Changes in Inflammatory Biomarkers When Switching from Three-Drug Regimens to Dolutegravir Plus Lamivudine in People Living with HIV.

It is not clear if there is a difference between three-drug regimens (3DR) and two-drug regimens (2DR) in terms of suppression of chronic inflammation. We compared C-reactive protein (CRP), CD4+/CD8+ ratio, lipid profiles measured in daily clinical practice before and after the switch to dolutegravir plus lamivudine (DTG/3TC) to examine the difference in the anti-inflammatory effect of 3DR and 2DR. In this single-center retrospective observational study, individuals who were on abacavir/lamivudine/dolutegravir (ABC/3TC/DTG), tenofovir alafenamide/emtricitabine (TAF/FTC) plus DTG, or bictegravir/emtricitabine/tenofovir alafenamide (B/F/TAF) before switching to DTG/3TC were eligible. A total of 119 individuals were enrolled in the study. The median (interquartile range) time since diagnosis of HIV infection was 12 (7-16) years. Overall, inflammation markers such as CD4+/CD8+ ratio, CD4+, CRP, and lipid profiles did not change. Analysis of only individuals who switched from ABC/3TC/DTG, TAF-based regimens also showed no significant changes in inflammatory markers. Since viremia raises inflammatory markers, differences in antiviral efficacy may make a difference in the suppression of chronic inflammation, but in conclusion we did not find any change in inflammatory markers by changing from 3DR to 2DR in daily clinical practice.

Predicting Drug Interactions with Human Equilibrative Nucleoside Transporters 1 and 2 Using Functional Knockout Cell Lines and Bayesian Modeling.

Equilibrative nucleoside transporters (ENTs) 1 and 2 facilitate nucleoside transport across the blood-testis barrier (BTB). Improving drug entry into the testes with drugs that use endogenous transport pathways may lead to more effective treatments for diseases within the reproductive tract. In this study, CRISPR/CRISPR-associated protein 9 was used to generate HeLa cell lines in which ENT expression was limited to ENT1 or ENT2. We characterized uridine transport in these cell lines and generated Bayesian models to predict interactions with the ENTs. Quantification of [3H]uridine uptake in the presence of the ENT-specific inhibitor S-(4-nitrobenzyl)-6-thioinosine (NBMPR) demonstrated functional loss of each transporter. Nine nucleoside reverse-transcriptase inhibitors and 37 nucleoside/heterocycle analogs were evaluated to identify ENT interactions. Twenty-one compounds inhibited uridine uptake and abacavir, nevirapine, ticagrelor, and uridine triacetate had different IC50 values for ENT1 and ENT2. Total accumulation of four identified inhibitors was measured with and without NBMPR to determine whether there was ENT-mediated transport. Clofarabine and cladribine were ENT1 and ENT2 substrates, whereas nevirapine and lexibulin were ENT1 and ENT2 nontransported inhibitors. Bayesian models generated using Assay Central machine learning software yielded reasonably high internal validation performance (receiver operator characteristic > 0.7). ENT1 IC50-based models were generated from ChEMBL; subvalidations using this training data set correctly predicted 58% of inhibitors when analyzing activity by percent uptake and 63% when using estimated-IC50 values. Determining drug interactions with these transporters can be useful in identifying and predicting compounds that are ENT1 and ENT2 substrates and can thereby circumvent the BTB through this transepithelial transport pathway in Sertoli cells. SIGNIFICANCE STATEMENT: This study is the first to predict drug interactions with equilibrative nucleoside transporter (ENT) 1 and ENT2 using Bayesian modeling. Novel CRISPR/CRISPR-associated protein 9 functional knockouts of ENT1 and ENT2 in HeLa S3 cells were generated and characterized. Determining drug interactions with these transporters can be useful in identifying and predicting compounds that are ENT1 and ENT2 substrates and can circumvent the blood-testis barrier through this transepithelial transport pathway in Sertoli cells.

Effects of Reverse Transcriptase Inhibitors on Proliferation, Apoptosis, and Migration in Breast Carcinoma Cells.

