Structural and Functional Analyses of Inhibition of Human Dihydroorotate Dehydrogenase by Antiviral Furocoumavirin.

Natural products are important sources of seed compounds for drug discovery. However, it has become difficult in recent years to discover new compounds with valuable pharmacological activities. On the other hand, among the vast number of natural products that have been isolated so far, a considerable number of compounds with specific biological activities are thought to be overlooked in screening that uses biological activity as an index. Therefore, it is conceivable that such overlooked useful compounds may be found by screening compound libraries that have been amassed previously through specific assays. Previously, NPD723, a member of the Natural Products Depository library comprised of a mixture of natural and non-natural products developed at RIKEN, and its metabolite H-006 were found to inhibit growth of various cancer cells at low nanomolar half-maximal inhibitory concentration. Subsequent analysis revealed that H-006 strongly inhibited human dihydroorotate dehydrogenase (DHODH), the rate-limiting enzyme in the de novo pyrimidine biosynthetic pathway. Here, we elucidated the crystal structure of the DHODH-flavin mononucleotide-orotic acid-H-006 complex at 1.7 Å resolution to determine that furocoumavirin, the S-enantiomer of H-006, was the actual inhibitor. The overall mode of interaction of furocoumavirin with the inhibitor binding pocket was similar to that described for previously reported tight-binding inhibitors. However, the structural information together with kinetic characterizations of site-specific mutants identified key unique features that are considered to contribute to the sub-nanomolar inhibition of DHODH by furocoumavirin. Our finding identified new chemical features that could improve the design of human DHODH inhibitors.

In Silico Comparison of Bioactive Compounds Characterized from Azadirachta indica with an FDA-Approved Drug against Schistosomal Agents: New Insight into Schistosomiasis Treatment.

The burden of human schistosomiasis, a known but neglected tropical disease in Sub-Saharan Africa, has been worrisome in recent years. It is becoming increasingly difficult to tackle schistosomiasis with praziquantel, a drug known to be effective against all Schistosoma species, due to reports of reduced efficacy and resistance. Therefore, this study seeks to investigate the antischistosomal potential of phytochemicals from Azadirachta indica against proteins that have been implicated as druggable targets for the treatment of schistosomiasis using computational techniques. In this study, sixty-three (63) previously isolated and characterized phytochemicals from A. indica were identified from the literature and retrieved from the PubChem database. In silico screening was conducted to assess the inhibitory potential of these phytochemicals against three receptors (Schistosoma mansoni Thioredoxin glutathione reductase, dihydroorotate dehydrogenase, and Arginase) that may serve as therapeutic targets for schistosomiasis treatment. Molecular docking, ADMET prediction, ligand interaction, MMGBSA, and molecular dynamics simulation of the hit compounds were conducted using the Schrodinger molecular drug discovery suite. The results show that Andrographolide possesses a satisfactory pharmacokinetic profile, does not violate the Lipinski rule of five, binds with favourable affinity with the receptors, and interacts with key amino acids at the active site. Importantly, its interaction with dihydroorotate dehydrogenase, an enzyme responsible for the catalysis of the de novo pyrimidine nucleotide biosynthetic pathway rate-limiting step, shows a glide score and MMGBSA of -10.19 and -45.75 Kcal/mol, respectively. In addition, the MD simulation shows its stability at the active site of the receptor. Overall, this study revealed that Andrographolide from Azadirachta indica could serve as a potential lead compound for the development of an anti-schistosomal drug.

An alternative conformation of the N-terminal loop of human dihydroorotate dehydrogenase drives binding to a potent antiproliferative agent.

Over the years, human dihydroorotate dehydrogenase (hDHODH), which is a key player in the de novo pyrimidine-biosynthesis pathway, has been targeted in the treatment of several conditions, including autoimmune disorders and acute myelogenous leukaemia, as well as in host-targeted antiviral therapy. A molecular exploration of its inhibitor-binding behaviours yielded promising candidates for innovative drug design. A detailed description of the enzymatic pharmacophore drove the decoration of well-established inhibitory scaffolds, thus gaining further in vitro and in vivo efficacy. In the present work, using X-ray crystallography, an atypical rearrangement was identified in the binding pose of a potent inhibitor characterized by a polar pyridine-based moiety (compound 18). The crystal structure shows that upon binding compound 18 the dynamics of a protein loop involved in a gating mechanism at the cofactor-binding site is modulated by the presence of three water molecules, thus fine-tuning the polarity/hydrophobicity of the binding pocket. These solvent molecules are engaged in the formation of a hydrogen-bond mesh in which one of them establishes a direct contact with the pyridine moiety of compound 18, thus paving the way for a reappraisal of the inhibition of hDHODH. Using an integrated approach, the thermodynamics of such a modulation is described by means of isothermal titration calorimetry coupled with molecular modelling. These structural insights will guide future drug design to obtain a finer Kd/logD7.4 balance and identify membrane-permeable molecules with a drug-like profile in terms of water solubility.

