrast the neurotoxic effect of glutamate. The present results highlight the chiral IASs as new NNRTIs with improved resistance profile against the mutant HIV-1 strains and reduced neurotoxic effects.

The clinical benefits of HIV-1 non-nucleoside reverse transcriptase (RT) inhibitors (NNRTIs) are hindered by their unsatisfactory pharmacokinetic (PK) properties along with the rapid development of drug-resistant variants. However, the clinical efficacy of these inhibitors can be improved by developing compounds with enhanced pharmacological profiles and heightened antiviral activity. We used computational and structure-guided design to develop two next-generation NNRTI drug candidates, compounds I and II, which are members of a class of catechol diethers. We evaluated the preclinical potential of these compounds in BALB/c mice because of their high solubility (510 µ g/ml for compound I and 82.9 µ g/ml for compound II), low cytotoxicity, and enhanced antiviral activity against wild-type (WT) HIV-1 RT and resistant variants. Additionally, crystal structures of compounds I and II with WT RT suggested an optimal binding to the NNRTI binding pocket favoring the high anti-viral potency. A single intraperitoneal dose of compounds I and II exhibited a prolonged serum residence time of 48 hours and concentration maximum ( C max ) of 4000- to 15,000-fold higher than their therapeutic/effective concentrations. These C max values were 4- to 15-fold lower than their cytotoxic concentrations observed in MT-2 cells. Compound II showed an enhanced area under the curve (0-last) and decreased plasma clearance over compound I and efavirenz, the standard of care NNRTI. Hence, the overall (PK) profile of compound II was excellent compared with that of compound I and efavirenz. Furthermore, both compounds were very well tolerated in BALB/c mice without any detectable acute toxicity. Taken together, these data suggest that compounds I and II possess improved anti-HIV-1 potency, remarkable in vivo safety, and prolonged in vivo circulation time, suggesting strong potential for further development as new NNRTIs for the potential treatment of HIV infection., (Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.)

Efavirenz is a non-nucleoside reverse transcriptase inhibitor (NNRTI) and an active constituent of the highly active antiretroviral therapy regime. It has significantly contributed in control and management of human immunodeficiency virus propagation. However, EFV administration has also led to severe adverse effects, several reports highlighted the role of EFV in mitochondrial dysfunction and toxicity but the molecular mechanism has been poorly understood. In present study, human hepatoma cells Huh 7.5 were treated with clinically relevant concentrations of EFV and parameters like cytotoxicity, mitochondrial transmembrane potential, mitochondrial morphology, cytochrome c release, mitochondria-mediated apoptosis, mtDNA and mtRNA levels and EFV distribution into mitochondrial compartment were evaluated to understand sequence of events leading to cell death in EFV-treated cells. EFV at its clinically relevant concentration was significantly toxic after 48 and 72 h of treatments. EFV-mediated toxicity is initiated with the permeabilization of mitochondrial outer membrane and change in mitochondrial membrane potential (Δψm) which triggers a series of events like cytochrome c release, alteration in mitochondrial morphology and mitochondria-mediated apoptosis. Total mitochondrial content is reduced after 48 h of EFV treatment at IC 50 concentration which is also reflected in reduced mitochondrial DNA and RNA levels. After detecting EFV in mitochondrial compartment after 12 h of incubation with EFV, we hypothesize that EFV being a lipophilic molecule is internalized into the mitochondrial compartment causing depolarization of Δψm which subsequently leads to a cascade of events causing cell death.

Efavirenz is a widely prescribed non-nucleoside reverse transcriptase inhibitor for the treatment of HIV infections. To test for potential long-term consequences of efavirenz on brain cells, cultured primary astrocytes were incubated with this substance or with its primary metabolite 8-hydroxy efavirenz for up to 7 days. Both, efavirenz and 8-hydroxy efavirenz caused time- and concentration-dependent cell toxicity and stimulated in subtoxic concentrations the glycolytic flux (glucose consumption and lactate release) in astrocytes. As 8-hydroxy efavirenz was less toxic than efavirenz and stimulated glycolysis in lower concentrations we tested for a potential hydroxylation of efavirenz to 8-hydroxy efavirenz in astrocytes. Analysis of media and cell lysates by HPLC-UV and mass spectrometry revealed that after 3 days of incubation viable astrocytes had accumulated about 17 and 7 % of the applied efavirenz and 8-hydroxy efavirenz, respectively. However, in cultures treated with efavirenz neither 8-hydroxy efavirenz nor any other known metabolite of efavirenz was detectable. These data demonstrate that cultured rat astrocytes efficiently accumulate, but not metabolize, efavirenz and 8-hydroxy efavirenz and that the observed chronic stimulation of glycolysis is mediated by both efavirenz and 8-hydroxy efavirenz.

