Effects of BMS-986094, a Guanosine Nucleotide Analogue, On

TOXICOLOGICAL SCIENCES, 153(2), 2016, 396–408

doi: 10.1093/toxsci/kfw135 Advance Access Publication Date: July 27, 2016 Research Article

Effects of BMS-986094, a Guanosine Nucleotide

Analogue, on Mitochondrial DNA Synthesis and Downloaded from https://academic.oup.com/toxsci/article/153/2/396/2579185 by guest on 23 September 2021 Function Bethany R. Baumgart,*,1 Faye Wang,* Jae Kwagh,* Chris Storck,* Catherine Euler,* Megan Fuller,* Damir Simic,* Suresh Sharma,† Jamie J. Arnold,† Craig E. Cameron,† Terry R. Van Vleet,* Oliver Flint,* Roderick T. Bunch,* Marc H. Davies,* Michael J. Graziano,* and Thomas P. Sanderson*

*Bristol-Myers Squibb, 777 Scudders Mill Road, Princeton, New Jersey 08536; and †The Pennsylvania State University, 201 Althouse Laboratory, University Park, Pennsylvania 16802

1To whom correspondence should be addressed at Bristol-Myers Squibb Pharmaceutical Company, 777 Scudders Mill Road, Princeton, NJ 08536. Fax: (812) 307-2432. E-mail: [email protected].

ABSTRACT BMS-986094, the prodrug of a guanosine nucleotide analogue (20-C-methylguanosine), was withdrawn from clinical trials due to serious safety issues. Nonclinical investigative studies were conducted as a follow up to evaluate the potential for BMS-986094-related mitochondrial-toxicity. In vitro, BMS-986094 was applied to human hepatoma cells (HepG2 and Huh-7) or cardiomyocytes (hiPSCM) up to 19 days to assess mitochondrial DNA content and speciﬁc gene expression. There were no mitochondrial DNA changes at concentrations 10 mM. Transcriptional effects, such as reductions in Huh-7 MT-ND1 and MT-ND5 mRNA content and hiPSCM MT-ND1, MT-COXII, and POLRMT protein expression levels, occurred only at cytotoxic concentrations (10 mM) suggesting these transcriptional effects were a consequence of the observed toxicity. Additionally, BMS-986094 has a selective weak afﬁnity for inhibition of RNA polymerases as opposed to DNA polymerases. In vivo, BMS- 986094 was given orally to cynomolgus monkeys for 3 weeks or 1 month at doses of 15 or 30 mg/kg/day. Samples of heart and kidney were collected for assessment of mitochondrial respiration, mitochondrial DNA content, and levels of high energy substrates. Although pronounced cardiac and renal toxicities were observed in some monkeys at 30 mg/kg/day treated for 3–4 weeks, there were no changes in mitochondrial DNA content or ATP/GTP levels. Collectively, these data suggest that BMS-986094 is not a direct mitochondrial toxicant.

Key words: mitochondria; nucleotide analogue; toxicity

Hepatitis C virus (HCV) infection affects around 130–200 million pegylated IFN and ribavirin for 24 or 48 weeks was the standard people worldwide (Scheel and Rice, 2013; Wendt et al, 2014). of care, yet these indirect antiviral agents are associated with Clinically, the disease usually manifests itself as chronic hepa- poor efficacy for some HCV genotypes and serious tolerability titis, which can progress into fibrosis, cirrhosis, and hepatocel- issues (Poveda et al, 2014). lular carcinoma (HCC) (Chan, 2014). Interferon (IFN)-based Advances in the understanding of HCV replication led to the therapies were the first treatment strategies available for HCV development of direct-acting antiviral (DAA) agents that target infection (Karbasi-Afshar, 2014). Until 2011, the combination of HCV-specific proteins (Poveda et al, 2014). First-generation DAA

396 BAUMGART ET AL. | 397

agents included protease inhibitors of the NS3/NS4a complex, DNA content and organelle function as a potential mechanism telaprevir and boceprevir (Poveda et al, 2014; Scheel and Rice, of myocardial and renal toxicities associated with BMS-986094. 2013; Wendt et al, 2014). Triple combination therapy of these agents with pegylated IFN and ribavirin achieved higher sus- MATERIALS AND METHODS tained virologic response rates, but worsened some of the pegylated IFN and ribavirin-related side effects, such as anemia, and Materials. BMS-986094 was synthesized by Bristol-Myers Squibb. In introduced new side effects such as dysgeusia and cutaneous vitro test compounds, azidothymidine (AZT) and diDeoxycytidine rash (Wendt et al, 2014). The large unmet medical need for (DDC), were purchased from Sigma-Aldrich (St. Louis, Missouri) chronic HCV infection resulted in extensive research efforts to and dissolved in sterile, distilled water or dimethyl sulfoxide identify and develop more effective treatments with improved (DMSO) as 20 mM stock solutions. safety and tolerance profiles. The resultant second-generation DAA agents include protease inhibitors of NS5a and nonnucleo- Animals. Male and female cynomolgus monkeys (approximately 3–4 years of age) were obtained from Charles River BRF, Inc side analogue and nucleos(t)ide analogue inhibitors of NS5b Downloaded from https://academic.oup.com/toxsci/article/153/2/396/2579185 by guest on 23 September 2021 (Poveda et al, 2014; Scheel and Rice, 2013; Wendt et al, 2014). (Houston, Texas) and Buckshire, Inc (Perkasie, Pennsylvania). Wherease antiviral agents demonstrate better efficacy than Monkeys were randomly assigned to different groups based on IFN-based treatments, some DAA agents (specifically nucleos(- body weight and individually housed in a temperature- t)ide analogues) have been linked to toxicity associated with in- controlled environment at 64–84 F, 30–70% humidity, and a hibition of nuclear DNA polymerase c and mitochondrial DNA 12-h light/dark cycle. During the study, water was provided ad depletion (Cossarizza and Moyle, 2004; Cote´ et al., 2002; Moyle, libitum and a daily ration of 12 biscuits was provided approxi- 2000a). Mitochondria contain the only nonnuclear DNA found mately 4 h post-dose. Monkeys were maintained in accordance within human cells encoding subunits of mitochondrial en- with the Guide for the Care and Use of Laboratory Animals and zymes, transfer RNA (tRNA), and ribosomal RNA (rRNA) (Moyle, all experiments were approved by the Institutional Animal Care 2000a). In their active triphosphate form, nucleoside and nu- and Use Committee. cleotide analogues compete with naturally occurring nucleo- Experimental design. In a 1-month investigative study in mon- tides for incorporation into the growing viral DNA chain (Moyle, keys, 5 males and 5 females were randomly assigned by body 2000a). Mitochondrial DNA is replicated via nuclear-encoded weight to each of 3 groups to assess myocardial and renal toxic- DNA polymerase c (Cossarizza and Moyle, 2004). The absence of ity biomarkers and potential mechanisms for multi-organ toxic- a30-hydroxyl group on nucleotide analogues prohibits phospho- ity. Dosing formulations of vehicle alone or 15 or 30 mg/kg/day diester bond formation resulting in terminated replication BMS-986094 in carrier were administered orally once daily for 1 (Moyle, 2000a). Reduction of mitochondrial DNA weakens syn- month. The test article carrier and vehicle were 95% Capmul thesis of enzymes and subunits important for oxidative phos- (medium chain monoglyceride) (Abitec Corporation, Janesville, phorylation, the process by which mitochondria produce most WI) and 5% Tween 80 (w/w) (Avantor Performance Materials, cellular energy in the form of adenosine triphosphate (ATP) Center Valley, Pennsylvania), respectively. The first 3 males and (Cossarizza and Moyle, 2004; Pereira et al, 2009). Mitochondrial the first 3 females in each group were scheduled for euthanasia dysfunction or damage can lead to impaired oxidative phos- at the end of the 1-month dosing period. The remaining animals phorylation, accumulation of intracellular lipids, and increased in each group were retained for a 1-month recovery evaluation. lactic acid (Lewis and Dalakas, 1995). Adverse effects associated In a 3-week investigative study in monkeys, 5 males and 5 with mitochondrial toxicity such as pancreatitis, lactic acidosis females were assigned to each of 3 groups to assess BMS-986094 with liver failure, hepatic steatosis, and myopathy have been re- metabolites in target and nontarget tissues. Dosing formula- ported among HCV patients treated with DAA agents (Moyle, tions of vehicle alone or 15 or 30 mg/kg/day BMS-986094 in car- 2000b; Fleischer et al, 2004). rier were administered orally once daily for 3 weeks. The test BMS-986094, a second generation DAA, is a guanosine nu- article carrier and vehicle were 95% Capmul and 5% Tween 80 cleotide inhibitor of HCV NS5b. In rat and monkey toxicology (w/w), respectively. The first 3 males and the first 3 females in studies, skeletal muscle, heart, and kidney toxicities occurred at each group were euthanatized at the end of the 3-week dosing nontolerated doses of BMS-986094 for up to 6 months in dur- period. The remaining animals in each group were retained for ation. Minimal skeletal muscle degeneration (correlating with a 3-week recovery evaluation. increased aminotransferases and creatine kinase) occurred at tolerated doses suggesting that clinical dosing of BMS-986094 Ex vivo mitochondrial isolation and respiration. Samples of heart could proceed safely with appropriate clinical monitoring. In a (left ventricle) and kidney (cortex) were collected from all avail- Phase I clinical study, BMS-986094, either alone or in combin- able monkeys following 3 weeks or 1 month of dosing. Samples ation with ribavirin, for 7 days was well tolerated in treatment- were placed in ice cold mitochondrial isolation medium (MIM) naive HCV genotype 1-infected patients. However, dosing in a (300 mM sucrose [Sigma-Aldrich], 10 mM sodium [Na] HEPES Phase II study was immediately terminated after a patient [Sigma-Aldrich], 0.2 mM ethylene diamine tetraacetic acid receiving BMS-986094 for 40 days experienced rapidly progres- [EDTA] [Sigma-Aldrich]) until processed for mitochondrial isola- sive heart failure characterized by decreased ventricular ejec- tion. The heart was trimmed of fat, placed in a dry Petri dish, tion fraction, electrocardiographic changes, and acute renal and minced. After mincing, 10 ml of a trypsin (Sigma-Aldrich) failure. This was the first indication of cardiotoxicity associated solution was added to the dish and incubated for 15 min at 4 C. DAA treatment (Ahmad et al., 2015). After incubation, trypsin inhibitor (10 ml) (Sigma-Aldrich) solu- Based on the unexpected cardiac toxicity with BMS-986094 tion was added to the dish. The contents were transferred to a in the clinic, a series of nonclinical investigative studies were 50-ml conical tube, inverted gently several times, the tissue conducted to elucidate the mechanism of toxicity with the po- allowed to settle, and the supernatant removed. The tissue was tential for BMS-986094-induced mitochondrial toxicity being a then resuspended in 10 ml of cold MIM with bovine serum albu- concern. The objective of this work was to assess mitochondrial min (BSA) (300 mM sucrose, 10 mM Na HEPES, 0.2 mM EDTA, 398 | TOXICOLOGICAL SCIENCES, 2016, Vol. 153, No. 2

