{"gene":"DHODH","run_date":"2026-06-09T23:54:42","timeline":{"discoveries":[{"year":2021,"finding":"DHODH operates in parallel to mitochondrial GPX4 (but independently of cytosolic GPX4 or FSP1) to inhibit ferroptosis in the mitochondrial inner membrane by reducing ubiquinone to ubiquinol, a radical-trapping antioxidant that suppresses mitochondrial lipid peroxidation.","method":"Genetic inactivation (DHODH KO), pharmacological inhibition (brequinar), metabolite supplementation (dihydroorotate/orotate), subcellular fractionation, lipid peroxidation assays, in vitro and in vivo tumor models","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — multiple orthogonal methods including KO, pharmacological inhibition, substrate/product rescue, and in vivo validation in a single rigorous study","pmids":["33981038"],"is_preprint":false},{"year":2011,"finding":"DHODH inhibition (e.g., leflunomide) suppresses transcriptional elongation of genes required for neural crest development and melanoma growth, distinct from its pyrimidine synthesis function.","method":"Chemical genetic screen in zebrafish, mammalian neural crest stem cell self-renewal assays, transcriptional elongation assays, melanoma xenograft studies, BRAF(V600E) transgenic zebrafish model","journal":"Nature","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (zebrafish genetics, mammalian cell assays, xenograft), replicated across model systems in a single rigorous study","pmids":["21430780"],"is_preprint":false},{"year":2006,"finding":"Human DHODH inhibitors bind competitively at the ubiquinone (CoQ) binding site; X-ray crystal structures revealed a dual binding mode of inhibitors within the same cocrystal, dependent on chemical substitution, with in vitro activity correlating with binding mode.","method":"High-resolution X-ray crystallography of DHODH-inhibitor complexes, in vitro enzymatic inhibition assays","journal":"Journal of medicinal chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structures with functional correlation, single lab but direct structural and enzymatic validation","pmids":["16480261"],"is_preprint":false},{"year":2018,"finding":"DHODH inhibition by (R)-HZ05 (a chiral tetrahydroindazole) increases p53 synthesis by causing S-phase accumulation and pyrimidine depletion; the crystal structure of the (R)-HZ05/DHODH complex confirmed binding mode and chiral specificity.","method":"Target deconvolution, X-ray crystal structure of DHODH/(R)-HZ05 complex, cell cycle analysis, p53 synthesis measurement, in vivo tumor growth assays","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structure plus functional cellular and in vivo validation in a single study","pmids":["29549331"],"is_preprint":false},{"year":2016,"finding":"ML390 binds directly to DHODH and inhibits it, inducing differentiation in HoxA9-overexpressing AML cells; the X-ray crystal structure of ML390 bound to DHODH elucidated binding interactions.","method":"Phenotypic high-throughput screen, genetic resistance mapping, X-ray crystallography of DHODH-ML390 complex","journal":"ACS medicinal chemistry letters","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structure with genetic target identification and cellular differentiation phenotype","pmids":["27994748"],"is_preprint":false},{"year":2012,"finding":"Biallelic DHODH loss-of-function mutations cause Miller syndrome by reducing pyrimidine biosynthesis (DHOdehase activity); complementation in auxotrophic yeast confirmed reduced pyrimidine synthesis, and in vitro enzymatic assays confirmed reduced DHOdehase activity for 11 disease-associated missense mutations. Mouse embryo in situ analysis showed Dhodh is strongly expressed in pharyngeal arch and limb bud, consistent with the developmental defects.","method":"Yeast complementation assay, in vitro enzymatic activity assay, urine metabolite analysis (orotic acid and dihydroorotate levels), in situ hybridization in mouse embryos","journal":"Human molecular genetics","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — multiple orthogonal methods (yeast complementation, enzymatic assay, metabolomics, in situ) across multiple families","pmids":["22692683"],"is_preprint":false},{"year":2007,"finding":"Human DHODH contains a functional mitochondrial targeting signal; expression of human DHODH fused to the U. maydis mitochondrial targeting signal complements pyr4 (DHODH) deletion auxotrophy in U. maydis, confirming mitochondrial localization and functional conservation.","method":"Genetic complementation of auxotrophic yeast pyr4 mutants, functional brequinar sensitivity assay in humanized U. maydis strains","journal":"Applied and environmental microbiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic complementation with functional drug sensitivity readout, single lab","pmids":["17369345"],"is_preprint":false},{"year":2020,"finding":"DHODH is an essential host factor for SARS-CoV-2 and other RNA virus replication; DHODH inhibition blocks viral replication in a DHODH-dependent manner (confirmed by DHODH knockout cells showing low viral replication and by uridine rescue experiments).","method":"DHODH inhibitor treatment (S312/S416/leflunomide/teriflunomide), DHODH knockout cell lines, in vivo antiviral efficacy, EC50 determination","journal":"Protein & cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KO cells plus pharmacological inhibition plus in vivo validation, single lab","pmids":["32754890"],"is_preprint":false},{"year":2020,"finding":"PTC299 (a DHODH inhibitor) inhibits SARS-CoV-2 replication in a DHODH-dependent and dose-dependent manner, and also suppresses IL-6, IL-17A, IL-17F, and VEGF production in tissue culture, consistent with DHODH's requirement for immunomodulatory cytokine production.","method":"Tissue culture SARS-CoV-2 replication assay, cytokine measurement by ELISA, selectivity index determination","journal":"Virus research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — pharmacological inhibition with DHODH-dependency confirmed, dose-response, multiple cytokine readouts, single lab","pmids":["33249060"],"is_preprint":false},{"year":2019,"finding":"DHODH inhibition with leflunomide substantially impairs colorectal cancer liver metastatic colonization and hypoxic growth by blocking pyrimidine nucleotide biosynthesis driven by gluconeogenic enzyme PCK1 under hypoxia.","method":"Patient-derived xenograft (PDX) in vivo selection, metabolite profiling, leflunomide pharmacological inhibition, in vivo metastasis models","journal":"eLife","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo PDX models plus metabolomics, single lab","pmids":["31841108"],"is_preprint":false},{"year":2017,"finding":"DHODH inactivation/deficiency in melanoma cells induces S-phase cell cycle arrest, autophagy via the AMPK-Ulk1 axis, and apoptosis in BCL-2-deficient cells; JNK phosphorylation of BCL-2 abrogates BCL-2/Beclin1 interaction to permit autophagy. BCL-2 rescues apoptosis but promotes cell cycle arrest and inhibits autophagy.","method":"DHODH siRNA knockdown, leflunomide treatment, Western blot for AMPK/Ulk1/JNK/BCL-2/Beclin1, flow cytometry for cell cycle and apoptosis, autophagy