Novel method to quantify phenotypic markers of HIV-associated neurocognitive disorder in a murine SCID model.

Despite combined antiretroviral therapy (cART), HIV infection in the CNS persists with reported increases in activation of macrophages (MΦ), microglia, and surrounding astrocytes/neurons, conferring HIV-induced inflammation. Chronic inflammation results in HIV-associated neurocognitive disorders (HAND) with reported occurrence of up to half of individuals with HIV infection. The existing HAND mouse model used by laboratories including ours, and the effect of novel agents on its pathology present with labor-intensive and time-consuming limitations since brain sections and immunohistochemistry assays have to be performed and analyzed. A novel flow cytometry-based system to objectively quantify phenotypic effects of HIV using a SCID mouse HAND model was developed which demonstrated that the HIV-infected mice had significant increases in astrogliosis, loss of neuronal dendritic marker, activation of murine microglia, and human macrophage explants compared to uninfected control mice. HIV p24 could also be quantified in the brains of the infected mice. Correlation of these impairments with HIV-induced brain inflammation and previous behavioral abnormalities studies in mice suggests that this model can be used as a fast and relevant throughput methodology to quantify preclinical testing of novel treatments for HAND.

Enhanced antiretroviral therapy in rhesus macaques improves RT-SHIV viral decay kinetics.

Using an established nonhuman primate model, rhesus macaques were infected intravenously with a chimeric simian immunodeficiency virus (SIV) consisting of SIVmac239 with the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase from clone HXBc2 (RT-SHIV). The impacts of two enhanced (four- and five-drug) highly active antiretroviral therapies (HAART) on early viral decay and rebound were determined. The four-drug combination consisted of an integrase inhibitor, L-870-812 (L-812), together with a three-drug regimen comprising emtricitabine [(-)-FTC], tenofovir (TFV), and efavirenz (EFV). The five-drug combination consisted of one analog for each of the four DNA precursors {using TFV, (-)-FTC, (-)-ß-D-(2R,4R)-1,3-dioxolane-2,6-diaminopurine (amdoxovir [DAPD]), and zidovudine (AZT)}, together with EFV. A cohort treated with a three-drug combination of (-)-FTC, TFV, and EFV served as treated controls. Daily administration of a three-, four-, or five-drug combination of antiretroviral agents was initiated at week 6 or 8 after inoculation and continued up to week 50, followed by a rebound period. Plasma samples were collected routinely, and drug levels were monitored using liquid chromatography-tandem mass spectrometry (LC-MS-MS). Viral loads were monitored with a standard TaqMan quantitative reverse transcriptase PCR (qRT-PCR) assay. Comprehensive analyses of replication dynamics were performed. RT-SHIV infection in rhesus macaques produced typical viral infection kinetics, with untreated controls establishing persistent viral loads of >10(4) copies of RNA/ml. RT-SHIV loads at the start of treatment (V0) were similar in all treated cohorts (P > 0.5). All antiretroviral drug levels were measureable in plasma. The four-drug and five-drug combination regimens (enhanced HAART) improved suppression of the viral load (within 1 week; P < 0.01) and had overall greater potency (P < 0.02) than the three-drug regimen (HAART). Moreover, rebound viremia occurred rapidly following cessation of any treatment. The enhanced HAART (four- or five-drug combination) showed significant improvement in viral suppression compared to the three-drug combination, but no combination was sufficient to eliminate viral reservoirs.

Why Certain Repurposed Drugs Are Unlikely to Be Effective Antivirals to Treat SARS-CoV-2 Infections.

Most repurposed drugs have proved ineffective for treating COVID-19. We evaluated median effective and toxic concentrations (EC50, CC50) of 49 drugs, mostly from previous clinical trials, in Vero cells. Ratios of reported unbound peak plasma concentrations, (Cmax)/EC50, were used to predict the potential in vivo efficacy. The 20 drugs with the highest ratios were retested in human Calu-3 and Caco-2 cells, and their CC50 was determined in an expanded panel of cell lines. Many of the 20 drugs with the highest ratios were inactive in human Calu-3 and Caco-2 cells. Antivirals effective in controlled clinical trials had unbound Cmax/EC50 ≥ 6.8 in Calu-3 or Caco-2 cells. EC50 of nucleoside analogs were cell dependent. This approach and earlier availability of more relevant cultures could have reduced the number of unwarranted clinical trials.