High telomerase activity in human breast cancer is associated with aggressive tumors resulting in decreased survival. Recent studies have shown that telomerase inhibitors may display anticancer properties in some human cancer cell lines. In the present study, we examined the effects of 4 reverse transcriptase inhibitors (RTIs), used for the treatment of HIV; Abacavir (AC), Lamivudine (LV), Stavudine (SV), and Tenofovir (TF) on proliferation, apoptosis, and migration in the normal human mammary epithelial cell line, hTERT-HME1, and the human breast cancer cell line, MCF-7. Cells were treated with AC, LV, SV, or TF alone or in combination with paclitaxel (PAC), a known drug used to treat breast cancer. Conduct of the thiazolyl blue tetrazolium bromide assay demonstrated that AC, SV, and TF had stronger cytotoxic effects on MCF-7 cells than in hTERT-HME1 cells. The combined treatment of RTIs and PAC caused high rates of cell death in MCF-7 and low rates of cell death in HTERT-HME1 by apoptosis. The percentages of apoptotic cells in the treatment of AC and SV in combination with PAC for 48 and 72 hours were higher than PAC. Significantly increased apoptosis and decreased migration levels were found in MCF-7 cells treated with AC and co-treatment of AC+PAC or SV+PAC than HME1 cells. These treatments can also prevent migration capacity more than PAC. Therefore, a combination strategy based on telomerase inhibitors such as AC or SV and anticancer drugs may be more effective in the treatment of certain breast cancers.

HIV Antivirals Affect Endothelial Activation and Endothelial-Platelet Crosstalk.

RATIONALE: People living with HIV on effective antiretroviral therapy are at increased risk of cardiovascular complications, possibly due to off-target drug effects. Some studies have associated antiretroviral therapy with increased risk of myocardial infarction and endothelial dysfunction, but a link between endothelial function and antiretrovirals has not been established. OBJECTIVE: To determine the effects of antiretrovirals in common clinical use upon in vitro endothelial function to better understand cardiovascular risk in people living with HIV. METHODS AND RESULTS: Human umbilical cord vein endothelial cells or human coronary artery endothelial cells were pretreated with the antiretrovirals abacavir sulphate (ABC), tenofovir disoproxil fumarate, or tenofovir alafenamide. Expression of adhesion molecules, ectonucleotidases (CD39 and CD73), tissue factor (TF), endothelial-derived microparticle (EMP) numbers and phenotype, and platelet activation were evaluated by flow cytometry. TF and ectonucleotidase activities were measured using colourimetric plate-based assays. ABC-treated endothelial cells had higher levels of ICAM (intercellular adhesion molecule)-1 and TF expression following TNF (tumor necrosis factor)-α stimulation. In contrast, tenofovir disoproxil fumarate and tenofovir alafenamide treatment gave rise to greater populations of CD39+CD73+ cells. These cell surface differences were also observed within EMP repertoires. ABC-treated cells and EMP had greater TF activity, while tenofovir disoproxil fumarate- and tenofovir alafenamide-treated cells and EMP displayed higher ectonucleotidase activity. Finally, EMP isolated from ABC-treated cells enhanced collagen-evoked platelet integrin activation and α-granule release. CONCLUSIONS: We report differential effects of antiretrovirals used in the treatment of HIV upon endothelial function. ABC treatment led to an inflammatory, prothrombotic endothelial phenotype that promoted platelet activation. In contrast, tenofovir disoproxil fumarate and tenofovir alafenamide conferred potentially cardioprotective properties associated with ectonucleotidase activity. These observations establish a link between antiretrovirals and specific functional effects that provide insight into cardiovascular disease in people living with HIV.

Nucleotide Analogues as Inhibitors of SARS-CoV-2 Polymerase, a Key Drug Target for COVID-19.

SARS-CoV-2 is responsible for the current COVID-19 pandemic. On the basis of our analysis of hepatitis C virus and coronavirus replication, and the molecular structures and activities of viral inhibitors, we previously demonstrated that three nucleotide analogues (the triphosphates of Sofosbuvir, Alovudine, and AZT) inhibit the SARS-CoV RNA-dependent RNA polymerase (RdRp). We also demonstrated that a library of additional nucleotide analogues terminate RNA synthesis catalyzed by the SARS-CoV-2 RdRp, a well-established drug target for COVID-19. Here, we used polymerase extension experiments to demonstrate that the active triphosphate form of Sofosbuvir (an FDA-approved hepatitis C drug) is incorporated by SARS-CoV-2 RdRp and blocks further incorporation. Using the molecular insight gained from the previous studies, we selected the active triphosphate forms of six other antiviral agents, Alovudine, Tenofovir alafenamide, AZT, Abacavir, Lamivudine, and Emtricitabine, for evaluation as inhibitors of the SARS-CoV-2 RdRp and demonstrated the ability of these viral polymerase inhibitors to be incorporated by SARS-CoV-2 RdRp, where they terminate further polymerase extension with varying efficiency. These results provide a molecular basis for inhibition of the SARS-CoV-2 RdRp by these nucleotide analogues. If sufficient efficacy of some of these FDA-approved drugs in inhibiting viral replication in cell culture is established, they may be explored as potential COVID-19 therapeutics.