Pivotal role of dihydroorotate dehydrogenase as a therapeutic target in adult T-cell leukemia.

OBJECTIVES: We aimed to determine the role of dihydroorotate dehydrogenase (DHODH) in pathogenesis of adult T-cell leukemia (ATL) caused by human T-cell leukemia virus type 1 (HTLV-1) and the effects of its inhibition on the de novo pyrimidine biosynthesis pathway. METHODS: Cell proliferation, viability, cycle, and apoptosis were analyzed using WST-8 assays, flow cytometry, and Hoechst 33342 staining. To elucidate the molecular mechanisms involved in the anti-ATL effects of DHODH knockdown and inhibition, RT-PCR and immunoblotting were conducted. RESULTS: HTLV-1-infected T-cell lines aberrantly expressed DHODH. Viral infection and the oncoprotein, Tax, enhanced DHODH expression, while knockdown of DHODH decreased HTLV-1-infected T-cell growth. In addition, BAY2402234, a DHODH inhibitor, exerted an anti-proliferative effect, which was reversed by uridine supplementation. BAY2402234 induced DNA damage and S phase arrest by downregulating c-Myc, CDK2, and cyclin A and upregulating p53 and cyclin E. It also induced caspase-mediated apoptosis by the upregulation of pro-apoptotic and downregulation of anti-apoptotic proteins. Furthermore, BAY2402234 induced caspase-independent ferroptosis and necroptosis. It decreased phosphorylation of IKK, IκBα, PTEN, Akt, and its downstream targets, suggesting that inhibition of NF-κB and Akt signaling is involved in its anti-ATL action. CONCLUSION: These findings highlight DHODH as a potential therapeutic target for treating ATL.

Discovery of a new class of potent pyrrolo[3,4-c]quinoline-1,3-diones based inhibitors of human dihydroorotate dehydrogenase: Synthesis, pharmacological and toxicological evaluation.

Twenty N-substituted pyrrolo[3,4-c]quinoline-1,3-diones 3a-t were synthesized by a cyclization reaction of Pfitzinger's quinoline ester precursor with the selected aromatic, heteroaromatic and aliphatic amines. The structures of all derivatives were confirmed by IR, 1H NMR, 13C NMR and HRMS spectra, while their purity was determined using HPLC techniques. Almost all compounds were identified as a new class ofpotent inhibitors against hDHODH among which 3a and 3t were the most active ones with the same IC50 values of 0.11 µM, about seven times better than reference drug leflunomide. These two derivatives also exhibited very low cytotoxic effects toward healthy HaCaT cells and the optimal lipophilic properties with logP value of 1.12 and 2.07 respectively, obtained experimentally at physiological pH. We further evaluated the comparative differences in toxicological impact of the three most active compounds 3a, 3n and 3t and reference drug leflunomide. The rats were divided into five groups and were treated intraperitoneally, control group (group I) with a single dose of leflunomide (20 mg/kg) group II and the other three groups, III, IV and V were treated with 3a, 3n and 3t (20 mg/kg bw) separately. The investigation was performed in liver, kidney and blood by examining serum biochemical parameters and parameters of oxidative stress.

DHODH inhibition represents a therapeutic strategy and improves abiraterone treatment in castration-resistant prostate cancer.

Castration-resistant prostate cancer (CRPC) is an aggressive disease with poor prognosis, and there is an urgent need for more effective therapeutic targets to address this challenge. Here, we showed that dihydroorotate dehydrogenase (DHODH), an enzyme crucial in the pyrimidine biosynthesis pathway, is a promising therapeutic target for CRPC. The transcript levels of DHODH were significantly elevated in prostate tumors and were negatively correlated with the prognosis of patients with prostate cancer. DHODH inhibition effectively suppressed CRPC progression by blocking cell cycle progression and inducing apoptosis. Notably, treatment with DHODH inhibitor BAY2402234 activated androgen biosynthesis signaling in CRPC cells. However, the combination treatment with BAY2402234 and abiraterone decreased intratumoral testosterone levels and induced apoptosis, which inhibited the growth of CWR22Rv1 xenograft tumors and patient-derived xenograft organoids. Taken together, these results establish DHODH as a key player in CRPC and as a potential therapeutic target for advanced prostate cancer.