assays","journal":"Oncotarget","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic and pharmacological inhibition with multiple downstream pathway readouts, single lab","pmids":["29348830"],"is_preprint":false},{"year":2020,"finding":"DHODH directly binds the NH2-terminal of β-catenin, interrupting the GSK3β–β-catenin interaction, preventing β-catenin degradation and causing its nuclear accumulation and activation of downstream genes (CCND1, E2F3, Nanog, OCT4) in esophageal squamous cell carcinoma; this regulation is independent of DHODH catalytic activity.","method":"Co-immunoprecipitation, proximity ligation assay, catalytically inactive DHODH mutant expression, β-catenin nuclear fractionation, downstream gene expression analysis","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — Co-IP plus catalytic mutant validation plus downstream signaling, single lab","pmids":["33060568"],"is_preprint":false},{"year":2022,"finding":"ATM (ataxia telangiectasia mutated) kinase is required for DHODH-inhibition-induced poly(A)+ RNA nuclear accumulation adjacent to nuclear speckles; this phenomenon is abrogated by exogenous uridine, linking intracellular pyrimidine depletion to nuclear poly(A)+ RNA metabolism via the mitochondrial respiratory chain complex III.","method":"Original mRNA metabolic screening, fluorescence in situ hybridization for poly(A)+ RNA, ATM inhibitor/genetic knockdown, uridine rescue experiments","journal":"RNA biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — pharmacological and genetic approaches with uridine rescue, single lab","pmids":["36412986"],"is_preprint":false},{"year":2024,"finding":"DHODH inhibition reduces CDP-choline levels and attenuates phosphatidylcholine synthesis via the CDP-choline-dependent Kennedy pathway; compensatory increased phospholipid methylation from phosphatidylethanolamine produces very long chain PUFA-containing PCs, promoting ferroptosis and enhancing cancer cell sensitivity to T cell cytotoxicity.","method":"DHODH genetic inactivation, lipidomic and metabolomic analyses, Kennedy pathway inhibitor studies, CD8+ T cell co-culture assays, in vivo mouse tumor models with PD-1 blockade","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic KO plus lipidomics/metabolomics plus in vivo immune model, single lab","pmids":["40274823"],"is_preprint":false},{"year":2024,"finding":"PRR11 directly binds and stabilizes DHODH protein by suppressing E3 ubiquitin ligase HERC4-mediated polyubiquitination and degradation of DHODH at the K306 site, conferring ferroptosis resistance in glioblastoma in a DHODH-dependent manner.","method":"Co-immunoprecipitation, ubiquitination assay, site-directed mutagenesis (K306), DHODH knockdown/overexpression, lipid peroxidation assays, in vivo xenograft models","journal":"Redox biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, ubiquitination assay with site identification, and in vivo validation, single lab","pmids":["38838551"],"is_preprint":false},{"year":2024,"finding":"Mitochondrial dysfunction during cisplatin-induced AKI causes SIRT3 reduction and subsequent DHODH acetylation; SIRT3 overexpression or NMN supplementation attenuates DHODH acetylation and reduces ferroptosis, while Sirt3 knockout aggravates DHODH acetylation and ferroptosis. MitoQ pretreatment alleviated mitochondrial dysfunction, SIRT3 SUMOylation, and DHODH acetylation.","method":"Targeted metabolomics, DHODH overexpression/knockdown, SIRT3 overexpression/KO, CoQH2 measurement, lipid peroxidation assays, in vivo mouse AKI model, NMN supplementation","journal":"Advanced science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple genetic approaches plus metabolomics plus in vivo model, single lab","pmids":["39303219"],"is_preprint":false},{"year":2025,"finding":"USP24 deubiquitinase directly interacts with DHODH and deubiquitinates it, stabilizing DHODH protein and protecting cells from lipid peroxidation and ferroptosis; pharmacological USP24 inhibition synergizes with ferroptosis inducers via a DHODH-dependent pathway.","method":"Co-immunoprecipitation, ubiquitination assay, USP24 silencing, DHODH rescue experiments, in vitro and in vivo ferroptosis assays","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — Co-IP, ubiquitination assay, genetic rescue, in vivo validation, single lab","pmids":["40715045"],"is_preprint":false},{"year":2024,"finding":"DHODH sustains O-GlcNAcylation of neuropilin-1 (NRP1) and its membrane localization, mediating tumor cell macropinocytosis; DHODH-NRP1-driven macropinocytosis increases intracellular lysine and tryptophan, which promotes glutarylation of CIITA transcription factor to repress MHC class II expression and enable immune evasion.","method":"Genome-wide CRISPR-Cas9 screen, metabolic compound library screen, NRP1 O-GlcNAcylation assay, membrane localization studies, amino acid metabolomics, CIITA glutarylation assay, in vivo immune cell infiltration analysis","journal":"Immunity","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — CRISPR screen plus biochemical pathway validation plus in vivo immune model, single lab","pmids":["40816268"],"is_preprint":false},{"year":2024,"finding":"DHODH inhibition (brequinar) causes upregulation of MHC class I antigen presentation pathway genes and cell surface MHC class I expression in cancer cells; this effect is strictly dependent on pyrimidine nucleotide depletion and is mediated by RNA polymerase II elongation control via positive transcription elongation factor B (P-TEFb), independent of canonical antigen presentation pathway transcriptional regulators.","method":"Gene expression profiling, pyrimidine nucleotide depletion rescue experiments, P-TEFb inhibitor studies, flow cytometry for MHC class I, in vivo immunocompetent melanoma model with immune checkpoint blockade","journal":"eLife","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mechanistic dissection with pathway inhibitors plus in vivo immune model, single lab","pmids":["38973593"],"is_preprint":false},{"year":2021,"finding":"DHODH inhibition (PTC299) induces intra-S-phase arrest and apoptotic cell death in MDS cells, and enhances incorporation of decitabine (a cytidine analog) into DNA by inhibiting pyrimidine production, synergizing with hypomethylating agents; MYC target gene sets are markedly downregulated, and MYC overexpression largely attenuates growth inhibitory effects of PTC299.","method":"Cell viability assays with uridine rescue, RNA-seq, MYC overexpression rescue, decitabine incorporation assay, xenograft mouse models","journal":"Blood advances","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal mechanistic experiments with genetic rescue, single lab","pmids":["33496740"],"is_preprint":false},{"year":2022,"finding":"DHODH inhibition (brequinar) induces differentiation of myeloid leukemia cells by activating the ATR/Chk1 signaling pathway via pyrimidine depletion; this mechanism is shared with low-dose cytarabine-induced differentiation, and Chk1 genetic inactivation abolishes differentiation.","method":"Immunoblotting for ATR/Chk1 activation, flow cytometry for differentiation markers, pharmacologic Chk1 inhibition, genetic Chk1 