Evaluation of single and combination therapies with tenofovir disoproxil fumarate and emtricitabine in vitro and in a robust mouse model supporting high levels of hepatitis B virus replication.

Next-generation therapies for chronic hepatitis B virus (HBV) infection will involve combinations of established and/or experimental drugs. The current study investigated the in vitro and in vivo efficacy of tenofovir disoproxil fumarate (TDF) and/or emtricitabine [(-)-FTC] alone and in combination therapy for HBV infection utilizing the HepAD38 system (human hepatoblastoma cells transfected with HBV). Cellular pharmacology studies demonstrated increased levels of (-)-FTC triphosphate with coincubation of increasing concentrations of TDF, while (-)-FTC had no effect on intracellular tenofovir (TFV) diphosphate levels. Quantification of extracellular HBV by real-time PCR from hepatocytes demonstrated the anti-HBV activity with TDF, (-)-FTC, and their combination. Combination of (-)-FTC with TDF or TFV (ratio, 1:1) had a weighted average combination index of 0.7 for both combination sets, indicating synergistic antiviral effects. No cytotoxic effects were observed with any regimens. Using an in vivo murine model which develops robust HBV viremia in nude mice subcutaneously injected with HepAD38 cells, TDF (33 to 300 mg/kg of body weight/day) suppressed virus replication for up to 10 days posttreatment. At 300 mg/kg/day, (-)-FTC strongly suppressed virus titers to up to 14 days posttreatment. Combination therapy (33 mg/kg/day each drug) sustained suppression of virus titer/ml serum (<1 log(10) unit from pretreatment levels) at 14 days posttreatment, while single-drug treatments yielded virus titers 1.5 to 2 log units above the initial virus titers. There was no difference in mean alanine aminotransferase values or mean wet tumor weights for any of the groups, suggesting a lack of drug toxicity. TDF-(-)-FTC combination therapy provides more effective HBV suppression than therapy with each drug alone.

Tenofovir renal proximal tubular toxicity is regulated by OAT1 and MRP4 transporters.

Tenofovir disoproxil fumarate (TDF) is an oral prodrug and acyclic nucleotide analog of adenosine monophosphate that inhibits HIV-1 (HIV) reverse transcriptase. A growing subset of TDF-treated HIV(+) individuals presented with acute renal failure, suggesting tenofovir-associated kidney-specific toxicity. Our previous studies using an HIV transgenic mouse model (TG) demonstrated specific changes in renal proximal tubular mitochondrial DNA (mtDNA) abundance. Nucleosides are regulated in biological systems via transport and metabolism in cellular compartments. In this study, the role(s) of organic anion transporter type 1 (OAT1) and multidrug-resistant protein type 4 (MRP4) in transport and regulation of tenofovir in proximal tubules were assessed. Renal toxicity was assessed in kidney tissues from OAT1 knockout (KO) or MRP4 KO compared with wild-type (WT, C57BL/6) mice following treatment with TDF (0.11 mg/day), didanosine (ddI, a related adenosine analog, 0.14 mg/day) or vehicle (0.1 M NaOH) daily gavage for 5 weeks. Laser-capture microdissection (LCM) was used to isolate renal proximal tubules for molecular analyses. mtDNA abundance and ultrastructural pathology were analyzed. mtDNA abundance in whole kidneys from both KO and WT was unchanged regardless of treatment. Renal proximal tubular mtDNA abundance from OAT1 KO also remained unchanged, suggesting prevention of TDF toxicity due to loss of tenofovir transport into proximal tubules. In contrast, renal proximal tubules from MRP4 KO exhibited increased mtDNA abundance following TDF treatment compared with WT littermates, suggesting compensation. Renal proximal tubules from TDF-treated WT and MRP4 KO exhibited increased numbers of irregular mitochondria with sparse, fragmented cristae compared with OAT1 KO. Treatment with ddI had a compensatory effect on mtDNA abundance in OAT1 KO but not in MRP4 KO. Both OAT1 and MRP4 have a direct role in transport and efflux of tenofovir, regulating levels of tenofovir in proximal tubules. Disruption of OAT1 activity prevents tenofovir toxicity but loss of MRP4 can lead to increased renal proximal tubular toxicity. These data help to explain mechanisms of human TDF renal toxicity.

Studies on the Efficacy, Potential Cardiotoxicity and Monkey Pharmacokinetics of GLP-26 as a Potent Hepatitis B Virus Capsid Assembly Modulator.