A library of nucleotide analogues terminate RNA synthesis catalyzed by polymerases of coronaviruses that cause SARS and COVID-19.

SARS-CoV-2, a member of the coronavirus family, is responsible for the current COVID-19 worldwide pandemic. We previously demonstrated that five nucleotide analogues inhibit the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp), including the active triphosphate forms of Sofosbuvir, Alovudine, Zidovudine, Tenofovir alafenamide and Emtricitabine. We report here the evaluation of a library of nucleoside triphosphate analogues with a variety of structural and chemical features as inhibitors of the RdRps of SARS-CoV and SARS-CoV-2. These features include modifications on the sugar (2' or 3' modifications, carbocyclic, acyclic, or dideoxynucleotides) or on the base. The goal is to identify nucleotide analogues that not only terminate RNA synthesis catalyzed by these coronavirus RdRps, but also have the potential to resist the viruses' exonuclease activity. We examined these nucleotide analogues for their ability to be incorporated by the RdRps in the polymerase reaction and to prevent further incorporation. While all 11 molecules tested displayed incorporation, 6 exhibited immediate termination of the polymerase reaction (triphosphates of Carbovir, Ganciclovir, Stavudine and Entecavir; 3'-OMe-UTP and Biotin-16-dUTP), 2 showed delayed termination (Cidofovir diphosphate and 2'-OMe-UTP), and 3 did not terminate the polymerase reaction (2'-F-dUTP, 2'-NH2-dUTP and Desthiobiotin-16-UTP). The coronaviruses possess an exonuclease that apparently requires a 2'-OH at the 3'-terminus of the growing RNA strand for proofreading. In this study, all nucleoside triphosphate analogues evaluated form Watson-Crick-like base pairs. The nucleotide analogues demonstrating termination either lack a 2'-OH, have a blocked 2'-OH, or show delayed termination. Thus, these nucleotide analogues are of interest for further investigation to evaluate whether they can evade the viral exonuclease activity. Prodrugs of five of these nucleotide analogues (Cidofovir, Abacavir, Valganciclovir/Ganciclovir, Stavudine and Entecavir) are FDA-approved medications for treatment of other viral infections, and their safety profiles are well established. After demonstrating potency in inhibiting viral replication in cell culture, candidate molecules can be rapidly evaluated as potential therapies for COVID-19.

Detection of Abacavir-Induced Structural Alterations in Human Leukocyte Antigen-B*57 : 01 Using Phage Display.

The interaction of human leukocyte antigen (HLA) with specific drugs is associated with delayed-type hypersensitivity reactions, which cause severe cutaneous toxicity. Such interactions induce structural alterations in HLA complexes via several different mechanisms such as the hapten theory, p-i concept, and altered peptide repertoire model, leading to the activation of cytotoxic T cells. To date, comprehensive detection of such structural alterations in preclinical studies has been difficult. Here, we evaluated structural alterations in HLA complexes focusing on the interaction between the HLA-B*57 : 01 allele and abacavir (an anti-human immunodeficiency virus drug), representing a model of abacavir hypersensitivity syndrome induced by changes in the peptide repertoire on the HLA molecule. We employed a phage display method using a commercially available antibody library to screen specific phage antibodies able to recognize HLA-B*57 : 01. The affinity of selected phage antibodies increased because of structural alterations in HLA-B*57 : 01 following exposure to abacavir, indicating that specific phage antibodies can identify drug-mediated structural changes in HLA complexes. We also identified an unreported structural change in HLA-B*57 : 01 using the phage display method, whereby abacavir increased the expression of peptide-deficient HLA-B*57 : 01 on the cell surface. These results suggest that phage display technology is a useful method for detecting structural changes in HLA complexes. This technology represents a potential novel strategy for predicting HLA-associated hypersensitivity reactions by drugs in pre-clinical studies.