inactivation, primary AML sample ex vivo assays, uridine rescue","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — pharmacological and genetic perturbation with multiple mechanistic readouts, single lab","pmids":["35790845"],"is_preprint":false},{"year":2024,"finding":"DHODH inhibition reduces mitochondrial respiration and CoQH2 production; DHODH upregulation by E2 (estradiol) protects cardiomyocytes from ferroptosis in heart failure via CoQ-related biological processes; CoQ siRNA knockdown abolishes the protective effect of DHODH upregulation.","method":"TAC mouse heart failure model, E2 treatment, siRNA knockdown of CoQ, DHODH overexpression/knockdown, ferroptosis marker assays, echocardiography, mitochondrial membrane potential measurement","journal":"Frontiers in bioscience","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic rescue experiments with CoQ knockdown plus in vivo model, single lab","pmids":["39082362"],"is_preprint":false},{"year":2019,"finding":"DHODH inhibition (brequinar/leflunomide) exerts anti-rotavirus activity through targeting DHODH to deplete the pyrimidine nucleotide pool, confirmed by restoration of viral replication with exogenous uridine supplementation.","method":"DHODH inhibitor treatment, uridine rescue experiment, Caco2 cell line and primary human intestinal organoids, clinical rotavirus strain testing","journal":"Antiviral research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mechanistic rescue experiment with uridine in multiple cell systems, single lab","pmids":["30974126"],"is_preprint":false},{"year":2021,"finding":"DHODH inhibition reduces mitochondrial respiration, promotes glycolysis, enhances GLUT4 translocation to the cytoplasmic membrane, and activates p53 to increase GDF15 expression; in db/db mice, DHODH inhibitors elevate circulating GDF15, reduce food intake, delay pancreatic β cell death, and improve metabolic balance.","method":"Seahorse metabolic assay, GLUT4 translocation imaging, p53 activation assay, GDF15 ELISA, db/db mouse model","journal":"iScience","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal metabolic assays plus in vivo model, single lab","pmids":["34113829"],"is_preprint":false},{"year":2024,"finding":"PACS2 downregulation upregulates DHODH expression and reverses high-glucose/palmitic acid-induced ferroptosis in cardiomyocytes by modulating mitochondrial function; this effect is mediated through CPT1A, as a CPT1A agonist with PACS2 silencing exacerbates ferroptosis.","method":"PACS2 siRNA knockdown, CPT1A agonist treatment, ferroptosis marker measurement (iron ions, lipid peroxides, ROS), mitochondrial function assays, streptozotocin/HFD mouse model","journal":"Cardiovascular diabetology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, indirect evidence for DHODH placement via upstream PACS2/CPT1A axis without direct DHODH mechanistic experiments","pmids":["39633391"],"is_preprint":false},{"year":2025,"finding":"OTUB1 deubiquitylase stabilizes DDX3X RNA-binding protein by suppressing its polyubiquitination; DDX3X in turn stabilizes DHODH mRNA, thereby upregulating DHODH protein and de novo pyrimidine synthesis to confer gemcitabine resistance in pancreatic cancer.","method":"Co-immunoprecipitation (OTUB1-DDX3X), ubiquitination assay, DDX3X knockdown, DHODH mRNA stability assay, in vivo murine tumoroid model with OTUB1 inhibitor","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — Co-IP, ubiquitination assay, mRNA stability, and in vivo validation, single lab","pmids":["41053036"],"is_preprint":false},{"year":2023,"finding":"MK-4 (menaquinone-4/vitamin K2) attenuates ferroptosis after subarachnoid hemorrhage by upregulating DHODH through activation of SIRT1 and promoting SIRT1 nuclear entry; DHODH inhibitor (brequinar) or SIRT1 inhibitor (selisistat) partially abolished MK-4's protective effects.","method":"SAH mouse model, Western blot for DHODH and SIRT1, immunofluorescence for SIRT1 localization, DHODH/SIRT1 inhibitor rescue experiments, ferroptosis marker assays","journal":"Free radical biology & medicine","confidence":"Low","confidence_rationale":"Tier 3 / Weak — pharmacological rescue experiments with inhibitors, indirect SIRT1→DHODH pathway, single lab","pmids":["38042225"],"is_preprint":false},{"year":2024,"finding":"CIRBP (cold-inducible RNA-binding protein) regulates DHODH protein expression; Cirbp-KO donor hearts show decreased DHODH expression during transplantation, compromising DHODH-mediated ubiquinone reduction, exacerbating lipid peroxidation and triggering ferroptosis.","method":"Cirbp-KO rat heart transplantation model, RNA-Seq, cardiac proteomics, DHODH expression analysis, lipid peroxidation measurement, CIRBP overexpression rescue","journal":"The Journal of clinical investigation","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo KO and overexpression with proteomics and functional ferroptosis readout, single lab","pmids":["38690728"],"is_preprint":false}],"current_model":"DHODH is a flavin-dependent mitochondrial inner membrane enzyme that catalyzes the fourth (rate-limiting) step of de novo pyrimidine biosynthesis—oxidizing dihydroorotate to orotate coupled to reduction of ubiquinone (CoQ) to ubiquinol (CoQH2); the CoQH2 produced serves as a radical-trapping antioxidant that suppresses mitochondrial lipid peroxidation and ferroptosis in parallel with mitochondrial GPX4, while DHODH also modulates transcriptional elongation, β-catenin/Wnt signaling, tumor macropinocytosis via NRP1 O-GlcNAcylation, MHC-I antigen presentation through P-TEFb, and mitochondrial metabolism, making it a multi-functional node linking pyrimidine synthesis to ferroptosis defense, immune evasion, and differentiation."},"narrative":{"mechanistic_narrative":"DHODH is a flavin-dependent mitochondrial inner-membrane enzyme that catalyzes the oxidation of dihydroorotate to orotate in de novo pyrimidine biosynthesis, coupling this reaction to the reduction of ubiquinone to ubiquinol; its activity functions as a metabolic node linking nucleotide supply to redox defense, differentiation, and immune signaling [PMID:33981038, PMID:22692683]. Beyond supplying pyrimidines, the ubiquinol generated by DHODH acts as a radical-trapping antioxidant that suppresses mitochondrial lipid peroxidation and ferroptosis in parallel with mitochondrial GPX4 but independently of cytosolic GPX4 or FSP1 [PMID:33981038]. Inhibitors bind competitively at the ubiquinone site, and high-resolution structures define their binding modes and chiral specificity [PMID:16480261, PMID:29549331, PMID:27994748]. Biallelic loss-of-function mutations that reduce DHOdehase activity cause Miller syndrome, consistent with strong embryonic expression in pharyngeal arch and limb bud [PMID:22692683]. DHODH activity is tuned post-translationally: HERC4-mediated ubiquitination at K306 (antagonized by PRR11) and USP24-mediated deubiquitination control DHODH stability and ferroptosis sensitivity, while SIRT3-regulated acetylation modulates its function under stress [PMID:38838551, PMID:40715045, PMID:39303219]. Pyrimidine depletion downstream of DHODH inhibition arrests cells in S phase and drives