While treatment options are available for hepatitis B virus (HBV), there is currently no cure. Anti-HBV nucleoside analogs and interferon-alpha 2b rarely clear HBV covalently closed circular DNA (cccDNA), requiring lifelong treatment. Recently, we identified GLP-26, a glyoxamide derivative which modulates HBV capsid assembly. The impact of GLP-26 on viral replication and integrated DNA was assessed in an HBV nude mouse model bearing HBV transfected AD38 xenografts. At day 45 post-infection, GLP-26 reduced HBV titers by 2.3-3 log10 versus infected placebo-treated mice. Combination therapy with GLP-26 and entecavir reduced HBV log10 titers by 4.6-fold versus placebo. Next, we examined the pharmacokinetics (PK) in cynomolgus monkeys administered GLP-26 via IV (1 mg/kg) or PO (5 mg/kg). GLP-26 was found to have 34% oral bioavailability, with a mean input time of 3.17 h. The oral dose produced a mean peak plasma concentration of 380.7 ng/mL, observed 0.67 h after administration (~30-fold > in vitro EC90 corrected for protein binding), with a mean terminal elimination half-life of 2.4 h and a mean area under the plasma concentration versus time curve of 1660 ng·hr/mL. GLP-26 was 86.7% bound in monkey plasma. Lastly, GLP-26 demonstrated a favorable toxicity profile confirmed in primary human cardiomyocytes. Thus, GLP-26 warrants further preclinical development as an add on to treatment for HBV infection.

Comparison of anti-SARS-CoV-2 activity and intracellular metabolism of remdesivir and its parent nucleoside.

Remdesivir, a monophosphate prodrug of nucleoside analog GS-441524, is widely used for the treatment of moderate to severe COVID-19. It has been suggested to use GS-441524 instead of remdesivir in the clinic and in new inhalation formulations. Thus, we compared the anti-SARS-CoV-2 activity of remdesivir and GS-441524 in Vero E6, Vero CCL-81, Calu-3, Caco-2 â€‹cells, and anti-HCoV-OC43 activity in Huh-7 â€‹cells. We also compared the cellular pharmacology of these two compounds in Vero E6, Vero CCL-81, Calu-3, Caco-2, Huh-7, 293T, BHK-21, 3T3 and human airway epithelial (HAE) cells. Overall, remdesivir exhibited greater potency and superior intracellular metabolism than GS-441524 except in Vero E6 and Vero CCL-81 â€‹cells.

Pharmacokinetics of Ruxolitinib in HIV Suppressed Individuals on Antiretroviral Agent Therapy from the ACTG A5336 Study.

Ruxolitinib is a US Food and Drug Administration-approved orally administered Janus kinase (1/2) inhibitor that reduces cytokine-induced inflammation. As part of a randomized, phase 2, open-label trial, ruxolitinib (10 mg twice daily) was administered to HIV-positive, virologically suppressed individuals (33 men, 7 women) on antiretroviral therapy (ART) for 5 weeks. Herein, we report the population PK subsequently determined from this study. Plasma concentrations of ruxolitinib (294 samples) and antiretroviral agents were measured at week 1 (N = 39 participants) and week 4 or 5 (N = 37). Ruxolitinib PK was adequately described with a 2-compartment model with first-order absorption and elimination with distribution volumes normalized to mean body weight (91.5 kg) and a separate typical clearance for participants administered efavirenz (a known cytochrome P450 3A4 inducer). Participants administered an ART regimen with efavirenz had an elevated typical apparent oral clearance versus the integrase inhibitor regimen group (22.5 vs 12.9 L/hr; N = 14 vs 25). Post hoc predicted apparent oral clearance was likewise more variable and higher (P < .0001) in those administered efavirenz. There was  an ≈25% variation in ruxolitinib plasma exposures between week 1 and week 4/5. ART plasma concentrations resembled those from PK studies without ruxolitinib. Therefore, integrase inhibitor-based ART regimens may be preferred over efavirenz-based regimens when ruxolitinib is administered to HIV-positive individuals.

Transgenic cardiac-targeted overexpression of human thymidylate kinase.