Repositioning Food and Drug Administration-Approved Drugs for Inhibiting Biliverdin IXß Reductase B as a Novel Thrombocytopenia Therapeutic Target.

Biliverdin IXß reductase B (BLVRB) has recently been proposed as a novel therapeutic target for thrombocytopenia through its reactive oxygen species (ROS)-associated mechanism. Thus, we aim at repurposing drugs as new inhibitors of BLVRB. Based on IC50 (<5 µM), we have identified 20 compounds out of 1496 compounds from the Food and Drug Administration (FDA)-approved library and have clearly mapped their binding sites to the active site. Furthermore, we show the detailed BLVRB-binding modes and thermodynamic properties (ΔH, ΔS, and KD) with nuclear magnetic resonance (NMR) and isothermal titration calorimetry together with complex structures of eight water-soluble compounds. We anticipate that the results will serve as a novel platform for further in-depth studies on BLVRB effects for related functions such as ROS accumulation and megakaryocyte differentiation, and ultimately treatments of platelet disorders.

Suppression of neuronal cholesterol biosynthesis impairs brain functions through insulin-like growth factor I-Akt signaling.

Some relationship between abnormal cholesterol content and impairment of insulin/insulin-like growth factor I (IGF-1) signaling has been reported in the pathogenesis of Alzheimer's disease (AD). However, the underlying mechanism of this correlation remains unclear. It is known that 3-ß hydroxycholesterol Δ 24 reductase (DHCR24) catalyzes the last step of cholesterol biosynthesis. To explore the function of cholesterol in the pathogenesis of AD, we depleted cellular cholesterol by targeting DHCR24 with siRNA (siDHCR24) or U18666A, an inhibitor of DHCR24, and studied the effect of the loss of cholesterol on the IGF-1-Akt signaling pathway in vitro and in vivo. Treatment with U18666A reduced the cellular cholesterol level and blocked the anti-apoptotic function of IGF-1 by impairing the formation of caveolae and the localization of IGF-1 receptor in caveolae of the PC12 cells. Downregulation of the DHCR24 expression induced by siRNA against DHCR24 also yielded similar results. Furthermore, the phosphorylation levels of IGF-1 receptor, insulin receptor substrate (IRS), Akt, and Bad in response to IGF-1 were all found to decrease in the U18666A-treated cells. Rats treated with U18666A via intracerebral injection also exhibited a significant decrease in the cholesterol level and impaired activities of IGF-1-related signaling proteins in the hippocampus region. A significant accumulation of amyloid ß and a decrease in the expression of neuron-specific enolase (NSE) was also observed in rats with U18666A. Finally, the Morris water maze experiment revealed that U18666A-treated rats showed a significant cognitive impairment. Our findings provide new evidence strongly supporting that a reduction in cholesterol level can result in neural apoptosis via the impairment of the IGF-1-Akt survival signaling in the brain.

Antimalarial Inhibitors Targeting Epigenetics or Mitochondria in Plasmodium falciparum: Recent Survey upon Synthesis and Biological Evaluation of Potential Drugs against Malaria.

Despite many efforts, malaria remains among the most problematic infectious diseases worldwide, mainly due to the development of drug resistance by P. falciparum. Over the past decade, new essential pathways have been emerged to fight against malaria. Among them, epigenetic processes and mitochondrial metabolism appear to be important targets. This review will focus on recent evolutions concerning worldwide efforts to conceive, synthesize and evaluate new drug candidates interfering selectively and efficiently with these two targets and pathways. The focus will be on compounds/scaffolds that possess biological/pharmacophoric properties on DNA methyltransferases and HDAC's for epigenetics, and on cytochrome bc1 and dihydroorotate dehydrogenase for mitochondrion.

Identification of 3,4-Dihydro-2H,6H-pyrimido[1,2-c][1,3]benzothiazin-6-imine Derivatives as Novel Selective Inhibitors of Plasmodium falciparum Dihydroorotate Dehydrogenase.