differentiation, p53 induction, and antitumor responses through ATR/Chk1 and MYC-dependent programs, and is also exploited as an antiviral mechanism against SARS-CoV-2 and other RNA viruses [PMID:29549331, PMID:33496740, PMID:35790845, PMID:32754890]. DHODH additionally influences transcriptional elongation and antigen presentation via P-TEFb, supports tumor macropinocytosis through NRP1 O-GlcNAcylation, and binds β-catenin to regulate Wnt signaling independently of its catalytic activity [PMID:21430780, PMID:38973593, PMID:40816268, PMID:33060568].","teleology":[{"year":2006,"claim":"Establishing where inhibitors act on DHODH was needed for rational drug design; structures showed inhibitors occupy the ubiquinone binding pocket.","evidence":"High-resolution X-ray crystallography of DHODH-inhibitor complexes with enzymatic correlation","pmids":["16480261"],"confidence":"High","gaps":["Does not address non-catalytic functions","Single-lab structural study"]},{"year":2007,"claim":"Whether human DHODH function depends on mitochondrial targeting was unresolved; complementation showed a functional mitochondrial targeting signal and conserved enzymatic role.","evidence":"Genetic complementation of auxotrophic U. maydis pyr4 mutants with humanized DHODH and brequinar sensitivity","pmids":["17369345"],"confidence":"Medium","gaps":["Heterologous system","Does not address regulation in human cells"]},{"year":2011,"claim":"It was unknown whether DHODH had functions beyond pyrimidine synthesis; inhibition was shown to suppress transcriptional elongation of neural crest and melanoma genes.","evidence":"Chemical genetic screen in zebrafish, mammalian stem cell assays, melanoma xenografts","pmids":["21430780"],"confidence":"High","gaps":["Molecular link between DHODH and elongation machinery not fully defined","Catalytic vs non-catalytic contribution unclear"]},{"year":2012,"claim":"The cause of Miller syndrome was unknown; biallelic DHODH loss-of-function mutations reducing enzymatic activity were shown to be causative.","evidence":"Yeast complementation, in vitro enzymatic assays of 11 missense mutations, urine metabolomics, mouse embryo in situ hybridization","pmids":["22692683"],"confidence":"High","gaps":["Tissue-specific basis of craniofacial/limb defects not mechanistically resolved"]},{"year":2016,"claim":"Whether DHODH inhibition could overcome AML differentiation blockade was untested; ML390 directly inhibited DHODH and induced differentiation in HoxA9 AML.","evidence":"Phenotypic HTS, genetic resistance mapping, X-ray crystallography of DHODH-ML390","pmids":["27994748"],"confidence":"High","gaps":["Downstream differentiation signaling not detailed in this study"]},{"year":2018,"claim":"How DHODH inhibition triggers tumor suppression was incompletely understood; (R)-HZ05 was shown to cause S-phase accumulation, pyrimidine depletion, and increased p53 synthesis.","evidence":"Target deconvolution, crystal structure, cell cycle and p53 synthesis assays, in vivo tumor models","pmids":["29549331"],"confidence":"High","gaps":["Generality across p53-mutant contexts not established"]},{"year":2020,"claim":"Whether DHODH-driven pyrimidine supply is required for RNA virus replication and inflammatory cytokine production was tested; inhibition blocked SARS-CoV-2 replication DHODH-dependently and suppressed cytokines.","evidence":"DHODH inhibitors, knockout cells, uridine rescue, cytokine ELISA, in vivo antiviral efficacy","pmids":["32754890","33249060"],"confidence":"Medium","gaps":["Host-vs-viral target specificity in vivo limited","Cytokine effects mechanistically indirect"]},{"year":2020,"claim":"Whether DHODH has catalysis-independent signaling roles was unknown; DHODH was shown to bind β-catenin and stabilize it by blocking GSK3β interaction, independent of enzymatic activity.","evidence":"Co-IP, proximity ligation, catalytically inactive mutant, nuclear fractionation, downstream gene expression","pmids":["33060568"],"confidence":"Medium","gaps":["Single lung/esophageal context","Structural basis of binding undefined","No reciprocal in vivo genetic validation"]},{"year":2021,"claim":"A non-pyrimidine role for DHODH in cell death was unresolved; DHODH was shown to suppress ferroptosis via ubiquinol generation in the mitochondrial inner membrane, parallel to mitochondrial GPX4.","evidence":"DHODH KO, brequinar, dihydroorotate/orotate rescue, fractionation, lipid peroxidation assays, in vivo tumor models","pmids":["33981038"],"confidence":"High","gaps":["Relative contribution vs GPX4 across tissues","Coupling of catalytic flux to local CoQH2 pool not quantified"]},{"year":2021,"claim":"Mechanisms of DHODH inhibition in MDS and metabolic disease were sought; pyrimidine depletion was shown to induce S-phase arrest with MYC suppression and to reprogram metabolism toward glycolysis with p53/GDF15 activation.","evidence":"RNA-seq, MYC overexpression rescue, decitabine incorporation, Seahorse, GLUT4 imaging, db/db and xenograft models","pmids":["33496740","34113829"],"confidence":"Medium","gaps":["Single-lab studies","Direct vs indirect link to p53/GDF15 not fully dissected"]},{"year":2022,"claim":"How DHODH inhibition drives differentiation and affects nuclear RNA was probed; pyrimidine depletion activated ATR/Chk1-dependent differentiation and caused ATM-dependent nuclear poly(A)+ RNA accumulation.","evidence":"Immunoblotting, Chk1 genetic inactivation, FISH for poly(A)+ RNA, ATM inhibition, uridine rescue","pmids":["35790845","36412986"],"confidence":"Medium","gaps":["Mechanistic link between pyrimidine pools and RNA export incomplete","Single-lab findings"]},{"year":2024,"claim":"Post-translational control of DHODH stability and its ferroptosis output was clarified; PRR11/HERC4 ubiquitination at K306, SIRT3-dependent acetylation, and CIRBP-dependent expression were shown to govern DHODH-mediated antioxidant defense.","evidence":"Co-IP, ubiquitination assays, K306 mutagenesis, SIRT3 KO, CoQH2 measurement, in vivo xenograft/AKI/heart-transplant models","pmids":["38838551","39303219","38690728"],"confidence":"Medium","gaps":["Each regulator validated in a single disease context","Crosstalk between modifications unexplored"]},{"year":2024,"claim":"How DHODH influences immune visibility of tumors was unknown; it was shown to repress MHC-I via P-TEFb-dependent elongation, sustain NRP1 O-GlcNAcylation to drive macropinocytosis and MHC-II repression, and reshape phospholipids to modulate ferroptosis and T-cell killing.","evidence":"CRISPR screen, gene expression profiling, P-TEFb inhibition, O-GlcNAcylation and glutarylation assays, lipidomics, CD8+ T-cell co-culture, in vivo immunocompetent models with checkpoint blockade","pmids":["38973593","40816268","40274823"],"confidence":"Medium","gaps":["Distinct mechanistic arms not integrated","Each demonstrated in single model systems"]},{"year":2025,"claim":"Upstream control of DHODH abundance in therapy resistance was further defined; USP24 deubiquitinates and stabilizes DHODH to protect against ferroptosis, and an OTUB1–DDX3X axis stabilizes DHODH mRNA to confer gemcitabine resistance.","evidence":"Co-IP, ubiquitination assays, mRNA