In silico, in ovo and in vitro antiviral efficacy of phosphorylated derivatives of abacavir: an experimental approach.

Outstanding increase of oral absorption, bioavailability, and antiviral efficacy of phosphorylated nucleosides and basic antiviral influence of abacavir is the central idea for the development of new series of phosphorylated abacavir (ABC) derivatives. The designed compounds were primarily screened for antiviral nature against HN protein of NDV and VP7 protein of BTV using the molecular environment approach. Out of all the designed compounds, the compounds which are having higher binding energies against these two viral strains were prompted for the synthesis of the target compounds (5A-K). Among the synthesized title compounds (5A-K), the compounds which have exhibited higher dock scores akin to the rest of the compounds were then selected and screened for the antiviral activity against NDV and BTV infected embryonated eggs and BHK 21 cell lines through the in ovo and in vitro approaches. The results revealed that all the designed compounds have formed higher binding energies against both the targets. Among all, the compounds which are selected based on their dock scores such as 5A, 5F, 5G, 5H, 5I, and 5K against NDV and 5J, 5E, 5I, 5C, 5A, and 5K against BTV have shown significant antiviral activity against HN protein of NDV, VP7 protein of Bluetongue virus in both NDV- and BTV-treated embryonated eggs and BHK 21 cell lines. Hence, it is concluded that, the best lead compounds will stand as the potential antiviral agents and prompted them as virtuous therapeutics against NDV and BTV in future.

Abacavir induces the transcriptional activity of YY1 and other oncogenic transcription factors in gastric cancer cells.

Yin Yang 1 (YY1) is a ubiquitous transcription factor with both transcriptional activating and repressing functions. Targeting YY1 is considered as a potential therapeutic strategy for several malignancies. Telomerase Reverse Transcriptase (TERT) is also considered as a potential target for cancer therapeutics. To enable the large-scale screening and identification of potential YY1 targeting drugs, a gastric cancer cell line-based drug screening assay was developed. In a YY1 targeted drug repurpose screen, abacavir sulfate, a nucleoside analog reverse transcriptase inhibitor, known to target TERT was identified to show the feature of activating YY1 mediated transcription. We further explored i) the molecular targets of abacavir, ii) activation pattern of pathways regulated by abacavir in gastric tumors, and iii) therapeutic potential of abacavir for gastric cancer cells. Oncogenic signaling pathways like MYC, HIF1-α, ERK, WNT, E2F, NFκB and NRF1/2 were also found to be highly activated by abacavir. Abacavir was found to have less impact on the viability of gastric cancer cells. Across gastric tumors, we observed the co-activation of TERT, alternative lengthening of telomere (ALT), DNA repair, and the oncogenic pathways MYC, E2F/DP1, ERK, YY1, HIF1α, and NFκB specific gene-sets, in a subset of gastric tumors. The observed connectivity among TERT, DNA repair, and multiple oncogenic pathways indicate the need for the development of combinatorial therapeutics for the gastric tumors with the activated TERT.

Phosphorylated abacavir analogue (ABC-1) has ameliorative action against Newcastle disease virus induced pathogenesis in chicken.

Newcastle disease virus (NDV) causes huge economic loss to the poultry industry due to high mortality and morbidity. The present study aimed to assess the protective role of novel phosphorylated analogue ABC-1 in vivo in NDV-infected chickens through the inhibition of fusion protein. Both NDV-induced oxidative damage and protective role of novel phosphorylated ABC-1 were evaluated in vital organs such as the liver and lung of chickens. Enzyme linked immunosorbent assay (ELISA) results showed that protein oxidation and nitration levels were significantly raised in NDV-infected tissues compared to healthy controls, whereas these levels were reduced significantly (P < 0.05) in birds treated with phosphorylated compounds compared to the NDV-infected group alone. Additional investigation with double immunofluorescence showed that the large amount of immuno colocalization and Western blot analysis also confirmed this observation through its band pattern in NDV-infected birds compared to healthy birds, whereas these alterations were reduced in treatment with novel phosphorylated ABC-1. The expression of fusion glycoprotein was studied by immuno colocalization, PCR, and flow cytometry, and results demonstrated that the novel phosphorylated analogues reduced the expression of fusion glycoprotein. These results put forth that novel phosphorylated ABC-1 protects chickens from NDV-induced pathogenesis, protein oxidation/nitration, and exerts potent antiviral activity.