1 mg/ml bovine serum albumin [BSA] [Sigma-Aldrich]), trans- Measurement of high energy substrates by ultra performance liquid ferred to a glass homogenization mortar, and homogenized chromatography. Samples of heart (left ventricle), kidney (cortex), with a grooved teflon dounce homogenizer attached to a hand- liver (left lateral lobe), and diaphragm were collected from all held power drill (Black & Decker, New Britain, Connecticut) (set- available animals following 3 weeks of dosing. Samples were ting 1; 5 strokes). The kidney was decapsulated and split placed in citric acid buffer (62.5 mM citric acid [Avantor bilaterally in a Petri dish containing MIM. The cortex was Performance Materials], 2.25 mg/mL K2EDTA [Sigma-Aldrich]) removed and cut into small pieces. The tissue was placed into a and homogenized using a handheld homogenizer. Homogenate glass homogenization mortar containing cold MIM-BSA and samples were analyzed for ATP (Sigma-Aldrich), GTP (Sigma- homogenized with a grooved Teflon dounce homogenizer Aldrich), guanosine monophosphate (GMP), and inosine mono- attached to a handheld power drill (setting 1; 3 strokes). Each phosphate (IMP). Sample extraction was carried out using an l homogenate sample was placed in a 50-ml polycarbonate cen- extraction solution (300 l) (1 M perchloric acid [HClO4] [Sigma- Aldrich] and 5 mM DTT), vortex-mixed briefly, and flash frozen trifuge tube (Beckman Coulter, Brea, California) and mitochon- in liquid nitrogen. Sample neutralization was performed using 2 dria isolated by differential centrifugation using an Optima L- Downloaded from https://academic.oup.com/toxsci/article/153/2/396/2579185 by guest on 23 September 2021 M potassium bicarbonate (KHCO ) (150 ll) (Sigma-Aldrich). 90K UltraCentrifuge (Beckman Coulter). A Bradford assay, using 3 Samples were centrifuged at 1200 g for 1 h at 4 C and the BSA Protein Assay Standards (Thermo Scientific, Waltham, resultant supernatant resolved using a Waters Acquity ultra Massachusetts) and Pierce Protein Assay Reagent (660 nm) performance liquid chromatography (UPLC) H-Class System (Thermo Scientific), was performed on all samples (diluted 1:100 (Waters Corporation, Milford, Connecticut) with a BEH C18 in MIM) using a SpectraMax Plus 384 microplate reader column (2.1 50 mm; particle size 1.7-micron) (Waters (Molecular Devices, Sunnyvale, California) and SoftMax Pro soft- Corporation) and quantified by photodiode array (PDA) detec- ware. The values obtained were used to normalize respiration tion. An isocratic mobile phase consisting of 2.4% phosphoric data. Oxygen consumption was measured using an RC650 6-cell acid (EMD Millipore, Billerica, Massachusetts), pH 4, diluted in Respirometer (Strathkelvin Instruments, North Lanarkshire, water, was used to elute the nucleotides/nucleosides from the Scotland). Warm mitochondrial incubation buffer (MIB) (125 mM column. Data were recorded, analyzed, and stored using potassium chloride [KCl] [Sigma-Aldrich], 20 mM Empower Software, version 3 (Waters Corporation). HEPES [Sigma-Aldrich], 3 mM magnesium [Mg]-acetate tetrahy- drate [Sigma-Aldrich], 0.4 mM ethylene glycol tetraacetic acid Cell culture. Human hepatocellular carcinoma (HepG2) cells were [EGTA] [Sigma-Aldrich], 0.3 mM dithiothreitol [DTT] [Sigma- obtained from the American Tissue Type Collection (ATCC,