stability assays, genetic silencing/rescue, in vivo tumoroid and ferroptosis models","pmids":["40715045","41053036"],"confidence":"Medium","gaps":["Single-lab studies","Tissue specificity of regulators unclear"]},{"year":null,"claim":"How DHODH's catalytic ubiquinol output is mechanistically partitioned among pyrimidine synthesis, ferroptosis defense, transcriptional elongation, and its catalysis-independent protein-binding roles remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unified model coupling enzymatic flux to non-canonical functions","Structural basis of β-catenin and signaling interactions undefined","Tissue-specific weighting of these roles unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016491","term_label":"oxidoreductase activity","supporting_discovery_ids":[0,5,2]},{"term_id":"GO:0016787","term_label":"hydrolase activity","supporting_discovery_ids":[5]},{"term_id":"GO:0016209","term_label":"antioxidant activity","supporting_discovery_ids":[0]}],"localization":[{"term_id":"GO:0005739","term_label":"mitochondrion","supporting_discovery_ids":[0,6]}],"pathway":[{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[0,5,9]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[0,14,16]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[3,19,20]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[17,18,13]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[5]}],"complexes":[],"partners":["CTNNB1","PRR11","HERC4","USP24","SIRT3","DDX3X","NRP1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q02127","full_name":"Dihydroorotate dehydrogenase (quinone), mitochondrial","aliases":["Dihydroorotate oxidase"],"length_aa":395,"mass_kda":42.9,"function":"Catalyzes the conversion of dihydroorotate to orotate with quinone as electron acceptor. 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A phase I clinical trial.","date":"2022","source":"Frontiers in medicine","url":"https://pubmed.ncbi.nlm.nih.gov/36325389","citation_count":5,"is_preprint":false},{"pmid":"36412986","id":"PMC_36412986","title":"Inhibition of mitochondrial complex III or dihydroorotate dehydrogenase (DHODH) triggers formation of poly(A)+ RNA foci adjacent to nuclear speckles following activation of ATM (ataxia telangiectasia mutated).","date":"2022","source":"RNA biology","url":"https://pubmed.ncbi.nlm.nih.gov/36412986","citation_count":4,"is_preprint":false},{"pmid":"39306285","id":"PMC_39306285","title":"Orally bioavailable RORγ/DHODH dual host-targeting small molecules with broad-spectrum antiviral activity.","date":"2024","source":"Antiviral research","url":"https://pubmed.ncbi.nlm.nih.gov/39306285","citation_count":4,"is_preprint":false},{"pmid":"28286324","id":"PMC_28286324","title":"Effects of dihydroorotate dehydrogenase (DHODH) inhibitors on the growth of Theileria equi and Babesia caballi in vitro.","date":"2017","source":"Experimental parasitology","url":"https://pubmed.ncbi.nlm.nih.gov/28286324","citation_count":4,"is_preprint":false},{"pmid":"28941392","id":"PMC_28941392","title":"Liquid phase combinatorial synthesis of 1,2,5-trisubstituted benzimidazole derivatives as human DHODH inhibitors.","date":"2017","source":"Bioorganic chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/28941392","citation_count":4,"is_preprint":false},{"pmid":"40513779","id":"PMC_40513779","title":"DHODH Blockade Induces Ferroptosis in Neuroblastoma by Modulating the Mevalonate Pathway.","date":"2025","source":"Molecular & cellular proteomics : MCP","url":"https://pubmed.ncbi.nlm.nih.gov/40513779","citation_count":3,"is_preprint":false},{"pmid":"40613295","id":"PMC_40613295","title":"The VDAC3/DHODH Axis Ameliorates Sepsis-induced Myocardial Injury by Regulating Ferroptosis.","date":"2025","source":"Frontiers in bioscience (Landmark edition)","url":"https://pubmed.ncbi.nlm.nih.gov/40613295","citation_count":3,"is_preprint":false},{"pmid":"38136869","id":"PMC_38136869","title":"Cloning and Characterization of Yak DHODH Gene and Its Functional Studies in a Bisphenol S-Induced Ferroptosis Model of Fetal Fibroblasts.","date":"2023","source":"Animals : an open access journal from MDPI","url":"https://pubmed.ncbi.nlm.nih.gov/38136869","citation_count":3,"is_preprint":false},{"pmid":"41053036","id":"PMC_41053036","title":"The deubiquitylase OTUB1 drives gemcitabine resistance in pancreatic cancer by enhancing pyrimidine metabolism through modulating DHODH mRNA stability.","date":"2025","source":"Cell death & disease","url":"https://pubmed.ncbi.nlm.nih.gov/41053036","citation_count":3,"is_preprint":false},{"pmid":"39766063","id":"PMC_39766063","title":"DHODH Inhibition Suppresses MYC and Inhibits the Growth of Medulloblastoma in a Novel In Vivo Zebrafish Model.","date":"2024","source":"Cancers","url":"https://pubmed.ncbi.nlm.nih.gov/39766063","citation_count":3,"is_preprint":false},{"pmid":"37066260","id":"PMC_37066260","title":"DHODH inhibition enhances the efficacy of immune checkpoint blockade by increasing cancer cell antigen presentation.","date":"2024","source":"bioRxiv : the preprint server for biology","url":"https://pubmed.ncbi.nlm.nih.gov/37066260","citation_count":3,"is_preprint":false},{"pmid":"37150227","id":"PMC_37150227","title":"Kinetic and structural studies of Mycobacterium tuberculosis dihydroorotate dehydrogenase reveal new insights into class 2 DHODH inhibition.","date":"2023","source":"Biochimica et biophysica acta. General subjects","url":"https://pubmed.ncbi.nlm.nih.gov/37150227","citation_count":3,"is_preprint":false},{"pmid":"36242990","id":"PMC_36242990","title":"SAR studies toward discovery of emvododstat (PTC299), a potent dihydroorotate dehydrogenase (DHODH) inhibitor.","date":"2022","source":"European journal of medicinal chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/36242990","citation_count":3,"is_preprint":false},{"pmid":"39620199","id":"PMC_39620199","title":"Virtual screening and molecular dynamics studies of novel small molecules targeting Schistosoma mansoni DHODH: identification of potential inhibitors.","date":"2024","source":"In silico pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/39620199","citation_count":3,"is_preprint":false},{"pmid":"39475586","id":"PMC_39475586","title":"Intrinsic Natural Resistance of Various Plant Pathogens to Ipflufenoquin, a New DHODH (Dihydroorotate Dehydrogenase)-Inhibiting Fungicide, in Relation to an Unaltered Amino Acid Sequence of the Target Site.","date":"2025","source":"Plant disease","url":"https://pubmed.ncbi.nlm.nih.gov/39475586","citation_count":3,"is_preprint":false},{"pmid":"40885318","id":"PMC_40885318","title":"Flubendazole inhibits cervical carcinoma by targeting DHODH to induce ferroptosis and mitophagy.","date":"2025","source":"Biochemical pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/40885318","citation_count":2,"is_preprint":false},{"pmid":"41239499","id":"PMC_41239499","title":"An immunometabolic prodrug strategy overcomes DHODH inhibitor resistance in refractory melanoma.","date":"2025","source":"Journal of experimental & clinical cancer research : CR","url":"https://pubmed.ncbi.nlm.nih.gov/41239499","citation_count":2,"is_preprint":false},{"pmid":"39377943","id":"PMC_39377943","title":"The DHODH inhibitor teriflunomide impedes cell proliferation and enhances chemosensitivity to daunorubicin (DNR) in T-cell acute lymphoblastic leukemia.","date":"2024","source":"Annals of hematology","url":"https://pubmed.ncbi.nlm.nih.gov/39377943","citation_count":2,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":50883,"output_tokens":6767,"usd":0.127077,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":15941,"output_tokens":4605,"usd":0.097415,"stage2_stop_reason":"end_turn"},"total_usd":0.224492,"stage1_batch_id":"msgbatch_01AsR7g8dGTEiEw9U83bJeWn","stage2_batch_id":"msgbatch_01YV5ZyxtFNUZEjxRcpd9jzb","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2021,\n      \"finding\": \"DHODH operates in parallel to mitochondrial GPX4 (but independently of cytosolic GPX4 or FSP1) to inhibit ferroptosis in the mitochondrial inner membrane by reducing ubiquinone to ubiquinol, a radical-trapping antioxidant that suppresses mitochondrial lipid peroxidation.