The Role of Conformational Dynamics in Abacavir-Induced Hypersensitivity Syndrome.

Abacavir is an antiretroviral drug used to reduce human immunodeficiency virus (HIV) replication and decrease the risk of developing acquired immune deficiency syndrome (AIDS). However, its therapeutic value is diminished by the fact that it is associated with drug hypersensitivity reactions in up to 8% of treated patients. This hypersensitivity is strongly associated with patients carrying human leukocyte antigen (HLA)-B*57:01, but not patients carrying closely related alleles. Abacavir's specificity to HLA-B*57:01 is attributed to its binding site within the peptide-binding cleft and subsequent influence of the repertoire of peptides that can bind HLA-B*57:01. To further our understanding of abacavir-induced hypersensitivity we used molecular dynamics (MD) to analyze the dynamics of three different peptides bound to HLA-B*57:01 in the presence and absence of abacavir or abacavir analogues. We found that abacavir and associated peptides bind to HLA-B*57:01 in a highly diverse range of conformations that are not apparent from static crystallographic snapshots, but observed no difference in either the conformations, nor degree of flexibility when compared to abacavir-unbound systems. Our results support hypersensitivity models in which abacavir-binding alters the conformational ensemble of neopeptides, so as to favour exposed peptide surfaces that are no longer recognized as self by circulating CD8+ T cells, and are conducive to TCR binding. Our findings highlight the need to also consider the role of dynamics in understanding drug-induced hypersensitivities at the molecular and mechanistic level. This additional insight can help inform the chemical modification of abacavir to prevent hypersensitivity reactions in HLA-B*57:01+ HIV patients whilst retaining potent antiretroviral activity.

Safety and tolerability of Triumeq in amyotrophic lateral sclerosis: the Lighthouse trial.

Background: Neuroinflammation and human endogenous retroviruses (HERV) are thought to have a role in the pathophysiology of amyotrophic lateral sclerosis (ALS). Therapy directed against endogenous retroviruses has demonstrated positive effects during in vitro and biomarker studies. Consequently, the present study was undertaken to assess the safety and tolerability of long-term antiretroviral therapy (ART), Triumeq (abacavir, lamivudine, and dolutegravir) exposure in patients with ALS, and efficacy against biomarkers of disease progression. Methods: Patients were observed during a 10-week lead-in period before receiving Triumeq treatment for 24 weeks at four specialist ALS centers. The primary outcomes were safety and tolerability. Secondary outcomes included HERV-K expression levels, urinary p75ECD levels, neurophysiological parameters, and clinical indicators. The ENCALS prediction model was applied to provide an estimate of the cohort survival. The trial was registered (NCT02868580). Findings: 40 patients with ALS received Triumeq and 35 (88%) completed treatment. There were no drug-related serious adverse events; one patient was withdrawn from the study due to a drug-associated increase in liver enzymes. A favorable response on HERV-K expression levels was observed, accompanied by a decline in ALSFRS-R progression rate of 21.8% (95% CI -4.8%-48.6%) and the amount of urinary p75ECD measured. One patient died five months after stopping treatment, while five were expected to have died during the treatment period (interquartile range 2-8). Interpretation: Long-term Triumeq exposure was safe and well tolerated in this cohort. There was suggestive indication for a possible biological response in some pharmacodynamic and clinical biomarkers. A larger international phase 3 trial will be deployed to assess the effect of Triumeq on overall survival and disease progression. Funding: Funding was provided by the FightMND Foundation; MND Research Institute of Australia; MND Association, United Kingdom, and GSK. ViiV Healthcare provided the Triumeq.

Trends in HIV-1 Drug Resistance Mutations from a U.S. Reference Laboratory from 2006 to 2017.