The prevalence of HIV-associated neurocognitive disorders (HAND) remains high despite combination antiretroviral therapy (cART). There is evidence that neural stem cells (NSCs) can migrate to sites of brain injury such as those caused by inflammation and oxidative stress, which are pathological features of HAND. Thus, reductions in NSCs may contribute to HAND pathogenesis. Since the HIV non-nucleoside reverse transcriptase inhibitor efavirenz (EFV) has previously been associated with cognitive deficits and promotion of oxidative stress pathways, we examined its effect on NSCs in vitro as well as in C57BL/6J mice. Here we report that EFV induced a decrease in NSC proliferation in vitro as indicated by MTT assay, as well as BrdU and nestin immunocytochemistry. In addition, EFV decreased intracellular NSC adenosine triphosphate (ATP) stores and NSC mitochondrial membrane potential (MMP). Further, we found that EFV promoted increased lactate dehydrogenase (LDH) release, activation of p38 mitogen-activated protein kinase (MAPK), and increased Bax expression in cultured NSCs. Moreover, EFV reduced the quantity of proliferating NSCs in the subventricular zone (SVZ) of C57BL/6J mice as suggested by BrdU, and increased apoptosis as measured by active caspase-3 immunohistochemistry. If these in vitro and in vivo models translate to the clinical syndrome, then a pharmacological or cell-based therapy aimed at opposing EFV-mediated reductions in NSC proliferation may be beneficial to prevent or treat HAND in patients receiving EFV.

Efavirenz is highly effective at suppressing HIV-1, and the WHO guidelines list it as a component of the first-line antiretroviral (ARV) therapies for treatment-naïve patients. Though the pharmacological basis is unclear, efavirenz is commonly associated with a risk for neuropsychiatric adverse events (NPAEs) when taken at the prescribed dose. In many patients these NPAEs appear to subside after several weeks of treatment, though long-term studies show that in some patients the NPAEs persist. In a recent study focusing on the abuse potential of efavirenz, its receptor psychopharmacology was reported to include interactions with a number of established molecular targets for known drugs of abuse, and it displayed a prevailing behavioral profile in rodents resembling an LSD-like activity. In this report, we discovered interactions with additional serotonergic targets that may be associated with efavirenz-induced NPAEs. The most robust interactions were with 5-HT3A and 5-HT6 receptors, with more modest interactions noted for the 5-HT2B receptor and monoamine oxidase A. From a molecular mechanistic perspective, efavirenz acts as a 5-HT6 receptor inverse agonist of Gs-signaling, 5-HT2A and 5-HT2C antagonist of Gq-signaling, and a blocker of the 5-HT3A receptor currents. Efavirenz also completely or partially blocks agonist stimulation of the M1 and M3 muscarinic receptors, respectively. Schild analysis suggests that efavirenz competes for the same site on the 5-HT2A receptor as two known hallucinogenic partial agonists (±)-DOI and LSD. Prolonged exposure to efavirenz reduces 5-HT2A receptor density and responsiveness to 5-HT. Other ARVs such as zidovudine, nevirapine and emtricitabine did not share the same complex pharmacological profile as efavirenz, though some of them weakly interact with the 5-HT6 receptor or modestly block GABAA currents., (Published by Elsevier Ltd.)

Mitochondrion is a cellular organelle that is present in most of the cells and is responsible for producing energy for the cell. Mitochondria have their own double-stranded DNA genome which is distinct from nuclear genome. The replication, recombination and repair of mtDNA are achieved by DNA polymerase-gamma which is encoded by POLG gene. Mutation in the mtDNA or POLG gene might lead to mitochondrial dysfunction and disease. Several mutations and polymorphisms in these regions have been associated to mitochondrial disorders. Nuceloside and nucelotide reverse transcriptase inhibitors (NRTIs) that form the basis of AIDS therapy have significantly increased the survival rate of HIV-infected individuals predisposing them to other side effects. One of the most common side effects of NRTI usage is mitochondrial toxicity leading to several mitochondrial disorders. Mutations in mtDNA have also been associated to the use of specific NRTIs leading to specific mitochondrial disorders. This review briefly summarizes the advances in mtDNA mutations and NRTI-caused mitochondrial toxicity and mutations.

Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTIs) are vital class of drugs in treating HIV-1 infection, but drug resistance and toxicity drive the need for effective new inhibitors with potent antiviral activity, less toxicity and improved physicochemical properties. In the present study, twelve novel 1-(4-chlorophenyl)-2-(3,4-dihydroquinolin-1(2H)-yl)ethyl phenylcarbamate derivatives were designed as inhibitor of HIV-1 RT using the ligand based drug design approach and in-silico evaluated for drug-likeness properties. Designed compounds were synthesized, characterized and in-vitro evaluated for RT inhibitory activity against wild HIV-1 RT. Among these, four compounds (6b, 6i, 6j and 6l) exhibited significant inhibition of HIV-1 RT (IC50 ⩽ 20 μM). Among four compounds, most active compounds 6b and 6j inhibited the RT activity with IC50 8.12 and 5.42 μM respectively. Docking studies of compounds 6b and 6j were performed against wild HIV-1 RT in order to predict their putative binding mode with selected target. Further, cytotoxicity and anti-HIV activity of compounds 6b and 6j were evaluated on T lymphocytes (C8166 cells). All the synthesized compounds were also evaluated for antifungal activity against Candida albicans and Aspergillus niger fungal strains., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The non-nucleoside reverse transcriptase inhibitor efavirenz is a widely prescribed antiretroviral drug used in combined antiretroviral therapy. Despite being an essential and life-saving medication, the required lifelong use of HIV drugs has been associated with a variety of adverse effects, including disturbances in lipid metabolism and increased cardiovascular risk. Efavirenz belongs to those HIV drugs for which cardiovascular and endothelial dysfunctions have been reported. It is here shown that elevated concentrations of efavirenz can inhibit endothelial meshwork formation on extracellular matrix gels by normal and immortalized human umbilical vein cells. This inhibition was associated with an increase in oxidative stress markers, endoplasmic reticulum (ER) stress markers, and autophagy. Induction of ER stress occurred at pharmacologically relevant concentrations of efavirenz and resulted in reduced proliferation and cell viability of endothelial cells, which worsened in the presence of elevated efavirenz concentrations. In combination with the HIV protease inhibitor nelfinavir, both oxidative stress and ER stress became elevated in endothelial cells. These data indicate that pharmacologically relevant concentrations of efavirenz can impair cell viability of endothelial cells and that these effects may be aggravated by either elevated concentrations of efavirenz or by a combined use of efavirenz with other oxidative stress-inducing medications.

There is a growing impetus to develop more accurate, predictive and relevant in vitro models of renal xenobiotic exposure. As part of the EU-FP7, Predict-IV project, a major aim was to develop models that recapitulate not only normal tissue physiology but also aspects of disease conditions that exist as predisposing risk factors for xenobiotic toxicity. Hypoxia, as a common micro-environmental alteration associated with pathophysiology in renal disease, was investigated for its effect on the toxicity profile of a panel of 14 nephrotoxins, using the human proximal tubular epithelial RPTECT/TERT1 cell line. Changes in ATP, glutathione and resazurin reduction, after 14 days of daily repeat exposure, revealed a number of compounds, including adefovir dipivoxil with enhanced toxicity in hypoxia. We observed intracellular accumulation of adefovir in hypoxia and suggest decreases in the efflux transport proteins MRP4, MRP5, NHERF1 and NHERF3 as a possible explanation. MRP5 and NHERF3 were also down-regulated upon treatment with the HIF-1 activator, dimethyloxalylglycine. Interestingly, adefovir dependent gene expression shifted from alterations in cell cycle gene expression to an inflammatory response in hypoxia. The ability to investigate aspects of disease states and their influence on renal toxin handling is a key advantage of in vitro systems developed here. They also allow for detailed investigations into mechanisms of compound toxicity of potential importance for compromised tissue exposure., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The prevention of mother-to-child transmission (MTCT) of HIV is a crucial component in HIV therapy. Nucleoside reverse transcriptase inhibitors (NRTIs), primarily 3'-azido-3'-thymidine (AZT [zidovudine]), have been used to treat both mothers and neonates. While AZT is being replaced with less toxic drugs in treating mothers in MTCT prevention, it is still commonly used to treat neonates. Problems related to mitochondrial toxicity and potential mutagenesis associated with AZT treatment have been reported in treated cohorts. Yet little is known concerning the metabolism and potential toxicity of AZT on embryonic and neonatal tissues, especially considering that the enzymes of nucleoside metabolism change dramatically as many tissues convert from hyperplastic to hypertrophic growth during this period. AZT is known to inhibit thymidine phosphorylation and potentially alter deoxynucleoside triphosphate (dNTP) pools in adults. This study examines the effects of AZT on dNTP pools, mRNA expression of deoxynucleoside/deoxynucleotide metabolic enzymes, and mitochondrial DNA levels in a neonatal rat model. Results show that AZT treatment dramatically altered dNTP pools in the first 7 days of life after birth, which normalized to age-matched controls in the second and third weeks. Additionally, AZT treatment dramatically increased the mRNA levels of many enzymes involved in deoxynucleotide synthesis and mitochondrial biogenesis during the first week of life, which normalized to age-matched controls by the third week. These results were correlated with depletion of mitochondrial DNA noted in the second week. Taken together, results demonstrated that AZT treatment has a powerful effect on the deoxynucleotide synthesis pathways that may be associated with toxicity and mutagenesis., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