\",\n      \"method\": \"Genetic inactivation (DHODH KO), pharmacological inhibition (brequinar), metabolite supplementation (dihydroorotate/orotate), subcellular fractionation, lipid peroxidation assays, in vitro and in vivo tumor models\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — multiple orthogonal methods including KO, pharmacological inhibition, substrate/product rescue, and in vivo validation in a single rigorous study\",\n      \"pmids\": [\"33981038\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"DHODH inhibition (e.g., leflunomide) suppresses transcriptional elongation of genes required for neural crest development and melanoma growth, distinct from its pyrimidine synthesis function.\",\n      \"method\": \"Chemical genetic screen in zebrafish, mammalian neural crest stem cell self-renewal assays, transcriptional elongation assays, melanoma xenograft studies, BRAF(V600E) transgenic zebrafish model\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (zebrafish genetics, mammalian cell assays, xenograft), replicated across model systems in a single rigorous study\",\n      \"pmids\": [\"21430780\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Human DHODH inhibitors bind competitively at the ubiquinone (CoQ) binding site; X-ray crystal structures revealed a dual binding mode of inhibitors within the same cocrystal, dependent on chemical substitution, with in vitro activity correlating with binding mode.\",\n      \"method\": \"High-resolution X-ray crystallography of DHODH-inhibitor complexes, in vitro enzymatic inhibition assays\",\n      \"journal\": \"Journal of medicinal chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structures with functional correlation, single lab but direct structural and enzymatic validation\",\n      \"pmids\": [\"16480261\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"DHODH inhibition by (R)-HZ05 (a chiral tetrahydroindazole) increases p53 synthesis by causing S-phase accumulation and pyrimidine depletion; the crystal structure of the (R)-HZ05/DHODH complex confirmed binding mode and chiral specificity.\",\n      \"method\": \"Target deconvolution, X-ray crystal structure of DHODH/(R)-HZ05 complex, cell cycle analysis, p53 synthesis measurement, in vivo tumor growth assays\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure plus functional cellular and in vivo validation in a single study\",\n      \"pmids\": [\"29549331\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"ML390 binds directly to DHODH and inhibits it, inducing differentiation in HoxA9-overexpressing AML cells; the X-ray crystal structure of ML390 bound to DHODH elucidated binding interactions.\",\n      \"method\": \"Phenotypic high-throughput screen, genetic resistance mapping, X-ray crystallography of DHODH-ML390 complex\",\n      \"journal\": \"ACS medicinal chemistry letters\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure with genetic target identification and cellular differentiation phenotype\",\n      \"pmids\": [\"27994748\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Biallelic DHODH loss-of-function mutations cause Miller syndrome by reducing pyrimidine biosynthesis (DHOdehase activity); complementation in auxotrophic yeast confirmed reduced pyrimidine synthesis, and in vitro enzymatic assays confirmed reduced DHOdehase activity for 11 disease-associated missense mutations. Mouse embryo in situ analysis showed Dhodh is strongly expressed in pharyngeal arch and limb bud, consistent with the developmental defects.\",\n      \"method\": \"Yeast complementation assay, in vitro enzymatic activity assay, urine metabolite analysis (orotic acid and dihydroorotate levels), in situ hybridization in mouse embryos\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — multiple orthogonal methods (yeast complementation, enzymatic assay, metabolomics, in situ) across multiple families\",\n      \"pmids\": [\"22692683\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Human DHODH contains a functional mitochondrial targeting signal; expression of human DHODH fused to the U. maydis mitochondrial targeting signal complements pyr4 (DHODH) deletion auxotrophy in U. maydis, confirming mitochondrial localization and functional conservation.\",\n      \"method\": \"Genetic complementation of auxotrophic yeast pyr4 mutants, functional brequinar sensitivity assay in humanized U. maydis strains\",\n      \"journal\": \"Applied and environmental microbiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic complementation with functional drug sensitivity readout, single lab\",\n      \"pmids\": [\"17369345\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"DHODH is an essential host factor for SARS-CoV-2 and other RNA virus replication; DHODH inhibition blocks viral replication in a DHODH-dependent manner (confirmed by DHODH knockout cells showing low viral replication and by uridine rescue experiments).\",\n      \"method\": \"DHODH inhibitor treatment (S312/S416/leflunomide/teriflunomide), DHODH knockout cell lines, in vivo antiviral efficacy, EC50 determination\",\n      \"journal\": \"Protein & cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO cells plus pharmacological inhibition plus in vivo validation, single lab\",\n      \"pmids\": [\"32754890\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"PTC299 (a DHODH inhibitor) inhibits SARS-CoV-2 replication in a DHODH-dependent and dose-dependent manner, and also suppresses IL-6, IL-17A, IL-17F, and VEGF production in tissue culture, consistent with DHODH's requirement for immunomodulatory cytokine production.\",\n      \"method\": \"Tissue culture SARS-CoV-2 replication assay, cytokine measurement by ELISA, selectivity index determination\",\n      \"journal\": \"Virus research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — pharmacological inhibition with DHODH-dependency confirmed, dose-response, multiple cytokine readouts, single lab\",\n      \"pmids\": [\"33249060\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"DHODH inhibition with leflunomide substantially impairs colorectal cancer liver metastatic colonization and hypoxic growth by blocking pyrimidine nucleotide biosynthesis driven by gluconeogenic enzyme PCK1 under hypoxia.