Trends in resistance to antiretroviral drugs for HIV-1 may inform clinical support and drug development. We evaluated drug resistance mutation (DRM) trends for nucleoside reverse transcriptase inhibitor (NRTI), non-nucleoside reverse transcriptase inhibitor (NNRTI), protease inhibitor (PI), and integrase strand transfer inhibitor (INSTI) in a large U.S. reference laboratory database. DRMs with a Stanford HIV Drug Resistance Database mutation score ≥10 from deidentified subtype B NRTI/NNRTI/PI specimens (2006-2017; >10,000/year) and INSTI specimens (2010-2017; >1,000/year) were evaluated. Sequences with NRTI, NNRTI, or PI single- or multiclass DRMs declined from 48.9% to 39.3%. High-level dual- and triple-class resistance declined from 43.3% (2006) to 17.1% (2017), while sequences with only single-class DRMs increased from 40.0% to 52.9%. The prevalence of DRMs associated with earlier treatment regimens declined, while prevalence of some DRMs associated with newer regimens increased. M184V/I decreased from 48.3% to 29.4%. K103N/S/T declined from 42.5% in 2012 to 36.4% in 2017. Rilpivirine and etravirine DRMs E138A/Q/R and E138K increased from 4.9% and 0.4% to 9.7% and 1.7%, respectively. Sequences with ≥1 darunavir DRM declined from 18.1% to 4.8% by 2017. INSTI DRM Q148H/K/R declined from 39.3% (2010) to 13.8% (2017). Prevalence of elvitegravir-associated DRMs T66A/I/K, E92Q, S147G, and the dolutegravir-associated DRM R263K increased. For a subset of patients with serial testing, 50% (2,646/5,290) of those who initially had no reportable DRM subsequently developed ≥1 DRM for NRTI/NNRTI/PI and 49.7% (159/320) for INSTI. These trends may inform the need for baseline genotypic resistance testing. The detection of treatment-emergent DRMs in serially tested patients confirms the value of genotypic testing following virologic failure.

Synthesis and Characterization of Specific Reverse Transcriptase Inhibitors for Mammalian LINE-1 Retrotransposons.

Retrotransposons are a type of transposable element (TE) that have amplified to astonishing numbers in mammalian genomes, comprising more than a third of the human and mouse genomes. Long interspersed element class 1 (LINE-1 or L1) retrotransposons are abundant and currently active retroelements in the human and mouse genomes. Similarly, long terminal repeat (LTR)-containing retrotransposons are abundant in both genomes, although only active in mice. LTR- and LINE-1-retroelements use different mechanisms for retrotransposition, although both involve the reverse transcription of an intermediate retroelement-derived RNA. Retrotransposon activity continues to effect the germline and somatic genomes, generating interindividual variability over evolution and potentially influencing cancer and brain physiology, respectively. However, relatively little is known about the functional consequences of retrotransposition. In this study, we have synthesized and characterized reverse transcriptase inhibitors specific for mammalian LINE-1 retrotransposons, which might help deciphering the functional impact of retrotransposition in vivo.

Synthesis and anti-HIV activity of L-2',3'-Dideoxy-4'-selenonucleosides (L-4'-Se-ddNs).

Based on the potent anti-HIV activity of L-2',3'-dideoxycytidine (L-ddC), L-2',3'-dideoxy-4'-selenonucleosides (L-4'-Se-ddNs) have been synthesized from natural chiral template, L-glutamic acid, using Pummerer-type condensation as a key step. All synthesized compounds were assayed for anti-HIV-1 activity, but none of them did show any significant antiviral activity up to 100 µM, probably due to conformational differences between L-ddC and L-4'-Se-ddC, induced by the bulky selenium atom, which might play an important role in phosphorylation by cellular kinase.

Morphine counteracts the antiviral effect of antiretroviral drugs and causes upregulation of p62/SQSTM1 and histone-modifying enzymes in HIV-infected astrocytes.