Thymidylate kinase (TMPK) is a nucleoside monophosphate kinase that catalyzes phosphorylation of thymidine monophosphate to thymidine diphosphate. TMPK also mediates phosphorylation of monophosphates of thymidine nucleoside analog (NA) prodrugs on the pathway to their active triphosphate antiviral or antitumor moieties. Novel transgenic mice (TG) expressing human (h) TMPK were genetically engineered using the alpha-myosin heavy chain promoter to drive its cardiac-targeted overexpression. In '2 by 2' protocols, TMPK TGs and wild-type (WT) littermates were treated with the NA zidovudine (a deoxythymidine analog, 3'-azido-3'deoxythymidine (AZT)) or vehicle for 35 days. Alternatively, TGs and WTs were treated with a deoxycytidine NA (racivir, RCV) or vehicle. Changes in mitochondrial DNA (mtDNA) abundance and mitochondrial ultrastructure were defined quantitatively by real-time PCR and transmission electron microscopy, respectively. Cardiac performance was determined echocardiographically. Results showed TMPK TGs treated with either AZT or RCV exhibited decreased cardiac mtDNA abundance. Cardiac ultrastructural changes were seen only with AZT. AZT-treated TGs exhibited increased left ventricle (LV) mass. In contrast, LV mass in RCV-treated TGs and WTs remained unchanged. In all cohorts, LV end-diastolic dimension remained unchanged. This novel cardiac-targeted overexpression of hTMPK helps define the role of TMPK in mitochondrial toxicity of antiretrovirals.

Murine cardiac mtDNA: effects of transgenic manipulation of nucleoside phosphorylation.

Mitochondrial toxicity results from pyrimidine nucleoside reverse transcriptase inhibitors (NRTIs) for HIV/AIDS. In the heart, this can deplete mitochondrial (mt) DNA and cause cardiac dysfunction (eg, left ventricle hypertrophy, LVH). Four unique transgenic, cardiac-targeted overexpressors (TGs) were generated to determine their individual impact on native mitochondrial biogenesis and effects of NRTI administration on development of mitochondrial toxicity. TGs included cardiac-specific overexpression of native thymidine kinase 2 (TK2), two pathogenic TK2 mutants (H121N and I212N), and a mutant of mtDNA polymerase, pol-gamma (Y955C). Each was treated with antiretrovirals (AZT-HAART, 3 or 10 weeks, zidovudine (AZT) + lamivudine (3TC) + indinavir, or vehicle control). Parameters included left ventricle (LV) performance (echocardiography), LV mtDNA abundance (real-time PCR), and mitochondrial fine structure (electron microscopy, EM) as a function of duration of treatment and presence of TG. mtDNA abundance significantly decreased in Y955C TG, increased in TK2 native and I212N TGs, and was unchanged in H121N TGs at 10 weeks regardless of treatment. Y955C and I212N TGs exhibited LVH during growth irrespective of treatment. Y955C TGs exhibited cardiomyopathy (CM) at 3 and 10 weeks irrespective of treatment, whereas H121N and I212N TGs exhibited CM only after 10 weeks AZT-HAART. EM features were consistent with cardiac dysfunction. mtDNA abundance and cardiac functional changes were related to TG expression of mitochondrially related genes, mutations thereof, and NRTIs.

Tenofovir renal toxicity targets mitochondria of renal proximal tubules.

Tenofovir disoproxil fumarate (TDF) is an analog of adenosine monophosphate that inhibits HIV reverse transcriptase in HIV/AIDS. Despite its therapeutic success, renal tubular side effects are reported. The mechanisms and targets of tenofovir toxicity were determined using '2 x 2' factorial protocols, and HIV transgenic (TG) and wild-type (WT) littermate mice with or without TDF (5 weeks). A parallel study used didanosine (ddI) instead of TDF. At termination, heart, kidney, and liver samples were retrieved. Mitochondrial DNA (mtDNA) abundance, and histo- and ultrastructural pathology were analyzed. Laser-capture microdissection (LCM) was used to isolate renal proximal tubules for molecular analyses. Tenofovir increased mtDNA abundance in TG whole kidneys, but not in their hearts or livers. In contrast, ddI decreased mtDNA abundance in the livers of WTs and TGs, but had no effect on their hearts or kidneys. Histological analyses of kidneys showed no disruption of glomeruli or proximal tubules with TDF or ddI treatments. Ultrastructural changes in renal proximal tubules from TDF-treated TGs included an increased number and irregular shape of mitochondria with sparse fragmented cristae. LCM-captured renal proximal tubules from TGs showed decreased mtDNA abundance with tenofovir. The results indicate that tenofovir targets mitochondrial toxicity on the renal proximal tubule in an AIDS model.