Plasmodium falciparum's resistance to available antimalarial drugs highlights the need for the development of novel drugs. Pyrimidine de novo biosynthesis is a validated drug target for the prevention and treatment of malaria infection. P. falciparum dihydroorotate dehydrogenase (PfDHODH) catalyzes the oxidation of dihydroorotate to orotate and utilize ubiquinone as an electron acceptor in the fourth step of pyrimidine de novo biosynthesis. PfDHODH is targeted by the inhibitor DSM265, which binds to a hydrophobic pocket located at the N-terminus where ubiquinone binds, which is known to be structurally divergent from the mammalian orthologue. In this study, we screened 40,400 compounds from the Kyoto University chemical library against recombinant PfDHODH. These studies led to the identification of 3,4-dihydro-2H,6H-pyrimido[1,2-c][1,3]benzothiazin-6-imine and its derivatives as a new class of PfDHODH inhibitor. Moreover, the hit compounds identified in this study are selective for PfDHODH without inhibition of the human enzymes. Finally, this new scaffold of PfDHODH inhibitors showed growth inhibition activity against P. falciparum 3D7 with low toxicity to three human cell lines, providing a new starting point for antimalarial drug development.

Efficacy and safety of dihydroorotate dehydrogenase (DHODH) inhibitors "leflunomide" and "teriflunomide" in Covid-19: A narrative review.

Dihydroorotate dehydrogenase (DHODH) is rate-limiting enzyme in biosynthesis of pyrimidone which catalyzes the oxidation of dihydro-orotate to orotate. Orotate is utilized in the biosynthesis of uridine-monophosphate. DHODH inhibitors have shown promise as antiviral agent against Cytomegalovirus, Ebola, Influenza, Epstein Barr and Picornavirus. Anti-SARS-CoV-2 action of DHODH inhibitors are also coming up. In this review, we have reviewed the safety and efficacy of approved DHODH inhibitors (leflunomide and teriflunomide) against COVID-19. In target-centered in silico studies, leflunomide showed favorable binding to active site of MPro and spike: ACE2 interface. In artificial-intelligence/machine-learning based studies, leflunomide was among the top 50 ligands targeting spike: ACE2 interaction. Leflunomide is also found to interact with differentially regulated pathways [identified by KEGG (Kyoto Encyclopedia of Genes and Genomes) and reactome pathway analysis of host transcriptome data] in cogena based drug-repurposing studies. Based on GSEA (gene set enrichment analysis), leflunomide was found to target pathways enriched in COVID-19. In vitro, both leflunomide (EC50 41.49 ± 8.8 µmol/L) and teriflunomide (EC50 26 µmol/L) showed SARS-CoV-2 inhibition. In clinical studies, leflunomide showed significant benefit in terms of decreasing the duration of viral shredding, duration of hospital stay and severity of infection. However, no advantage was seen while combining leflunomide and IFN alpha-2a among patients with prolonged post symptomatic viral shredding. Common adverse effects of leflunomide were hyperlipidemia, leucopenia, neutropenia and liver-function alteration. Leflunomide/teriflunomide may serve as an agent of importance to achieve faster virological clearance in COVID-19, however, findings needs to be validated in bigger sized placebo controlled studies.

Combination of consensus and ensemble docking strategies for the discovery of human dihydroorotate dehydrogenase inhibitors.

The inconsistencies in the performance of the virtual screening (VS) process, depending on the used software and structural conformation of the protein, is a challenging issue in the drug design and discovery field. Varying performance, especially in terms of early recognition of the potential hit compounds, negatively affects the whole process and leads to unnecessary waste of the time and resources. Appropriate application of the ensemble docking and consensus-scoring approaches can significantly increase reliability of the VS results. Dihydroorotate dehydrogenase (DHODH) is a key enzyme in the pyrimidine biosynthesis pathway. It is considered as a valuable therapeutic target in cancer, autoimmune and viral diseases. Based on the conducted benchmark study and analysis of the effect of different combinations of the applied methods and approaches, here we suggested a structure-based virtual screening (SBVS) workflow that can be used to increase the reliability of VS.

Cell-specific transcriptional control of mitochondrial metabolism by TIF1γ drives erythropoiesis.

Transcription and metabolism both influence cell function, but dedicated transcriptional control of metabolic pathways that regulate cell fate has rarely been defined. We discovered, using a chemical suppressor screen, that inhibition of the pyrimidine biosynthesis enzyme dihydroorotate dehydrogenase (DHODH) rescues erythroid differentiation in bloodless zebrafish moonshine (mon) mutant embryos defective for transcriptional intermediary factor 1 gamma (tif1γ). This rescue depends on the functional link of DHODH to mitochondrial respiration. The transcription elongation factor TIF1γ directly controls coenzyme Q (CoQ) synthesis gene expression. Upon tif1γ loss, CoQ levels are reduced, and a high succinate/α-ketoglutarate ratio leads to increased histone methylation. A CoQ analog rescues mon's bloodless phenotype. These results demonstrate that mitochondrial metabolism is a key output of a lineage transcription factor that drives cell fate decisions in the early blood lineage.

