James J. Kohler

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 × 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.

Antiretroviral nucleosides, deoxynucleotide carrier and mitochondrial DNA: evidence supporting the DNA pol γ 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 γ 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 γ in vitro, and are corroborated by molecular models. Disrupted pools of nucleotide precursors and inhibition of DNA pol γ by specific NRTIs are mechanistically important in mitochondrial toxicity.

DECREASED mtDNA, OXIDATIVE STRESS, CARDIOMYOPATHY, AND DEATH FROM TRANSGENIC CARDIAC TARGETED HUMAN MUTANT POLYMERASE γ *

POLG is the human gene that encodes the catalytic subunit of DNA polymerase γ (Pol γ), the replicase for human mitochondrial DNA (mtDNA). A POLG Y955C point mutation causes human chronic progressive external ophthalmoplegia (CPEO), a mitochondrial disease with eye muscle weakness and mtDNA defects. Y955C POLG was targeted transgenically (TG) to the murine heart. Survival was determined in four TG (+/−) lines and wild-type (WT) littermates (−/−). Left ventricle (LV) performance (echocardiography and MRI), heart rate (electrocardiography), mtDNA abundance (real time PCR), oxidation of mtDNA (8-OHdG), histopathology and electron microscopy defined the phenotype. Cardiac targeted Y955C POLG yielded a molecular signature of CPEO in the heart with cardiomyopathy (CM), mitochondrial oxidative stress, and premature death. Increased LV cavity size and LV mass, bradycardia, decreased mtDNA, increased 8-OHdG, and cardiac histopathological and mitochondrial EM defects supported and defined the phenotype. This study underscores the pathogenetic role of human mutant POLG and its gene product in mtDNA depletion, mitochondrial oxidative stress, and CM as it relates to the genetic defect in CPEO. The transgenic model pathophysiologically links human mutant Pol γ, mtDNA depletion, and mitochondrial oxidative stress to the mtDNA replication apparatus and to CM.

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.

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‐γ. 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.

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 H2O2, 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 H2O2 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 H2O2, LV mass and LV cavity volume resembled corresponding values from vehicle-treated WTs. AZT worsens cardiac dysfunction and increases mitochondrial H2O2 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 H2O2 abundance, oxidative stress from H2O2 is crucial pathogenetically in AZT-induced mitochondrial CM.

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.

Impact on Genetic Networks in Human Macrophages by a CCR5 Strain of Human Immunodeficiency Virus Type 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) impacts multiple lineages of hematopoietic cells, including lymphocytes and macrophages, either by direct infection or indirectly by perturbations of cell networks, leading to generalized immune deficiency. We designed a study to discover, in primary human macrophages, sentinel genetic targets that are impacted during replication over the course of 7 days by a CCR5-using virus. Expression of mRNA and proteins in virus- or mock-treated macrophages from multiple donors was evaluated. Hierarchical agglomerative cluster analysis grouped into distinct temporal expression patterns >900 known human genes that were induced or repressed at least fourfold by virus. Expression of more than one-third of the genes was induced rapidly by day 2 of infection, while other genes were induced at intermediate (day 4) or late (day 7) time points. More than 200 genes were expressed exclusively in either virus- or mock-treated macrophage cultures, independent of the donor, providing an unequivocal basis to distinguish an effect by virus. HIV-1 altered levels of mRNA and/or protein for diverse cellular programs in macrophages, including multiple genes that can contribute to a transition in the cell cycle from G1 to G2/M, in contrast to expression in mock-treated macrophages of genes that maintain G0/G1. Virus treatment activated mediators of cell cycling, including PP2A, which is impacted by Vpr, as well as GADD45 and BRCA1, potentially novel targets for HIV-1. The results identify interrelated programs conducive to optimal HIV-1 replication and expression of genes that can contribute to macrophage dysfunction.

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.

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-γ (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.