Aldrich], 5 mM potassium phosphate [KH2PO4] [Sigma-Aldrich], Manassas, Virginia) and cultured using Dulbecco’s modified 2 mg/mL BSA, 10 mM Na pyruvate [Sigma-Aldrich], 5 mM malic eagle medium (DMEM) (Life Technologies) supplemented with acid [Sigma-Aldrich]) and approximately 0.5–1 mg of mitochon- 10% fetal bovine serum (FBS), 2 mM L-glutamine (Life drial protein was added to each cell. State IV respiration was Technologies), 100 mM minimal essential media (MEM) sodium measured for approximately 2 min. To induce State III respira- pyruvate (Life Technologies), 100 U/ml penicillin (Life tion, 1 mM adenosine 50-diphosphate (ADP) (Sigma-Aldrich) was Technologies), and 100 mg/ml streptomycin (Life Technologies). added to each cell. State III respiration was measured until a Human differentiated hepatocyte-derived cellular carcinoma consistent slope was reached; approximately 2 min. If the State (Huh-7) cells were grown and maintained by the Department of III/State IV respiratory control ratio (RCR) was 2, the mitochon- Biochemistry and Molecular Biology at The Pennsylvania State dria membranes were considered to be intact. University. Huh-7 cells were cultured using DMEM supplemented with 10% FBS, 5 mM HEPES, 10 mM galactose (Sigma- Measurement of mitochondrial NADH dehydrogenase subunit 1 (MT- Aldrich), 1 mM Na pyruvate (Corning, Inc., Manassas, Virginia), ND1) content by PCR. Samples of heart (left ventricle), liver (left 100 U/ml penicillin, and 100ET AL.mg/ml streptomycin. Human lateral lobe), kidney (cortex), and diaphragm were collected induced pluripotent stem cell-derived cardiomyocytes (hiPSCM) from all available animals following 1 month of dosing. DNA were obtained from Cellular Dynamics International (CDI, isolation was performed using a MagMAX-96 DNA multi-sample Madison, Wisconsin) and cultured, according to the manufacturer’s manual, using iCell cardiomyocyte culture media (CDI). kit (Life Technologies, Grand Island, New York) and MagMAX All cultures were maintained in a sterile environment at 37 C Express Magnetic Particle Processor (Life Technologies) accord- with 5% CO and 95% humidity. ing to manufacturer’s instructions. DNA was quantified using a 2 NanoDrop 1000 spectrophotometer (Thermo Scientific). A reac- Cell growth and treatment. HepG2 cells were plated in triplicate in tion containing 5 ng kidney or heart DNA, 2.5 ng diaphragm 24-well plates (tissue culture treated polystyrene) at a density of DNA, or 10 ng liver DNA was amplified in a 7900HT fast real- 2 105 per well. After attachment, the cells were treated with time PCR system (Life Technologies) using universal conditions. BMS-986094 (0.2, 0.5, 1, 1.5, or 2 lM), DDC (0.1, 0.5, or 1 lM), or The monkey primer/probe sets (Nuclear GAPDH: forward vehicle (water) for 19 days. Samples of culture medium and cells 0 0 0 5 -CCATGGCATAGCCTGAGAAATCCT-3 , reverse 5 -CCCGGG were taken on Days 5, 9, 14, and 19 for analysis of cell viability 0 0 TTCCCTGTCTCTAG-3 , probe 5 -FAM-CCAAAGCAGCAGGG and mitochondrial DNA content. HepG2 cells were re-plated at a 0 0 TCTCCTCAFAM/BHQ-BHQ-3 ; MT-ND1: forward 5 -CCCACTCCA density of 2 105 per well and fresh treatment-containing 0 0 CTTATTCACCAGAACT-3 , reverse 5 -TGCTGCCCGGATT medium was added to the cultures on Days 5, 9, and 14. The 0 0 CATAGG-3 , probe 5 -FAM-TCACAACATGCTTCGCCACCAA- hiPSCM cells were plated in triplicate in 0.1% gelatin pre-coated 0 BHQ-3 ) were designed using RealTimeDesign Software 24-well plates at a density of 300 000 cells/ml per well. After (Biosearch Technologies, Petaluma, California). Cycling thresh- 7 days, the cells were treated with BMS-986094 (0.01, 0.1, 1, 5, 10, old (Ct) values were generated and target gene levels (MT-ND1) 25 or 50 lM), AZT (1, 20 or 200 lM), or vehicle (DMSO) for 10 days. normalized to GAPDH to generate DCt values. The comparative Samples of culture medium and cells were taken on Days 1, 3, 6, analyses were performed to calculate the relative quantity (RQ) and 10 for analysis of cell viability and mitochondrial DNA con- value for each treated sample. tent and basal cell oxygen consumption and mitochondrial BAUMGART ET AL. | 399

respiration measurements were taken. The hiPSCM cells were the membrane using a UV Crosslinker (Stratalinker 2400, also plated at a density of 200 000 cells/ml per well and treated Stratagene, La Jolla, California). The membrane was washed with BMS-986094 (0.1, 1, and 10 lM) or vehicle (DMSO) for 24 or twice with wash buffer (1 SSC and 0.1% sodium dodecyl sulfate 72 h. Samples of cells were taken for InCell Western analysis. [SDS] [Sigma-Aldrich]) for 30 min each at 65 C. The membrane Huh-7 cells were plated in triplicate at a density of 0.5 106 per was pre-hybridized in modified Church’s buffer (0.5 M sodium well and pre-treated with 50 ng/ml ethidium bromide (EtBr) for phosphate [Sigma-Aldrich], pH 7.2, 7% SDS, and 1 mM EDTA 24 h to deplete mitochondrial transcripts from cells. The cells [Sigma-Aldrich]) for 4 h at 65 C. The hybridization probe was were then treated with BMS-986094 (0.1, 1, or 10 lM; 10, 50, 100, denatured for 5 min at 95 C and chilled on ice for 1 min. 200, or 400 lM; and 1, 10, 100, 200, 400, 600, or 800 lM) or 20-C- Hybridization was performed in a modified Church’s buffer for methylguanosine (unmodified nucleoside) (0.1, 1, or 10 lM; 10, 16 h at 65 C. The membrane was washed twice in wash buffer 50, 100, 200, or 400 lM; and 1, 10, 100, 200, 400, 600, or 800 lM) for (1 SSC and 0.1% SDS) for 20 min each at 65 C and once at room 24 h. Samples of culture medium and cells were taken for temperature. Hybridization probes (Nuclear GAPDH: forward 50- 0 0 Northern blot analysis and IC50 of the 2 compounds. TGTGGTCATGAGTCCTTCCA-3 , reverse 5 - CGAGATCCCTCC Downloaded from https://academic.oup.com/toxsci/article/153/2/396/2579185 by guest on 23 September 2021 AAAATCAA-30; MT-ND1: forward 50-GCCAACCTCCTACTC-30, Measurement of cell viability. Cell viability was measured using reverse 50-GAGGGGGAATGCTGG-30; MT-ND5: forward 50-GCAGA the CellTiter-Glo Luminescent Cell Viability Assay (Promega, TGCCAACACA-30, reverse 50-GCTTCCGGCTGCTAG-30) were Madison, WI) and EnVision plate reader (Perkin-Elmer, made by PCR using DNA oligonucleotides and mtDNA or nuclear Waltham, Massachusetts) according to manufacturer’s instruc- DNA as a template. Probes for GAPDH were used as a positive tions. Bioluminescence was measured as Light Intensity Units control. In the PCR reaction, [a-32P]-dATP (1 mCi/mL, 3000 Ci/ (LU)/mg cell protein. mmol, [GE Healthcare]) was used with cold dNTPs (300 mM for each dCTP, dGTP, and dTTP and 10 mM for dATP) in a total vol- In vitro mitochondrial respiration. Basal cell oxygen consumption ume of 100 ml per reaction. The quality of PCR product was con- and mitochondrial respiratory function were measured in firmed by agarose gel electrophoresis and counts per minute hiPSCM cells treated with BMS-986094 (0.01, 0.1, 1, 5, 10, 25, or 50 (cpm) was determined using a LBK Wallac 1217 Rackbeta Liquid mM) for up to 10 days using a Seahorse XF96 Extracellular Flux Scintillation Counter (LKB Instruments, Mt Waverley, Victoria, Analyzer (Agilent, Santa Clara, California) according to manu- Australia). RNA was visualized by exposing the membrane to a facturer’s instructions. phosphor screen followed by scanning on a Typhoon 8600 scan- ner (GE Healthcare) in phosphor mode. Measurement of mitochondrial cytochrome c oxidase subunit II (MT-