The human DNA polymerase gamma (Pol γ) is responsible for DNA replication in mitochondria. Pol γ is particularly susceptible to inhibition by dideoxynucleoside-based inhibitors designed to fight viral infection. Here, we report crystal structures of the replicating Pol γ-DNA complex bound to either substrate or zalcitabine, an inhibitor used for HIV reverse transcriptase. The structures reveal that zalcitabine binds to the Pol γ active site almost identically to the substrate dCTP, providing a structural basis for Pol γ-mediated drug toxicity. When compared to the apo form, Pol γ undergoes intra- and inter-subunit conformational changes upon formation of the ternary complex with primer/template DNA and substrate. We also find that the accessory subunit Pol γB, which lacks intrinsic enzymatic activity and does not contact the primer/template DNA directly, serves as an allosteric regulator of holoenzyme activities. The structures presented here suggest a mechanism for processivity of the holoenzyme and provide a model for understanding the deleterious effects of Pol γ mutations in human disease. Crystal structures of the mitochondrial DNA polymerase, Pol γ, in complex with substrate or antiviral inhibitor zalcitabine provide a basis for understanding Pol γ-mediated drug toxicity., (© 2015 The Authors.)

The nucleoside reverse transcriptase inhibitors (NRTIs), used for treatment of the human immunodeficiency virus-1, compromise mitochondria in cardiomyocytes and other host cells, limiting the clinical use of these drugs. To explore underlying mechanisms, we overexpressed PGC-1α, a master regulator of mitochondrial biogenesis, twofold in H9c2 rat cardiomyocyte cultures, hypothesizing that this might protect the mitochondria from damage induced by the NRTI combination zidovudine (AZT) and didanosine (ddI). The experimental groups, evaluated during 16 passages (P) of drug exposure, included: PGC-1α-overexpressing cells with no exposure, or exposure to 50 µM AZT plus 50 µM ddI; and control cells with no exposure or exposure to the same doses of AZT and ddI. The AZT/ddI combination caused a growth inhibition of 15-20% in control cells, but none in PGC-1α cells. Apoptosis was highest in AZT/ddI-exposed control cells, and PGC-1α overexpression protected cells from AZT/ddI-induced apoptosis. At P3, P6, P8, and P12, uncoupled mitochondrial oxygen consumption rate, determined by Seahorse 24 XF Analyzer, as higher in AZT/ddI-exposed PGC-1α cells, compared to AZT/ddI-exposed control cells (p < 0.05 at all P). Complex I activity was higher in AZT/ddI-exposed PGC-1α overexpressing cells than that in AZT/ddI-exposed control cells (p < 0.05), and reactive oxygen species levels were lower in PGC-1α overexpressing cells than that in control cells (p < 0.05) when both were exposed to AZT/ddI. Taken together, these experiments show proof of concept that overexpression of PGC-1α protects cardiomyocytes from NRTI-induced toxicity, and suggest that a pharmaceutical agent with similar activity may protect against NRTI-induced mitochondrial toxicity.