Transgenic mitochondrial superoxide dismutase and mitochondrially targeted catalase prevent antiretroviral-induced oxidative stress and cardiomyopathy.

Transgenic mice (TG) were used to define mitochondrial oxidative stress and cardiomyopathy (CM) induced by zidovudine (AZT), an antiretroviral used to treat HIV/AIDS. Genetically engineered mice either depleted or overexpressed mitochondrial superoxide dismutase (SOD2(+/-) KOs and SOD2-OX, respectively) or expressed mitochondrially targeted catalase (mCAT). TGs and wild-type (WT) littermates were treated (oral AZT, 35 days). Cardiac mitochondrial H(2)O(2), aconitase activity, histology and ultrastructure were analyzed. Left ventricle (LV) mass and LV end-diastolic dimension were determined echocardiographically. AZT induced cardiac oxidative stress and LV dysfunction in WTs. Cardiac mitochondrial H(2)O(2) increased and aconitase was inactivated in SOD2(+/-) KOs, and cardiac dysfunction was worsened by AZT. Conversely, the cardiac function in SOD2-OX and mCAT hearts was protected. In SOD2-OX and mCAT TG hearts, mitochondrial H(2)O(2), LV mass and LV cavity volume resembled corresponding values from vehicle-treated WTs. AZT worsens cardiac dysfunction and increases mitochondrial H(2)O(2) in SOD2(+/-) KO. Conversely, both SOD2-OX and mCAT TGs prevent or attenuate AZT-induced cardiac oxidative stress and LV dysfunction. As dysfunctional changes are ameliorated by decreasing and worsened by increasing H(2)O(2) abundance, oxidative stress from H(2)O(2) is crucial pathogenetically in AZT-induced mitochondrial CM.

Mitochondrial DNA impairment in nucleoside reverse transcriptase inhibitor-associated cardiomyopathy.

Acquired immune deficiency syndrome (AIDS) is a global epidemic that continues to escalate. Recent World Health Organization estimates include over 33 million people currently diagnosed with HIV/AIDS. Another 20 million HIV-infected individuals died over the past quarter century. Antiretrovirals are effective treatments that changed the outcome of HIV infection from a fatal disease to a chronic illness. Cardiomyopathy (CM) is a bona fide component of HIV/AIDS with occurrence that is higher in HIV positive individuals. CM may result from individual or combined effects of HIV, immune reactions, or toxicities of prolonged antiretrovirals. Nucleoside reverse transcriptase inhibitors (NRTIs) are the cornerstone of antiretroviral therapy. Despite pharmacological benefits of NRTIs, NRTI side effects include increased risk for CM. Clinical observations and in vitro and in vivo studies support various mechanisms of CM. This perspective highlights some of the hypotheses and focuses on mitochondrial-associated pathways of NRTI- related CM.

Cardiac-targeted transgenic mutant mitochondrial enzymes: mtDNA defects, antiretroviral toxicity and cardiomyopathy.

Mitochondrial (mt) DNA biogenesis is critical to cardiac contractility. DNA polymerase gamma (Pol gamma) replicates mtDNA, whereas thymidine kinase 2 (TK2) monophosphorylates pyrimidines intramitochondrially. Point mutations in POLG and TK2 result in clinical diseases associated with mtDNA depletion and organ dysfunction. Pyrimidine analogs (NRTIs) inhibit Pol gamma and mtDNA replication. Cardiac "dominant negative" murine transgenes (TGs; Pol gamma Y955C, and TK2 H121N or I212N) defined the role of each in the heart. mtDNA abundance, histopathological features, histochemistry, mitochondrial protein abundance, morphometry, and echocardiography were determined for TGs in "2 x 2" studies with or without pyrimidine analogs. Cardiac mtDNA abundance decreased in Y955C TGs ( approximately 50%) but increased in H121N and I212N TGs (20-70%). Succinate dehydrogenase (SDH) increased in hearts of all mutants. Ultrastructural changes occurred in Y955C and H121N TGs. Histopathology demonstrated hypertrophy in H121N, LV dilation in I212N, and both hypertrophy and dilation in Y955C TGs. Antiretrovirals increased LV mass ( approximately 50%) for all three TGs which combined with dilation indicates cardiomyopathy. Taken together, these studies demonstrate three manifestations of cardiac dysfunction that depend on the nature of the specific mutation and antiretroviral treatment. Mutations in genes for mtDNA biogenesis increase risk for defective mtDNA replication, leading to LV hypertrophy.