COXII) content by PCR. DNA isolation from HepG2 and hiPSCM Determination of IC50. MT-ND5 and GAPDH bands were quantified cells was performed using the Qiagen DNeasy Blood & Tissue using ImageQuant 5.0 Software (GE Healthcare). Data were fit by Kit (Qiagen, Venlo, Limburg, Netherlands) according to manu- nonlinear regression using the program KaleidaGraph (Synergy facturer’s instructions. DNA was quantified using a NanoDrop ÀÁSoftware,ÂÃÂÃ Reading, Pennsylvania). To determine IC50, the 1000 spectrophotometer. A reaction containing 10 ng of DNA, MTND5 MTND5 GAPDH GAPDH EtBr ratio was plotted as a function of com- was amplified in a ABI PRISM 7900 Sequence Detection System pound concentration. The data were fit to an equation defining (Life Technologies) using universal conditions. The human pri- a hyperbola: Y ¼ A A½Compound þ C; where A is the ampli- mer/probe sets (Nuclear GAPDH: forward 50-GGTTTACATG IC50þCompound TTCCAATATGATTCCA-30, reverse 50-ATGGGATTTCCATTGATG tude, IC50 is the compound concentration yielding 50% inhibi- ACAAG-30, probe 50-FAM-ATGGCACCGTCAAGGCTGAGAACG-30; ÀÁtionÂÃ andÂÃ C is the endpoint (C ¼ 1, which is 0 0 0 MTND5 MTND5 MT-COXII: forward 5 -GCCCCCCAACAGGCATCA-3 , reverse 5 - GAPDH GAPDH EtBr . GATGCGAGTAATACGGATGTGTTT-30), probe 50-FAM-CCCGCT AAATCCCCTAGAAGTCCCACTC-30) were designed using ABI Measurement of mitochondrial RNA polymerase (POLRMT), MT-ND1, PRISM Primer Expression Software (Life Technologies). Cycling MT-COXII, RNA polymerase II (POLRII), RNA polymerase III (POLRIII), threshold (Ct) values were generated and target gene expression and DNA polymerase a (POLa) by InCell Western analysis. (COXII) was normalized to GAPDH and the vehicle control. The Determination of protein content from hiPSCM cells was per- comparative analyses were performed to calculate the relative formed using the InCell Western protocol provided by Li-Cor quantity (RQ) value for each treated sample. Biosciences (Lincoln, Nebraska) followed by an overnight primary antibody incubation at 4 C. All primary antibodies were Measurement of MT-ND1 and mitochondrial NADH dehydrogenase purchased from AbCam (Cambridge, Massachusetts) with the subunit 5 (MT-ND5) content by Northern blot analysis. Huh-7 Cells exception of anti-RNA polymerase III (Santa Cruz were lysed by adding TRI Reagent (Sigma-Aldrich) with Biotechnology, Dallas, Texas). IRDye 800CW-labeled secondary 5 chloroform (EMD Millipore). After centrifugation at 12 000 g antibodies from Licor were used. After final wash, plates were for 15 min at 4 C, RNA from the aqueous phase was precipitated scanned on the Odyssey Infrared Imaging System (Li-Cor using isopropyl and the resultant RNA pellets washed with 75% Biosciences). Each well was normalized for cell number using ethanol, centrifuged at 7500 g for 5 min at 4 C, and dissolved DRAQ5 (DNA stain) (Li-Cor Biosciences) and Sapphire700 (cellu- in RNase-free water (Life Technologies). The RNA was separated lar stain) (Li-Cor Biosciences) and protein levels were expressed on a 1% denaturing agarose gel (0.8 M formaldehyde [EMD as a percent of vehicle control. Millipore] in 1 MOPS [Sigma-Aldrich]) by running at 120 V for 2 h. The gel was washed twice in water for 30 min each followed Statistical analysis. Data are presented in all tables and figures as by soaking in 20 saline-sodium citrate (SSC) (1 SSC: 0.15 M mean 6 standard deviation. The data were examined for signifi- NaCl [Sigma-Aldrich] and 0.015 M sodium citrate [Sigma- cant changes with respect to the vehicle-treated control using Aldrich]) for 30 min. RNA was transferred to a nylon membrane GraphPad Prism 5 (La Jolla, California). Statistical comparisons (Hybond XL, GE Healthcare) using capillary blotting with employed are Student’s t-test or one-way ANOVA followed by a 20 SSC for 16 h at room temperature. RNA was crosslinked to Dunnett or Tukey–Kramer post-hoc comparisons of means. 400 | TOXICOLOGICAL SCIENCES, 2016, Vol. 153, No. 2