Today HIV-1 infection is recognized as a chronic disease with obligatory lifelong treatment to keep viral titers below detectable levels. The continuous intake of antiretroviral drugs however, leads to severe and even life-threatening side effects, supposedly by the deleterious impact of nucleoside-analogue type compounds on the functioning of the mitochondrial DNA polymerase. For detailed investigation of the yet partially understood underlying mechanisms, the availability of a versatile model system is crucial. We therefore set out to develop the use of Caenorhabditis elegans to study drug induced mitochondrial toxicity. Using a combination of molecular-biological and functional assays, combined with a quantitative analysis of mitochondrial network morphology, we conclude that anti-retroviral drugs with similar working mechanisms can be classified into distinct groups based on their effects on mitochondrial morphology and biochemistry. Additionally we show that mitochondrial toxicity of antiretroviral drugs cannot be exclusively attributed to interference with the mitochondrial DNA polymerase.

Administration of lersivirine, a nonnucleotide reverse transcriptase inhibitor, daily by oral gavage to Sprague-Dawley rats for up to 2 yr was associated with decreased survival, decreased body weights, and an increase in neoplasms and related proliferative lesions in the liver, thyroid, kidney, and urinary bladder. Thyroid follicular adenoma and carcinoma, the associated thyroid follicular hypertrophy/hyperplasia, hepatocellular adenoma/adenocarcinoma, altered cell foci, and hepatocellular hypertrophy were consistent with lersivirine-related induction of hepatic microsomal enzymes. Renal tubular adenoma and renal tubular hyperplasia were attributed to the lersivirine-related exacerbation of chronic progressive nephropathy (CPN), while urinary bladder hyperplasia and transitional cell carcinoma in the renal pelvis and urinary bladder were attributed to urinary calculi. Renal tubular neoplasms associated with increased incidence and severity of CPN, neoplasms of transitional epithelium attributed to crystalluria, and thyroid follicular and hepatocellular neoplasms related to hepatic enzyme induction have low relevance for human risk assessment., (© 2014 by The Author(s).)

Purpose of Review: Here we present fetal genotoxicity and mitochondrial toxicity, induced by nucleoside reverse transcriptase inhibitors (NRTIs), in HIV-1-infected pregnant women treated to prevent mother-to-child HIV-1 transmission, and in virus-free pregnant patas monkeys., Recent Findings: In the offspring of pregnant patas monkeys given human-equivalent NRTI protocols, aneuploidy was found in cultured bone marrow cells taken at birth, 1, and 3 years of age. In some newborn human infants, the offspring of HIV-1-infected mothers given zidovudine (AZT) therapy, aneuploidy, mitochondrial DNA (mtDNA) depletion, morphologically damaged mitochondria, and reduction in cardiac left ventricular muscle were observed. NRTI-exposed human and patas umbilical cords had similar levels of mtDNA depletion and mitochondrial morphological damage. NRTI-exposed patas offspring showed a compensatory increase in heart mtDNA, and a 50% loss of brain mtDNA at 1 year of age. Mitochondrial morphological damage and mtDNA loss were persistent in blood cells of NRTI-exposed infants up to 2 years of age, and in heart and brain from NRTI-exposed patas up to 3 years of age (human equivalent of 15 years)., Summary: Whereas use of NRTIs in human pregnancy protects many thousands of children worldwide, some HIV-1-uninfected infants born to HIV-1-infected mothers receiving antiretroviral drug therapy sustain toxicities that may have adverse consequences later in life.

Two series of fifteen N-substituted benzyl/phenyl-2-(3,4-dimethyl-5,5-dioxidopyrazolo[4,3-c][1,2]benzothiazin-2(4H)-yl)acetamides were screened for anti-HIV-1 activity and cytotoxicity. The compounds 6a, 6d, 6e, 6g and 6i from the series 6a-i of benzylamides and 7a, 7b, 7c, 7d and 7e from the series 7a-f of anilides were identified as effective anti-HIV-1 agents with EC50 values <20μM. Among these compounds that displayed anti-HIV-1 activity, 6a, 6e, 6g and 6i showed no toxicity in human PBM, CEM and Vero cells, with the exception of 6a which displayed toxicity in Vero cells. Molecular docking of these compounds provided insight into the molecular mechanism and it was found that 6e, 6g and 6i bound deeply in the NNRTI binding pocket of the HIV-1 reverse transcriptase, using RT-bound nevirapine X-ray data and molecular docking for validation, showing the potential of these new structures as inhibitors of this viral enzyme., (Copyright © 2015. Published by Elsevier Ltd.)