Mobile Health Intervention to Reduce HIV Transmission: A Randomized Trial of Behaviorally Enhanced HIV Treatment as Prevention (B-TasP).

OBJECTIVES: We conducted a randomized clinical trial to test a mobile health behavioral intervention designed to enhance HIV treatment as prevention (B-TasP) by simultaneously increasing combination antiretroviral therapies (cART) adherence and improving the sexual health of people living with HIV. METHODS: A cohort of sexually active men (n = 383) and women (n = 117) living with HIV were enrolled. Participants were baseline assessed and randomized to either (1) B-TasP adherence and sexual health intervention or (2) general health control intervention. Outcome measures included HIV RNA viral load, cART adherence monitored by unannounced pill counts, indicators of genital tract inflammation, and sexual behaviors assessed over 12 months. RESULTS: Eighty-six percent of the cohort was retained for 12-month follow-up. The B-TasP intervention demonstrated significantly lower HIV RNA, OR = 0.56, P = 0.01, greater cART adherence, Wald χ = 33.9, P = 0.01, and fewer indicators of genital tract inflammation, Wald χ = 9.36, P = 0.05, over the follow-up period. Changes in sexual behavior varied, with the B-TasP intervention showing lower rates of substance use in sexual contexts, but higher rates of condomless sex with non-HIV positive partners occurred in the context of significantly greater beliefs that cART reduces HIV transmission. CONCLUSIONS: Theory-based mobile health behavioral interventions can simultaneously improve cART adherence and sexual health in people living with HIV. Programs aimed to eliminate HIV transmission by reducing HIV infectiousness should be bundled with behavioral interventions to maximize their impact and increase their chances of success.

A brief overview of mechanisms of mitochondrial toxicity from NRTIs.

Nucleoside reverse transcriptase inhibitors (NRTIs) in combinations with other antiretrovirals (highly active antiretroviral therapy, HAART) are the cornerstones of AIDS therapy, turning HIV infection into a manageable clinical entity. Despite the initial positive impact of NRTIs, therapeutic experience revealed serious side effects that appeared to originate in the mitochondria and which ultimately manifested as dysfunction of that organelle. It may be reasonable to consider that as the AIDS epidemic continues and as survival with HIV infection is prolonged by treatment with HAART, long-term side effects of NRTIs may become increasingly common. This consideration may be underscored in children who are born to HIV-infected mothers who received NRTI therapy in utero during gestation. The long-term effect of that NRTI exposure in utero is not clear yet. This review examines some proposed mechanisms of NRTI mitochondrial toxicity, including genetic predisposition, defects in mitochondria DNA replication, the encompassing "DNA pol-gamma hypothesis," the relationship between mitochondrial nucleotide and NRTI pools, mitochondrial DNA mutation and dysfunction, and oxidative stresses related to HIV infection and NRTIs. Mechanisms of mitochondrial toxicity are reviewed with respect to key cell biological, pathological, and pharmacological events.

Antiretroviral nucleosides, deoxynucleotide carrier and mitochondrial DNA: evidence supporting the DNA pol gamma hypothesis.

DESIGN: Nucleoside reverse transcriptase inhibitors (NRTIs) exhibit mitochondrial toxicity. The mitochondrial deoxynucleotide carrier (DNC) transports nucleotide precursors (or phosphorylated NRTIs) into mitochondria for mitochondrial (mt)DNA replication or inhibition of mtDNA replication by NRTIs. Transgenic mice (TG) expressing human DNC targeted to murine myocardium served to define mitochondrial events from NRTIs in vivo and findings were corroborated by biochemical events in vitro. METHODS: Zidovudine (3'-azido-2',3'-deoxythymidine; ZDV), stavudine (2', 3'-didehydro-2', 3'-deoxythymidine; d4T), or lamivudine ((-)-2'-deoxy-3'-thiacytidine; 3TC) were administered individually to TGs and wild-type (WT) littermates (35 days) at human doses with drug-free vehicle as control. Left ventricle (LV) mass was defined echocardiographically, mitochondrial ultrastructural defects were identified by electron microscopy, the abundance of cardiac mtDNA was quantified by real time polymerase chain reaction, and mtDNA-encoded polypeptides were quantified. RESULTS: Untreated TGs exhibited normal LV mass with minor mitochondrial damage. NRTI monotherapy (either d4T or ZDV) increased LV mass in TGs and caused significant mitochondrial destruction. Cardiac mtDNA was depleted in ZDV and d4T-treated TG hearts and mtDNA-encoded polypeptides decreased. Changes were absent in 3TC-treated cohorts. In supportive structural observations from molecular modeling, ZDV demonstrated close contacts with K947 and Y951 in the DNA pol gamma active site that were absent in the HIV reverse transcriptase active site. CONCLUSIONS: NRTIs deplete mtDNA and polypeptides, cause mitochondrial structural and functional defects in vivo, follow inhibition kinetics with DNA pol gamma in vitro, and are corroborated by molecular models. Disrupted pools of nucleotide precursors and inhibition of DNA pol gamma by specific NRTIs are mechanistically important in mitochondrial toxicity.