RESULTS heart, kidney, liver, and diaphragm of monkeys treated with BMS-986094 for 3 weeks. There were no clear BMS-986094 Cardiac and Renal Function Were Altered Following Treatment With effects at any treatment level (Tables 2 and 3). These results BMS0986094 In Vivo suggest there was no inhibition to guanosine synthesis or mito- After approximately 3 weeks of BMS-986094 treatment, 3 ani- chondrial production of ATP. mals at 30 mg/kg/day were euthanatized (by sedation with ket- amine hydrochloride followed by anesthesia with intravenous Cell Viability Decreased Following Treatment 1 mM BMS-986094 In propofol and subsequent exsanguination) due to clinical signs Vitro of toxicity. Additional toxicology and pathology findings Cell viability was determined following treatment with BMS- included dose-dependent observations in the heart character- 986094 for 5, 9, 14, or 19 days in HepG2 cells and 1, 3, 6, and ized by time-dependent QT prolongation and T-wave changes 10 days in hiPSCM cells. In HepG2 cells, BMS-986094 at concen- (polarity shifts and decreases), increases in serum cardiac tro- trations 0.5 mM produced no effects for the entire 19 days of ponin I at 30 mg/kg/day, and degeneration of cardiomyocytes at treatment. A decrease in cell viability was observed after 5 days Downloaded from https://academic.oup.com/toxsci/article/153/2/396/2579185 by guest on 23 September 2021 15 mg/kg/day (data not shown). Time-dependent proteinuria at a concentration of 2 mM(Figure 4A). At or after 14 days, cell and increased output of kidney injury biomarkers (serum cal- viability was markedly reduced at concentrations 1 mM(Figure bindin, b2 microglobulin, and clusterin) were observed at 4A). In hiPSCM cells, BMS-986094 at concentrations 0.1 mM pro- 15 mg/kg/day, with macroscopic findings of pale discoloration duced no effects for the entire 10 days of treatment. Decreased and ultrastructural changes indicative of renal proximal tubular cell viability was observed after 3 days at concentrations 5 mM degeneration at 30 mg/kg/day (data not shown). All findings (Figure 4B). At or after 6 days, cell viability was markedly were reversible with the exception of QT prolongation which reduced at concentrations 1 mM(Figure 4B). was only partially reversible in males. Mitochondrial Respiration in hiPSCM Cells Was Reduced Following 3 Mitochondrial Respiration Was Unaffected by BMS-986094 Days of Treatment 10 mM of BMS-986094 or following 6 Days of Treatment In Vivo Treatment at BMS-986094 Concentrations 0.1 mM Mitochondrial respiration was analyzed by comparing the oxy- TherewerenoBMS-986094-relatedeffectsonbasalcelloxygen gen consumption slopes at resting state (basal rate, State IV) consumption at any concentration on Days 1 or 3. However, basal and after the addition of ADP (maximal rate, State III). Following cell oxygen consumption was reduced on Days 6 and 10, but only 1 month of treatment, there were no clear effects on heart or at concentrations 1mM(Figure 5A). This reduction of basal cell kidney respiratory ratios (State III/IV) in males or females (Table oxygen consumption is likely linked to the markedly reduced cell 1). A statistically significant increase in State III (maximal) and viability observed at or after 6 days at BMS-986094 concentrations State IV (basal) respiration in the heart mitochondria was 1 mM. Mitochondrial respiration was reduced on Days 1 and 3 at observed in males at 30 mg/kg/day (Figs. 1A and B), but was not BMS-986094 concentrations 10 mM(Figure 5B). Reduced mito- considered relevant given the variability and overlap with the chondrial respiration occurred after 6 and 10 days of BMS-986094 female controls. State III and State IV respiration values in the treatment only at concentrations 0.1 mM. Again, these reduc- heart at the end of the 1-month recovery (30 mg/kg/day) were tions in mitochondrial respiration were likely a secondary comparable to controls indicating reversibility (Figs. 2A and B). response to the reduced cell viability observed in hiPSCM cells at No meaningful changes in myocardial mitochondrial respira- or after 3 days at BMS-986094 concentrations 5 mM and at or tion were observed in BMS-986094-treated females. A slight after 6 days at BMS-986094 concentrations 1 mM. dose-related trend of increased State III and State IV respiration in the kidneys was observed in BMS-986 094-treated males and MT-COXII DNA Content Was Altered by BMS-986094 Treatment In females (although not significant) (Figs. 1C and D). The Vitro increases were reduced following the 1-month recovery period Following treatment with 1 mM DDC, a decrease in mitochon- (Figs. 2C and D). Respiration results from the 3-week investiga- drial content was observed in HepG2 cells after 5 days (Figure tive study were similar, therefore the data is not represented 6A). Concurrently, an increase in mitochondrial content was here. noted at 0.5, 1.5, and 2 mM BMS-986094 after 5 days (Figure 6B). However, there was no change in mitochondrial DNA content MT-ND1 DNA Content Was Unaltered by BMS-986094 Treatment In observed following treatment with 1 mM AZT in hiPSCM cells Vivo after 6 days (Figure 7A). A lack of a response with 200 mM AZT Following 1 month of treatment, there were no clear differences treatment has been noted before in cultured human muscle in mitochondrial DNA content in the hearts and kidneys of cells (Benbrik et al., 1997). Similar to HepG2 cells, an increase in males (Figure 3B) or females (Figure 3A). A statistically signifi- mitochondrial DNA content was noted at 10 mM BMS-986094 in cant increase in mitochondrial DNA content in the diaphragm hiPSCM cells after 3 days (Figure 7B). As cell viability was mark- of male monkeys at 15 mg/kg/day was attributed to biologic var- edly reduced at concentrations of BMS-986094 1 mM in hiPSCM iation due to the lack of a dose response or other statistically cells after 10 days (Figure 4B), only 1 viable Day 10 sample was significant differences between groups in other tissues in mon- collected (data not shown). As a result, this time point was keys treated for 1 month. Furthermore, there was no indication excluded from statistical analyses and comparisons. These of mitochondrial DNA content changes in any tissue in any of results indicate there were likely compensatory changes in the recovery monkeys suggesting no change in the quantity of mitochondrial DNA in heart and liver cells treated with BMS- mitochondrial DNA. 986094 due to decreased cell viability.

ATP, GTP, GMP, and IMP Levels Did Not Change With BMS-986094 BMS-986094 Treatment Leads to Reduced MT-ND1 and MT-ND5 Treatment In Vivo Transcription In Vitro The potential effects of BMS-986094 on tissue ATP, GTP, GMP, Mitochondrial transcription was detected and analyzed in Huh- and IMP levels were analyzed by comparing lmol/g amounts in 7 cells by Northern blot. MT-ND1 and MT-ND5 transcription BAUMGART ET AL. | 401 Downloaded from https://academic.oup.com/toxsci/article/153/2/396/2579185 by guest on 23 September 2021

FIG. 1. Mitochondrial respiration was assessed in monkeys treated with BMS-986094 at doses of 0, 15, or 30 mg/kg/day for 1 month. Samples of (A, B) heart and (C, D) kidney were collected from all available animals at the scheduled necropsy for mitochondrial isolation. Data are expressed as the mean 6 standard deviation (End of Dose [n ¼ 3/sex/group]; *P .05; **P .01). were reduced in Huh-7 cells exposed to BMS-986094 at 10 mM DISCUSSION for 24 h (Figs. 8A and B). Treatment with the unmodified guano- A number of nucleoside analogues have been developed as sine nucleotide analogue, 20-C-methylguanosine, at concentra- therapeutic agents for viral infections or cancer treatments tions 400 mM for 24 h also reduced transcription of MT-ND1 (Galmarini et al., 2002; Quan and Peter, 2004). Several have been and MT-ND5 (Figure 8C). This indicates both BMS-986094 (pro- linked to mitochondrial toxicities, including at least 2 guanosine drug) and 20-C-methylguanosine are incorporated into mito- analogues (Brinkman et al., 1998; Kakuda, 2000; Lewis and chondrial RNA and inhibit transcriptional activity. Furthermore, Dalakas, 1995). Target organs for these compounds are typically IC data indicated that BMS-986094 was 30-fold more 50 rich in mitochondria, and the mechanism of toxicity has been 0 effective at terminating transcription than 2 -C-methylguano- largely reported to be inhibition of the mitochondrial DNA poly- sine (Table 4). merase gamma (Kakuda, 2000). Inhibition of the mitochondrial DNA polymerase or incorporation of nucleoside analogues into POLRMT, MT-ND1, MT-COXII, POLRII, POLRIII Translation mitochondrial DNA results in cumulative damage to mitochon- Decreased Following Treatment of 10 mM BMS-986094 In Vitro drial genes and the resulting proteins. These changes produce Protein content was detected and analyzed in hiPSCM cells by permanent damage to mitochondrial genes/proteins, inefficient InCell Western assay. Following 24 h of treatment, MT-COXII or deficient respiration, and increased reactive oxygen genera- translation was reduced only at a concentration of 10 mM BMS- tion. Eventually, these changes result in the loss of mitochon- 986094 (Figure 9A). However, translation was decreased for dria, impaired energy production, and apoptosis. A more POLRMT, MT-ND1, MT-COXII, POLRII, and POLRIII following 72 h recently identified potential mechanism for mitochondrial tox- of treatment at a concentration of 10 mM BMS-986094 (Figure icity is via inhibition of mitochondrial RNA polymerase (inhibit- 9B). POLa protein levels were unaffected at all treatment con- ing transcription of mitochondrially encoded genes) (Arnold centrations. These results may suggest a selective affinity of et al., 2012). Similarly, inhibition of mitochondrial RNA polymer- BMS-986094 for inhibition of RNA polymerase as opposed to ase causes lasting effects on mitochondrial protein expression DNA polymerase at high concentrations of BMS-986094. that are not immediately alleviated by removal of the inhibitor. 402 | TOXICOLOGICAL SCIENCES, 2016, Vol. 153, No. 2 Downloaded from https://academic.oup.com/toxsci/article/153/2/396/2579185 by guest on 23 September 2021

FIG. 2. Mitochondrial respiration was assessed after a 1-month recovery period in monkeys treated with BMS-986094 at doses of 0, 15, or 30 mg/kg/day for 1 month. Samples of (A, B) heart and (C, D) kidney were collected from all available animals at the scheduled necropsy for mitochondrial isolation. Data are expressed as the mean 6 standard deviation (Recovery [n ¼ 2/sex/group]; *P .05; **P .01).