\",\n      \"method\": \"Patient-derived xenograft (PDX) in vivo selection, metabolite profiling, leflunomide pharmacological inhibition, in vivo metastasis models\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo PDX models plus metabolomics, single lab\",\n      \"pmids\": [\"31841108\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"DHODH inactivation/deficiency in melanoma cells induces S-phase cell cycle arrest, autophagy via the AMPK-Ulk1 axis, and apoptosis in BCL-2-deficient cells; JNK phosphorylation of BCL-2 abrogates BCL-2/Beclin1 interaction to permit autophagy. BCL-2 rescues apoptosis but promotes cell cycle arrest and inhibits autophagy.\",\n      \"method\": \"DHODH siRNA knockdown, leflunomide treatment, Western blot for AMPK/Ulk1/JNK/BCL-2/Beclin1, flow cytometry for cell cycle and apoptosis, autophagy assays\",\n      \"journal\": \"Oncotarget\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic and pharmacological inhibition with multiple downstream pathway readouts, single lab\",\n      \"pmids\": [\"29348830\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"DHODH directly binds the NH2-terminal of β-catenin, interrupting the GSK3β–β-catenin interaction, preventing β-catenin degradation and causing its nuclear accumulation and activation of downstream genes (CCND1, E2F3, Nanog, OCT4) in esophageal squamous cell carcinoma; this regulation is independent of DHODH catalytic activity.\",\n      \"method\": \"Co-immunoprecipitation, proximity ligation assay, catalytically inactive DHODH mutant expression, β-catenin nuclear fractionation, downstream gene expression analysis\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — Co-IP plus catalytic mutant validation plus downstream signaling, single lab\",\n      \"pmids\": [\"33060568\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"ATM (ataxia telangiectasia mutated) kinase is required for DHODH-inhibition-induced poly(A)+ RNA nuclear accumulation adjacent to nuclear speckles; this phenomenon is abrogated by exogenous uridine, linking intracellular pyrimidine depletion to nuclear poly(A)+ RNA metabolism via the mitochondrial respiratory chain complex III.\",\n      \"method\": \"Original mRNA metabolic screening, fluorescence in situ hybridization for poly(A)+ RNA, ATM inhibitor/genetic knockdown, uridine rescue experiments\",\n      \"journal\": \"RNA biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — pharmacological and genetic approaches with uridine rescue, single lab\",\n      \"pmids\": [\"36412986\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"DHODH inhibition reduces CDP-choline levels and attenuates phosphatidylcholine synthesis via the CDP-choline-dependent Kennedy pathway; compensatory increased phospholipid methylation from phosphatidylethanolamine produces very long chain PUFA-containing PCs, promoting ferroptosis and enhancing cancer cell sensitivity to T cell cytotoxicity.\",\n      \"method\": \"DHODH genetic inactivation, lipidomic and metabolomic analyses, Kennedy pathway inhibitor studies, CD8+ T cell co-culture assays, in vivo mouse tumor models with PD-1 blockade\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic KO plus lipidomics/metabolomics plus in vivo immune model, single lab\",\n      \"pmids\": [\"40274823\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"PRR11 directly binds and stabilizes DHODH protein by suppressing E3 ubiquitin ligase HERC4-mediated polyubiquitination and degradation of DHODH at the K306 site, conferring ferroptosis resistance in glioblastoma in a DHODH-dependent manner.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, site-directed mutagenesis (K306), DHODH knockdown/overexpression, lipid peroxidation assays, in vivo xenograft models\",\n      \"journal\": \"Redox biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, ubiquitination assay with site identification, and in vivo validation, single lab\",\n      \"pmids\": [\"38838551\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Mitochondrial dysfunction during cisplatin-induced AKI causes SIRT3 reduction and subsequent DHODH acetylation; SIRT3 overexpression or NMN supplementation attenuates DHODH acetylation and reduces ferroptosis, while Sirt3 knockout aggravates DHODH acetylation and ferroptosis. MitoQ pretreatment alleviated mitochondrial dysfunction, SIRT3 SUMOylation, and DHODH acetylation.\",\n      \"method\": \"Targeted metabolomics, DHODH overexpression/knockdown, SIRT3 overexpression/KO, CoQH2 measurement, lipid peroxidation assays, in vivo mouse AKI model, NMN supplementation\",\n      \"journal\": \"Advanced science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple genetic approaches plus metabolomics plus in vivo model, single lab\",\n      \"pmids\": [\"39303219\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"USP24 deubiquitinase directly interacts with DHODH and deubiquitinates it, stabilizing DHODH protein and protecting cells from lipid peroxidation and ferroptosis; pharmacological USP24 inhibition synergizes with ferroptosis inducers via a DHODH-dependent pathway.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, USP24 silencing, DHODH rescue experiments, in vitro and in vivo ferroptosis assays\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — Co-IP, ubiquitination assay, genetic rescue, in vivo validation, single lab\",\n      \"pmids\": [\"40715045\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"DHODH sustains O-GlcNAcylation of neuropilin-1 (NRP1) and its membrane localization, mediating tumor cell macropinocytosis; DHODH-NRP1-driven macropinocytosis increases intracellular lysine and tryptophan, which promotes glutarylation of CIITA transcription factor to repress MHC class II expression and enable immune evasion.\",\n      \"method\": \"Genome-wide CRISPR-Cas9 screen, metabolic compound library screen, NRP1 O-GlcNAcylation assay, membrane localization studies, amino acid metabolomics, CIITA glutarylation assay, in vivo immune cell infiltration analysis\",\n      \"journal\": \"Immunity\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — CRISPR screen plus biochemical pathway validation plus in vivo immune model, single lab\",\n      \"pmids\": [\"40816268\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"DHODH inhibition (brequinar) causes upregulation of MHC class I antigen presentation pathway genes and cell surface MHC class I expression in cancer cells; this effect is strictly dependent on pyrimidine nucleotide depletion and is mediated by RNA polymerase II elongation control via positive transcription elongation factor B (P-TEFb), independent of canonical antigen presentation pathway transcriptional regulators.