Accelerated neurological disorders are increasingly prominent among the HIV-infected population and are likely driven by the toxicity from long-term use of antiretroviral drugs. We explored potential side effects of antiretroviral drugs in HIV-infected primary human astrocytes and whether opioid co-exposure exacerbates the response. HIV-infected human astrocytes were exposed to the reverse transcriptase inhibitor, emtricitabine, alone or in combination with two protease inhibitors ritonavir and atazanavir (ERA) with and without morphine co-exposure. The effect of the protease inhibitor, lopinavir, alone or in combination with the protease inhibitor, abacavir, and the integrase inhibitor, raltegravir (LAR), with and without morphine co-exposure was also explored. Exposure with emtricitabine alone or ERA in HIV-infected astrocytes caused a significant decrease in viral replication and attenuated HIV-induced inflammatory molecules, while co-exposure with morphine negated the inhibitory effects of ERA, leading to increased viral replication and inflammatory molecules. Exposure with emtricitabine alone or in combination with morphine caused a significant disruption of mitochondrial membrane integrity. Genetic analysis revealed a significant increase in the expression of p62/SQSTM1 which correlated with an increase in the histone-modifying enzyme, ESCO2, after exposure with ERA alone or in combination with morphine. Furthermore, several histone-modifying enzymes such as CIITA, PRMT8, and HDAC10 were also increased with LAR exposure alone or in combination with morphine. Accumulation of p62/SQSTM1 is indicative of dysfunctional lysosomal fusion. Together with the loss of mitochondrial integrity and epigenetic changes, these effects may lead to enhanced viral titer and inflammatory molecules contributing to the neuropathology associated with HIV.

Pharmacological impact of antiretroviral therapy on platelet function to investigate human immunodeficiency virus-associated cardiovascular risk.

BACKGROUND AND PURPOSE: Some clinical studies have reported increased myocardial infarction in people living with human immunodeficiency virus (HIV) taking the antiretroviral abacavir sulphate (ABC). Given that clinical studies contain confounding variables (e.g., HIV-associated factors), we investigated the pharmacological effects of antiretrovirals on platelet function in HIV-negative volunteers in order to identify mechanisms of increased cardiovascular risk. EXPERIMENTAL APPROACH: Platelets were isolated from healthy volunteers and HIV-negative subjects enrolled on a Phase I clinical trial and platelet function evaluated using aggregometry and flow cytometry. In vivo platelet thromboembolism was monitored in anaesthetized mice. KEY RESULTS: Human platelet aggregation was unaffected by all antiretrovirals tested, but ABC treatment led uniquely to increased platelet granule release. ABC also interrupted NO-mediated inhibition of platelet aggregation and increased in vivo aggregation in mice. Another antiretroviral, tenofovir, did not affect platelet function. Furthermore, aggregation and activation of platelets isolated from 20 subjects taking clinically relevant doses of tenofovir were comparable to baseline samples. CONCLUSIONS AND IMPLICATIONS: ABC can enhance platelet activation, independently of variables that confound clinical studies, suggesting a potential pharmacological effect that is absent with tenofovir. Mechanistically, we propose that ABC enhances platelet degranulation and interrupts NO-mediated platelet inhibition. The interaction of ABC with NO signalling is demonstrated by ABC-mediated enhancement of aggregation in vivo and in vitro that persisted in the presence of NO. Although an association between ABC and platelet activation has not been confirmed in patients, these findings provide evidence of a mechanistic link between platelet activation and antiretroviral therapy.

Synthesis of 1',2'-methano-2',3'-dideoxynucleosides as potential antivirals.

The synthesis of constrained nucleosides has become an important tool to understand the SAR in the interaction between biological and synthetic nucleotides in the context of antisense oligonucleotide therapy. The incorporation of a cyclopropane into a furanose ring of a nucleoside induces some degree of constrain without affecting significantly the steric environment of a nucleoside. Here, we report a new, short and stereocontrolled synthesis of two constrained nucleosides analogues, 1',2'- methano-2',3'-dideoxyuridine 9, and the corresponding cytidine analog 12. X-ray crystallography revealed that the furanose ring in the constrained uridine and cytidine analogues was flattened with virtual loss of pseudorotation. The phosphoramidate esters of the novel constrained uridine and cytidine nucleosides, intended as prodrugs, were tested in cell-based assays for viral replication across the herpes virus family and HIV inhibition courtesy of Merck laboratories, Rahway. They were also tested in antiproliferative assays against colorectal and melanoma cell lines. Unfortunately, none of the compounds showed activity in these assays.

Alternative switching strategies based on regimens with a low genetic barrier: do clinicians have a choice nowadays?

Clinicians sometimes use switching strategies based on regimens such as RAL + ABC/3TC or RPV + ABC/3TC in order to resolve tolerability or safety issues associated with conventional recommended first-line strategies. Despite the low genetic barrier of these regimens, high safety and efficacy rates have been reported in retrospective studies.