TABLE 1. Mitochondrial respiratory control ratios in monkey tissues following 1 month of treatment with BMS-986094 and a 1-month recovery.

Treatment End of dose Treatment Recovery

Heart Kidney Heart Kidney

0 mg/kg Males 2.64 6 0.23 2.41 6 0.22 0 mg/kg Males 2.90 6 0.52 2.64 6 0.11 15 mg/kg Males 3.10 6 0.76 2.70 6 0.72 15 mg/kg Males 2.04# 2.17# 30 mg/kg Males 2.79 6 0.32a 2.78 6 0.68a 30 mg/kg Males 3.73# 2.97# 0 mg/kg Females 3.17 6 0.97 2.83 6 0.38 0 mg/kg Females 2.56 6 1.53 2.74 6 0.23 15 mg/kg Females 3.02 6 0.39 3.51 6 0.74 15 mg/kg Females 1.14# 2.92# 30 mg/kg Females 2.67 6 0.12 3.04 6 0.32 30 mg/kg Females 2.94 6 0.93 3.31 6 0.04 aMoribund animal, originally intended as recovery, evaluated 4 days after completion of the 1-month dosing period was added to this evaluation. *The Respiratory Control Ratio (RCR) was determined for mitochondria isolated from monkey heart and kidney following treatment with BMS-986094 at doses of 0, 15, or 30 mg/kg/day for 1 month and following a 1-month recovery period. Data are expressed as the mean 6 standard deviation (End of Dose [n ¼ 3]; Recovery [n ¼ 2]; #n ¼ 1 [no standard deviation]; *P .05; **P .01).

Data presented in this manuscript provide strong evidence that BMS-986094 inhibited isolated mitochondrial RNA polymer- BMS-986094 does not act through similar mechanisms and that ase and appeared to inhibit the transcription of 2 mitochondri- mitochondria are not a primary target of BMS-986094. ally encoded genes (MT-ND1 and MT-ND5) and translation of 5 It should also be noted that mechanisms of toxicity for nucleo- mitochondrially encoded proteins (POLRMT, MT-ND1, MT- side analogues may not be universal and care should be taken COXII, POLRII, and POLRIII) assessed in vitro, but these effects in generalizing a universal mitochondrial mechanism (Lund were observed only at concentrations that were cytotoxic (10 et al., 2007). mM). Both BMS-986094 and 20-C-methylguanosine (INX-08032) BAUMGART ET AL. | 403

in vivo administration of BMS-986094 at doses up to 30 mg/kg/ day was not sufficient to induce mitochondrial dysfunction. Evaluation of the heart and kidneys from BMS-986094- treated monkeys showed no evidence of inhibited mitochondrial respiration that would lead to decreased mitochondrial function. This is in contrast to the reported mechanism for nucleoside analogue-mediated mitochondrial toxicity, which produce permanent/lasting cumulative deficits to mitochondrial function (Zhou et al., 2001). BMS-986094 treatment produced statistically significant increases in mitochondrial respiration in heart mitochondria of male monkeys at 30 mg/kg/ day. However, the values were variable and overlapped with

that of the control females indicating this increase may not be Downloaded from https://academic.oup.com/toxsci/article/153/2/396/2579185 by guest on 23 September 2021 biologically significant. Similarly, the kidneys of BMS-986094- treated male and female monkeys exhibited mildly increased respiratory rates and, in some cases, slight increases in respiratory ratios. These respiration increases would not be possible if BMS-986094 were inhibiting mitochondrial function, such as the proposed mechanisms for nucleoside analogues. Rather, the reversible, slight increases in mitochondrial state III respiration and respiratory ratios (State III/IV) in the heart and/or kidneys of monkeys given 15 or 30 mg/kg/day BMS-986094 for 3–4 weeks likely reflected compensatory mechanisms to increase energy production in response to the pronounced clinical toxicity. However, based on the in vitro mitochondrial respiration assessments, an increase in mitochondrial resting membrane polar- ization or inefficient oxidative phosphorylation cannot be ruled out (Devin and Rigoulet, 2007; Ma et al., 2011; Stanley et al., 2005). BMS-986094 showed evidence of inhibiting mitochondrial respiration in hiPSCM cells on the first day of treatment at cytotoxic concentrations 25 mM and after 3 days of treatment at cytotoxic concentrations 10 mM. Reduced mitochondrial respiration was also observed after 6 days of treatment at BMS-986094

FIG. 3. Mitochondrial DNA content of the mitochondrial NADH dehydrogenase concentrations 0.1 mM. Whereas this indicates a level of mito- subunit I gene was assessed in (A) end of dose and (B) recovery monkeys treated chondrial dysfunction was induced, it is unclear whether these with BMS-986094 at doses of 0, 15, or 30 mg/kg/day for 1 month and following a changes are specific to BMS-986094 treatment or secondary to 1-month recovery period. Samples of heart and kidney were collected from all the markedly reduced cell viability observed in hiPSCM cells available animals, DNA was isolated, and PCR was performed. Means are at or after 6 days of BMS-986094 treatments at concentrations expressed as percent control 6 standard deviation (End of Dose [n ¼ 3/sex/ group]; Female Recovery [n ¼ 2/group]; Male Recovery [n ¼ 1/group]; *P .05; 1 mM. The discrepancy between the two sets of data likely **P .01). results from the measurements being from isolated mitochondria following in vivo treatments and cultured cells during in vitro treatments. Both types of assays have certain limita- are substrates of POLRMT and are incorporated into mitochon- tions. Whereas using cells in culture avoids not only the time- drial RNA leading to chain termination (Arnold et al., 2012). consuming and cumbersome mitochondrial isolation process, it BMS-986094 (IC50 9 mM) is more effective at terminating tran- also decreases the artifacts and potential damage associated scription than INX-08032 (IC50 201 mM). This difference is with isolated mitochondria (Brand and Nicholls, 2011). These likely a reflection of the poor cellular permeability of INX-08032 differences could help explain why BMS-986094 treatment and the associated higher intracellular concentration of the appears to be more potent in disrupting mitochondrial respira- BMS-986094 triphosphorylated metabolite (INX-09114). The tion in vitro compared to in vivo. potency of BMS-986094 to inhibit POLRMT and terminate tran- In vitro assessments of mitochondrial DNA content in scription may be relevant to the cardiac and renal toxicities HiPSCM or HepG2 cells treated with BMS-986094 for up to observed in monkeys administered doses of 15 and 30 mg/kg/ 19 days showed no clear decreases in mitochondrial DNA con- day because tissue concentrations (7to8mM) of BMS-986094 tent. Noted increases were likely the result of a compensatory and INX-09114 approximated the IC50 value (9 mM). However, mechanism and do not indicate direct mitochondrial toxicity. because the transcriptional and translational effects observed HepG2 cells have abundant mitochondria and represent a well- in these in vitro assessments did not precede cytotoxicity, they established model for evaluating mitochondrial depletion are most likely a consequence of the observed toxicity rather in vitro (Lund et al., 2007; Venhoff et al., 2007). Furthermore, liver than its cause. Moreover, in vivo effects on mitochondrial RNA and kidney samples collected from monkeys treated with BMS- polymerase or mitochondrial DNA polymerase should be evi- 986094 did not demonstrate any substantive changes in mito- dent in lasting effects on mitochondrial respiration, mitochon- chondrial DNA content. The technique utilized in this study to drial DNA content, and ATP levels, which were not observed in assess mitochondrial DNA content has been successfully used key target organs from monkeys treated with high oral doses of by others to show mitochondrial depletion in the tissues of BMS-986094 for up to 1 month. These results highlight that patients (Helene et al., 2002; Lebrecht et al., 2009) as well as 404 | TOXICOLOGICAL SCIENCES, 2016, Vol. 153, No. 2