Gentisyl alcohol and homogentisic acid: Plasmodium falciparum dihydroorotate dehydrogenase inhibitors isolated from fungi.

Two Indonesian fungi Aspergillus assiutensis BioMCC-f.T.7495 and Penicillium pedernalense BioMCC-f.T.5350 along with a Japanese fungus Hypomyces pseudocorticiicola FKI-9008 have been found to produce gentisyl alcohol (1), which inhibits Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH) with an IC50 value of 3.4 µM. Another Indonesian fungus, Penicillium citrinum BioMCC-f.T.6730, produced an analog of 1, homogentisic acid (4), which also inhibits PfDHODH with an IC50 value of 47.6 µM.

Targeting Acute Myelogenous Leukemia Using Potent Human Dihydroorotate Dehydrogenase Inhibitors Based on the 2-Hydroxypyrazolo[1,5-a]pyridine Scaffold: SAR of the Biphenyl Moiety.

The connection with acute myelogenous leukemia (AML) of dihydroorotate dehydrogenase (hDHODH), a key enzyme in pyrimidine biosynthesis, has attracted significant interest from pharma as a possible AML therapeutic target. We recently discovered compound 1, a potent hDHODH inhibitor (IC50 = 1.2 nM), able to induce myeloid differentiation in AML cell lines (THP1) in the low nM range (EC50 = 32.8 nM) superior to brequinar's phase I/II clinical trial (EC50 = 265 nM). Herein, we investigate the 1 drug-like properties observing good metabolic stability and no toxic profile when administered at doses of 10 and 25 mg/kg every 3 days for 5 weeks (Balb/c mice). Moreover, in order to identify a backup compound, we investigate the SAR of this class of compounds. Inside the series, 17 is characterized by higher potency in inducing myeloid differentiation (EC50 = 17.3 nM), strong proapoptotic properties (EC50 = 20.2 nM), and low cytotoxicity toward non-AML cells (EC30(Jurkat) > 100 µM).

Potent Antimalarials with Development Potential Identified by Structure-Guided Computational Optimization of a Pyrrole-Based Dihydroorotate Dehydrogenase Inhibitor Series.

Dihydroorotate dehydrogenase (DHODH) has been clinically validated as a target for the development of new antimalarials. Experience with clinical candidate triazolopyrimidine DSM265 (1) suggested that DHODH inhibitors have great potential for use in prophylaxis, which represents an unmet need in the malaria drug discovery portfolio for endemic countries, particularly in areas of high transmission in Africa. We describe a structure-based computationally driven lead optimization program of a pyrrole-based series of DHODH inhibitors, leading to the discovery of two candidates for potential advancement to preclinical development. These compounds have improved physicochemical properties over prior series frontrunners and they show no time-dependent CYP inhibition, characteristic of earlier compounds. Frontrunners have potent antimalarial activity in vitro against blood and liver schizont stages and show good efficacy in Plasmodium falciparum SCID mouse models. They are equally active against P. falciparum and Plasmodium vivax field isolates and are selective for Plasmodium DHODHs versus mammalian enzymes.

Targeting Chikungunya Virus Replication by Benzoannulene Inhibitors.

A benzo[6]annulene, 4-(tert-butyl)-N-(3-methoxy-5,6,7,8-tetrahydronaphthalen-2-yl) benzamide (1a), was identified as an inhibitor against Chikungunya virus (CHIKV) with antiviral activity EC90 = 1.45 µM and viral titer reduction (VTR) of 2.5 log at 10 µM with no observed cytotoxicity (CC50 = 169 µM) in normal human dermal fibroblast cells. Chemistry efforts to improve potency, efficacy, and drug-like properties of 1a resulted in a novel lead compound 8q, which possessed excellent cellular antiviral activity (EC90 = 270 nM and VTR of 4.5 log at 10 µM) and improved liver microsomal stability. CHIKV resistance to an analog of 1a, compound 1c, tracked to a mutation in the nsP3 macrodomain. Further mechanism of action studies showed compounds working through inhibition of human dihydroorotate dehydrogenase in addition to CHIKV nsP3 macrodomain. Moderate efficacy was observed in an in vivo CHIKV challenge mouse model for compound 8q as viral replication was rescued from the pyrimidine salvage pathway.