Human immunodeficiency virus type 1 (HIV-1) induces activation of multiple STATs in CD4+ cells of lymphocyte or monocyte/macrophage lineages.

Human immunodeficiency virus type 1 (HIV-1) impacts the activation state of multiple lineages of hematopoietic cells. Chronic HIV-1 infection among individuals with progressive disease can be associated with increased levels of activated signal transducers and activators of transcription (STATs) in peripheral blood mononuclear cells. To investigate interactions between HIV-1 and CD4(+) cells, activated, phosphorylated STAT proteins in nuclear extracts from lymphocytic and promonocytic cell lines as well as primary monocyte-derived macrophages were measured. Levels of activated STATs increased six- to tenfold in HUT78 and U937 cells within 2 h following exposure to virions. The response to virus was dose-dependent, but kinetics of activation was delayed relative to interleukin-2 or interferon-gamma. Activation of STAT1, STAT3, and STAT5 occurred with diverse viral envelope proteins, independent of coreceptor use or viral replication. Envelope-deficient virions had no effect on STAT activation. Monoclonal antibody engagement of CD4 identified a novel role for CD4 as a mediator in the activation of multiple STATs. Results provide a model for HIV-1 pathogenesis in infected and noninfected hematopoietic cells.

Approaches to hepatitis C treatment and cure using NS5A inhibitors.

Recent progress in the understanding of hepatitis C virus (HCV) biology and the availability of in vitro models to study its replication have facilitated the development of direct-acting antiviral agents (DAAs) that target specific steps in the viral replication cycle. Currently, there are three major classes of DAA in clinical development: NS3/4A protease inhibitors, NS5B polymerase inhibitors, and NS5A directed inhibitors. Several compounds thought to bind directly with NS5A are now in various clinical trial phases, including the most advanced, daclatasvir (BMS-790052), ledipasvir (GS-5885), and ABT-267. While many NS5A-targeted compounds demonstrate picomolar potency, the exact mechanism(s) of their action is still unclear. In the clinic, NS5A HCV inhibitors show promise as important components in DAA regimens and have multifunctionality. In addition to inhibiting viral replication, they may synergize with other DAAs, possibly by modulating different viral proteins, to help suppress the emergence of resistant viruses. Structure-based models have identified target interaction domains and spatial interactions that explain drug resistance for mutations at specific positions (eg, residues 93 and 31) within NS5A and potential binding partners. This review provides, insights into the unique complexity of NS5A as a central platform for multiple viral/host protein interactions, and possible mechanism(s) for the NS5A inhibitors currently undergoing clinical trials that target this nonstructural viral protein.

Asymmetric binding to NS5A by daclatasvir (BMS-790052) and analogs suggests two novel modes of HCV inhibition.

Symmetric, dimeric daclatasvir (BMS-790052) is the clinical lead for a class of picomolar inhibitors of HCV replication. While specific, resistance-bearing mutations at positions 31 and 93 of domain I strongly suggest the viral NS5A as target, structural mechanism(s) for the drugs' activities and resistance remains unclear. Several previous models suggested symmetric binding modes relative to the homodimeric target; however, none can fully explain SAR details for this class. We present semiautomated workflows to model potential receptor conformations for docking. Surprisingly, ranking docked hits with our library-derived 3D-pharmacophore revealed two distinct asymmetric binding modes, at a conserved poly-proline region between 31 and 93, consistent with SAR. Interfering with protein-protein interactions at this membrane interface can explain potent inhibition of replication-complex formation, resistance, effects on lipid droplet distribution, and virion release. These detailed interaction models and proposed mechanisms of action will allow structure-based design of new NS5A directed compounds with higher barriers to HCV resistance.