TABLE 2. High energy substrate levels in monkey tissues following 3 weeks of treatment with BMS-986094.

Treatment Tissue End of dose ATP (lmol/g) End of dose GTP (lmol/g) End of dose GMP (lmol/g) End of dose IMP (lmol/g)

0 mg/kg Males Heart 7.61 6 0.19 8.41 6 0.11 8.04 6 0.20 6.80 6 0.19 Kidney 7.49 6 0.55 7.55 6 0.64 8.47 6 0.36 7.13 6 0.19 Diaphragm 7.58 6 0.66 7.33 6 1.38 6.05 6 1.48 6.48 6 0.21 Liver 7.12 6 0.76 6.50 6 0.09 7.19 6 1.53 6.58 6 0.45 15 mg/kg Males Heart 7.58 6 0.14 8.42 6 0.18 8.16 6 0.18 6.88 6 0.09 Kidney 6.88 6 0.20 7.71 6 0.32 8.31 6 0.22 7.12 6 0.46 Diaphragm 7.67 6 0.38 8.21 6 0.08 7.10 6 0.27 6.47 6 0.06 Liver 6.38 6 0.27 5.71 6 0.92 7.84 6 0.48 6.72 6 0.49 30 mg/kg Males Heart 7.20 6 0.57 7.99 6 0.68 7.76 6 0.52 6.61 6 0.64 Kidney 7.17 6 0.25 7.60 6 0.35 7.66 6 0.25* 6.97 6 0.33 Diaphragm 6.57 6 1.21 7.11 6 1.31 6.09 6 1.60 5.61 6 1.22 Downloaded from https://academic.oup.com/toxsci/article/153/2/396/2579185 by guest on 23 September 2021 Liver 6.07 6 0.43 5.76 6 0.86 7.32 6 0.59 6.33 6 0.52 0 mg/kg Females Heart 7.29 6 0.60 8.02 6 0.65 7.68 6 0.55 6.42 6 0.48 Kidney 6.88 6 0.43 7.57 6 0.36 8.19 6 0.55 7.11 6 0.45 Diaphragm 7.70 6 0.50 7.83 6 0.33 6.66 6 0.24 6.07 6 0.87 Liver 7.01 6 0.24 5.99 6 1.21 7.81 6 0.59 6.69 6 0.76 15 mg/kg Females Heart 7.68 6 0.07 8.43 6 0.08 8.09 6 0.26 6.91 6 0.15 Kidney 6.97 6 0.06 7.75 6 0.13 8.39 6 0.14 7.30 6 0.17 Diaphragm 7.82 6 0.23 8.23 6 0.15 7.12 6 0.19 7.12 6 0.40 Liver 6.18 6 1.31 5.91 6 0.59 8.10 6 0.08 7.01 6 0.08 30 mg/kg Females Heart 7.56 6 0.53 8.43 6 0.50 8.14 6 0.35 6.17 6 0.81 Kidney 6.87 6 0.51 7.83 6 0.77 8.31 6 0.59 7.35 6 0.60 Diaphragm 7.73 6 0.92 7.69 6 0.69 6.22 6 0.76 6.47 6 0.69 Liver 5.65 6 0.79 5.90 6 0.65 7.70 6 0.28 6.47 6 0.17

*Nucleoside/Nucleotide levels were assessed in the heart, liver, kidney, and diaphragm of monkeys treated with BMS-986094 at doses of 0, 15, or 30 mg/kg/day for 3 weeks. Data are expressed as mean lmol/g of tissue 6 standard deviation (n ¼ 3; *P .05; **P .01).

TABLE 3. High energy substrate levels in monkey tissues following a 3-week recovery in monkeys treated with BMS-986094.

Treatment Tissue Recovery ATP (lmol/g) Recovery GTP (lmol/g) Recovery GMP (lmol/g) Recovery IMP (lmol/g)

0 mg/kg Males Heart 7.55 6 0.02 8.33 6 0.09 7.85 6 0.06 6.42 6 0.11 Kidney 7.33 6 0.70 7.92 6 0.10 8.46 6 0.22 7.25 6 0.00 Diaphragm 7.52 6 0.25 7.97 6 0.13 6.95 6 0.20 6.25 6 0.01 Liver 6.73 6 0.27 6.70 6 0.27 7.92 6 0.00 6.96 6 0.12 15 mg/kg Males Heart 8.18# 9.04# 8.35# 7.07# Kidney 6.48# 7.24# 7.74# 6.78# Diaphragm 7.44# 7.76# 6.60# 5.31# Liver 6.11# 5.09# 7.62# 6.59# 30 mg/kg Males Heart 7.58 6 0.00 8.37 6 0.06 7.92 6 0.08 6.72 6 0.14 Kidney 6.95 6 0.18 7.88 6 0.08 8.27 6 0.18 7.37 6 0.17 Diaphragm 7.81 6 0.11 8.21 6 0.04 7.08 6 0.01 6.65 6 0.10 Liver 5.73 6 1.10 5.48 6 0.11 7.99 6 0.09 6.98 6 0.22 0 mg/kg Females Heart 7.63 6 0.04 8.47 6 0.07 7.96 6 0.07 6.57ˆ Kidney 7.27 6 0.50 7.79 6 0.19 8.20 6 0.09 7.44 6 0.12 Diaphragm 7.86 6 0.24 8.26 6 0.15 6.98 6 0.42 6.72 6 0.40 Liver 6.73 6 0.02 5.82 6 1.15 7.96 6 0.00 6.86 6 0.21 15 mg/kg Females Heart 5.96 6 0.51 8.27 6 0.31 7.67 6 0.36 6.92ˆ Kidney 6.41 6 0.49 7.16 6 0.83 7.81 6 0.72 6.99 6 1.00 Diaphragm 7.67 6 0.33 7.96 6 0.08 6.65 6 0.13 6.56 6 0.23 Liver 6.31 6 0.43 6.02 6 1.03 7.68 6 0.58 6.76 6 0.50 30 mg/kg Females Heart 7.55 6 0.67 8.55 6 0.42 8.00 6 0.30 6.80 6 0.35 Kidney 6.66 6 0.30 7.07 6 0.99 8.00 6 0.26 7.08 6 0.24 Diaphragm 8.22 6 0.08 8.36 6 0.32 7.03 6 0.36 6.75 6 0.33 Liver 6.36 6 0.64 6.22 6 0.66 7.44 6 0.76 6.49 6 0.61