Effective deploying of a novel DHODH inhibitor against herpes simplex type 1 and type 2 replication.

Emergence of drug resistance and adverse effects often affect the efficacy of nucleoside analogues in the therapy of Herpes simplex type 1 (HSV-1) and type 2 (HSV-2) infections. Host-targeting antivirals could therefore be considered as an alternative or complementary strategy in the management of HSV infections. To contribute to this advancement, here we report on the ability of a new generation inhibitor of a key cellular enzyme of de novo pyrimidine biosynthesis, the dihydroorotate dehydrogenase (DHODH), to inhibit HSV-1 and HSV-2 in vitro replication, with a potency comparable to that of the reference drug acyclovir. Analysis of the HSV replication cycle in MEDS433-treated cells revealed that it prevented the accumulation of viral genomes and reduced late gene expression, thus suggesting an impairment at a stage prior to viral DNA replication consistent with the ability of MEDS433 to inhibit DHODH activity. In fact, the anti-HSV activity of MEDS433 was abrogated by the addition of exogenous uridine or of the product of DHODH, the orotate, thus confirming DHODH as the MEDS433 specific target in HSV-infected cells. A combination of MEDS433 with dipyridamole (DPY), an inhibitor of the pyrimidine salvage pathway, was then observed to be effective in inhibiting HSV replication even in the presence of exogenous uridine, thus mimicking in vivo conditions. Finally, when combined with acyclovir and DPY in checkerboard experiments, MEDS433 exhibited highly synergistic antiviral activity. Taken together, these findings suggest that MEDS433 is a promising candidate as either single agent or in combination regimens with existing direct-acting anti-HSV drugs to develop new strategies for treatment of HSV infections.

Inhibition of mitochondrial complex III induces differentiation in acute myeloid leukemia.

Although acute myeloid leukemia (AML) is a highly heterogeneous disease with diverse genetic subsets, one hallmark of AML blasts is myeloid differentiation blockade. Extensive evidence has indicated that differentiation induction therapy represents a promising treatment strategy. Here, we identified that the pharmacological inhibition of the mitochondrial electron transport chain (ETC) complex III by antimycin A inhibits proliferation and promotes cellular differentiation of AML cells. Mechanistically, we showed that the inhibition of dihydroorotate dehydrogenase (DHODH), a rate-limiting enzyme in de novo pyrimidine biosynthesis, is involved in antimycin A-induced differentiation. The activity of antimycin A could be reversed by supplement of excessive amounts of exogenous uridine as well as orotic acid, the product of DHODH. Furthermore, we also found that complex III inhibition exerts a synergistic effect in differentiation induction combined with DHODH inhibitor brequinar as well as with the pyrimidine salvage pathway inhibitor dipyridamole. Collectively, our study uncovered the link between mitochondrial complex III and AML differentiation and may provide further insight into the potential application of mitochondrial complex III inhibitor as a mono or combination treatment in differentiation therapy of AML.

Investigating dihydroorotate dehydrogenase inhibitor mediated mitochondrial dysfunction in hepatic in vitro models.

Inhibition of dihydroorotate dehydrogenase (DHODH), the rate-limiting enzymatic step in de novo pyrimidine synthesis, has broad immunosuppressive effects in vivo and shows promise as a therapeutic target for the treatment of malignancies, viral infections and auto-immune diseases. Whilst there are numerous DHODH inhibitors under development, leflunomide and teriflunomide are the only FDA approved compounds on the market, each of which have been issued with black-box warnings for hepatotoxicity. Mitochondrial dysfunction is a putative mechanism by which teriflunomide and leflunomide elicit their hepatotoxic effects, however it is as yet unclear whether this is shared by other nascent DHODH inhibitors. The present study aimed to evaluate the propensity for DHODH inhibitors to mediate mitochondrial dysfunction in two hepatic in vitro models. Initial comparisons of cytotoxicity and ATP content in HepaRG® cells primed for oxidative metabolism, in tandem with mechanistic evaluations by extracellular flux analysis identified multifactorial toxicity and moderate indications of respiratory chain dysfunction or uncoupling. Further investigations using HepG2 cells, a hepatic line with limited capability for phase I xenobiotic metabolism, identified leflunomide and brequinar as positive mitochondrial toxicants. Taken together, biotransformation of some DHODH inhibitor species may play a role in mediating or masking hepatic mitochondrial liabilities.