*Nucleoside/Nucleotide levels were assessed after a 3-week recovery period in the heart, liver, kidney, and diaphragm of monkeys treated with BMS-986094 at doses of 0, 15 or 30 mg/kg/day for 3 weeks. Data are expressed as mean lmol/g of tissue 6 standard deviation (n ¼ 2; #n ¼ 1 [no standard deviation];ˆonly one animal showing IMP [no standard deviation]; *P .05; **P .01). BAUMGART ET AL. | 405 Downloaded from https://academic.oup.com/toxsci/article/153/2/396/2579185 by guest on 23 September 2021

FIG. 4. Cell viability was determined in (A) HepG2 cells treated with BMS-986094 at concentrations of 0.2, 0.5, 1., 1.5, or 2 mM for 5, 9, 14, or 19 days and (B) hiPSCM cells treated with BMS-986094 at concentrations of 0.01, 0.1, 1, 5, 10, 25, or 50 mM for 1, 3, 6, and 10 days. Means are expressed as percent control 6 standard devia- n ¼ P P tion ( 6; * .05; ** .01). FIG. 6. Mitochondrial DNA content of the mitochondrial cytochrome c oxidase subunit II gene was assessed in HepG2 cells treated with (A) DDC at concentrations of 0.1, 0.5 or 1 mM or (B) BMS-986094 at concentrations of 0.2, 0.5, 1, 1.5, or 2 mM for 5, 9, 14, and 19 days. Cells were harvested, DNA was isolated, and PCR was performed. Means are expressed as percent control 6 standard deviation (n ¼ 3; *P .05; **P .01).

animals (Lebrecht et al., 2009) treated with mitochondrial toxicants, including nucleoside analogues. Evaluation of nucleoside levels, particularly adenosine triphosphate (ATP), in target tissues (heart, kidney, liver, and diaphragm) from BMS-986094-treated monkeys further suggests no effect of treatment on mitochondrial function. That is, there was no effect of BMS-986094 treatment on tissue ATP, GTP, GMP, or IMP levels, demonstrating intact production of energy, the major role of mitochondria. By comparison, in vitro and in vivo studies have consistently demonstrated decreases in ATP levels with mitochondrial toxicants (Begriche et al., 2011; Degli Esposti et al., 2012; Ikeda et al., 2014; Marin-Garcia et al., 2001; Nieminen et al., 1994). Furthermore, the lack of an effect on mitochondrial GTP levels suggests that BMS-986094 does not lead to GTP depletion and associated toxicity due to inhibition of GTP synthesis via inhibition of IMP dehydrogenase (IMPDH) as reported for some antiviral compounds (Graci and Cameron, 2006; Leyssen et al., 2005). Overall, this work provides strong evidence that BMS- 986094-mediated toxicity does not result from inhibition of mitochondrial function as reported for some other nucleoside analogues. Several potential mechanisms of BMS-986094- related toxicity have been ruled out including inhibition of FIG. 5. Basal cell oxygen consumption (A) and mitochondrial respiration (B) were assessed in hiPSCM cells treated with BMS-986094 at concentrations of mitochondrial DNA polymerase (and mitochondrial replication), 0.01, 0.1, 1, 5, 10, 25 or 50 mM for 1, 3, 6, and 10 days. Means are expresses as per- mechanisms leading to inhibition of mitochondrial ATP produc- cent control 6 standard deviation (n ¼ 3). tion; inhibition of IMPDH leading to GTP depletion; and 406 | TOXICOLOGICAL SCIENCES, 2016, Vol. 153, No. 2

mechanisms leading to decreased mitochondrial DNA content. Although BMS-986094 inhibits mitochondrial polymerase at high concentrations in vitro, there were no apparent effects on mitochondrial RNA polymerase or mitochondrial DNA polymerase in vivo. Collectively, the absence of reductions in mitochondrial respiration, mitochondrial DNA content, and ATP levels in the heart and kidneys of monkeys treated with BMS-986094 for up to 1 month at doses up to 30 mg/kg/day that produced cardiac and renal toxicity suggest there is no direct effect on mitochondrial function in vivo. However, we cannot rule out the potential for altered secondary mitochondrial energy utilization. Downloaded from https://academic.oup.com/toxsci/article/153/2/396/2579185 by guest on 23 September 2021 FUNDING This study was funded by Bristol-Myers Squibb.

CONFLICT OF INTEREST The authors take full responsibility for the content of this publication and confirm that it reflects their viewpoint and expertise. The authors did not receive financial compensation for author- ing the manuscript. B.R.B., F.W., J.K., C.E., R.T.B., M.J.G., and T.P.S. are employees of Bristol-Myers Squibb. R.T.B., M.J.G., and T.P.S. received equity from Bristol-Myers Squibb as part of employee compensation. S.S., J.J.A., and C.E.C. are employees of The Pennsylvania State University. C.S., M.F., D.S., T.R.V., O.F., and M.H.D. have no conflicting interests. C.S., M.F., D.S., T.R.V., O.F., and M.H.D. were employees of Bristol-Myers Squibb at the

FIG. 7. Mitochondrial DNA content of the mitochondrial cytochrome c oxidase time the reported work was performed. subunit II gene was assessed in hiPSCM cells treated with (A) AZT at concentrations of 1, 20, or 200 mM or (B) BMS-986094 at concentrations of 0.01, 0.1, 1, or ACKNOWLEDGMENTS 10 mM for 1, 3, and 6 days. Cells were harvested, DNA was isolated, and PCR was performed. Means are expressed as percent control 6 standard deviation (n ¼ 3; The authors wish to thank the Drug Safety Evaluation *P .05; **P .01). Toxicology, Pathology, and Veterinary Science staffs at

FIG. 8. Mitochondrial NADH dehydrogenase subunit 1 (MT-ND1) and 5 (MT-ND5) transcription was detected and analyzed by Northern blot. Huh-7 cells were pre- treated with 50 ng/ml EtBr for 24 h to deplete mitochondrial transcripts. The cells were then treated with (A) 20-C-methylguanosine at concentrations of 10, 50, 100, 200, or 400 mM or BMS-986094 at concentrations of 10, 50, 100, or 200 mM for 24 h; (B) 20-C-methylguanosine or BMS-986094 at concentrations of 0.1, 1, or 10 mM for 24 h; or (C) 20-C-methylguanosine at concentrations of 1, 10, 100, 200, 400, 600, or 800 mM for 24 h. Cells were harvested, RNA was isolated, and Northern blot analysis was performed. BAUMGART ET AL. | 407

TABLE 4. IC50 values for inhibition of mitochondrial transcription. (2015). Cardiac dysfunction associated with a nucleotide polymerase inhibitor for treatment of Hepatitis C. Hepatology. Compound IC (mM) 50 62, 409–416. INX-08032 (2’-C-methylguanosine) Trial 1 201 6 48 Arnold, J. J., Sharma, S. D., Feng, J. Y., Ray, A. S., Smidansky, E. D., INX-08032 (2’-C-methylguanosine) Trial 2 340 6 89 Kireeva, M. L., Cho, A., Perry, J., Vela, J. E., Park, Y., et al. (2012). BMS-986094 9 6 4 Sensitivity of mitochondrial transcription and resistance of RNA polymerase II dependent nuclear transcription to anti- The IC50 for BMS-986094 and the guanosine nucleotide analogue 20-C-methyl- viral ribonucleosides. PLoS Pathog. 8, e1003030. guanosine were determined. MT-ND5 and GAPDH bands from Northern blot Begriche, K., Massart, J., Robin, M., Borgne-Sanchez, A., and analysis were quantified using ImageQuant 5.0 Software (GE Healthcare) an data Fromenty, B. (2011). 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