\",\n      \"method\": \"Gene expression profiling, pyrimidine nucleotide depletion rescue experiments, P-TEFb inhibitor studies, flow cytometry for MHC class I, in vivo immunocompetent melanoma model with immune checkpoint blockade\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mechanistic dissection with pathway inhibitors plus in vivo immune model, single lab\",\n      \"pmids\": [\"38973593\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"DHODH inhibition (PTC299) induces intra-S-phase arrest and apoptotic cell death in MDS cells, and enhances incorporation of decitabine (a cytidine analog) into DNA by inhibiting pyrimidine production, synergizing with hypomethylating agents; MYC target gene sets are markedly downregulated, and MYC overexpression largely attenuates growth inhibitory effects of PTC299.\",\n      \"method\": \"Cell viability assays with uridine rescue, RNA-seq, MYC overexpression rescue, decitabine incorporation assay, xenograft mouse models\",\n      \"journal\": \"Blood advances\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal mechanistic experiments with genetic rescue, single lab\",\n      \"pmids\": [\"33496740\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"DHODH inhibition (brequinar) induces differentiation of myeloid leukemia cells by activating the ATR/Chk1 signaling pathway via pyrimidine depletion; this mechanism is shared with low-dose cytarabine-induced differentiation, and Chk1 genetic inactivation abolishes differentiation.\",\n      \"method\": \"Immunoblotting for ATR/Chk1 activation, flow cytometry for differentiation markers, pharmacologic Chk1 inhibition, genetic Chk1 inactivation, primary AML sample ex vivo assays, uridine rescue\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — pharmacological and genetic perturbation with multiple mechanistic readouts, single lab\",\n      \"pmids\": [\"35790845\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"DHODH inhibition reduces mitochondrial respiration and CoQH2 production; DHODH upregulation by E2 (estradiol) protects cardiomyocytes from ferroptosis in heart failure via CoQ-related biological processes; CoQ siRNA knockdown abolishes the protective effect of DHODH upregulation.\",\n      \"method\": \"TAC mouse heart failure model, E2 treatment, siRNA knockdown of CoQ, DHODH overexpression/knockdown, ferroptosis marker assays, echocardiography, mitochondrial membrane potential measurement\",\n      \"journal\": \"Frontiers in bioscience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic rescue experiments with CoQ knockdown plus in vivo model, single lab\",\n      \"pmids\": [\"39082362\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"DHODH inhibition (brequinar/leflunomide) exerts anti-rotavirus activity through targeting DHODH to deplete the pyrimidine nucleotide pool, confirmed by restoration of viral replication with exogenous uridine supplementation.\",\n      \"method\": \"DHODH inhibitor treatment, uridine rescue experiment, Caco2 cell line and primary human intestinal organoids, clinical rotavirus strain testing\",\n      \"journal\": \"Antiviral research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mechanistic rescue experiment with uridine in multiple cell systems, single lab\",\n      \"pmids\": [\"30974126\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"DHODH inhibition reduces mitochondrial respiration, promotes glycolysis, enhances GLUT4 translocation to the cytoplasmic membrane, and activates p53 to increase GDF15 expression; in db/db mice, DHODH inhibitors elevate circulating GDF15, reduce food intake, delay pancreatic β cell death, and improve metabolic balance.\",\n      \"method\": \"Seahorse metabolic assay, GLUT4 translocation imaging, p53 activation assay, GDF15 ELISA, db/db mouse model\",\n      \"journal\": \"iScience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal metabolic assays plus in vivo model, single lab\",\n      \"pmids\": [\"34113829\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"PACS2 downregulation upregulates DHODH expression and reverses high-glucose/palmitic acid-induced ferroptosis in cardiomyocytes by modulating mitochondrial function; this effect is mediated through CPT1A, as a CPT1A agonist with PACS2 silencing exacerbates ferroptosis.\",\n      \"method\": \"PACS2 siRNA knockdown, CPT1A agonist treatment, ferroptosis marker measurement (iron ions, lipid peroxides, ROS), mitochondrial function assays, streptozotocin/HFD mouse model\",\n      \"journal\": \"Cardiovascular diabetology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, indirect evidence for DHODH placement via upstream PACS2/CPT1A axis without direct DHODH mechanistic experiments\",\n      \"pmids\": [\"39633391\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"OTUB1 deubiquitylase stabilizes DDX3X RNA-binding protein by suppressing its polyubiquitination; DDX3X in turn stabilizes DHODH mRNA, thereby upregulating DHODH protein and de novo pyrimidine synthesis to confer gemcitabine resistance in pancreatic cancer.\",\n      \"method\": \"Co-immunoprecipitation (OTUB1-DDX3X), ubiquitination assay, DDX3X knockdown, DHODH mRNA stability assay, in vivo murine tumoroid model with OTUB1 inhibitor\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — Co-IP, ubiquitination assay, mRNA stability, and in vivo validation, single lab\",\n      \"pmids\": [\"41053036\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"MK-4 (menaquinone-4/vitamin K2) attenuates ferroptosis after subarachnoid hemorrhage by upregulating DHODH through activation of SIRT1 and promoting SIRT1 nuclear entry; DHODH inhibitor (brequinar) or SIRT1 inhibitor (selisistat) partially abolished MK-4's protective effects.\",\n      \"method\": \"SAH mouse model, Western blot for DHODH and SIRT1, immunofluorescence for SIRT1 localization, DHODH/SIRT1 inhibitor rescue experiments, ferroptosis marker assays\",\n      \"journal\": \"Free radical biology & medicine\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — pharmacological rescue experiments with inhibitors, indirect SIRT1→DHODH pathway, single lab\",\n      \"pmids\": [\"38042225\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"CIRBP (cold-inducible RNA-binding protein) regulates DHODH protein expression; Cirbp-KO donor hearts show decreased DHODH expression during transplantation, compromising DHODH-mediated ubiquinone reduction, exacerbating lipid peroxidation and triggering ferroptosis.\",\n      \"method\": \"Cirbp-KO rat heart transplantation model, RNA-Seq, cardiac proteomics, DHODH expression analysis, lipid peroxidation measurement, CIRBP overexpression rescue\",\n      \"journal\": \"The Journal of clinical investigation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo KO and overexpression with proteomics and functional ferroptosis readout, single lab\",\n      \"pmids\": [\"38690728\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"DHODH is a flavin-dependent mitochondrial inner membrane enzyme that catalyzes the fourth (rate-limiting) step of de novo pyrimidine biosynthesis—oxidizing dihydroorotate to orotate coupled to reduction of ubiquinone (CoQ) to ubiquinol (CoQH2); the CoQH2 produced serves as a radical-trapping antioxidant that suppresses mitochondrial lipid peroxidation and ferroptosis in parallel with mitochondrial GPX4, while DHODH also modulates transcriptional elongation, β-catenin/Wnt signaling, tumor macropinocytosis via NRP1 O-GlcNAcylation, MHC-I antigen presentation through P-TEFb, and mitochondrial metabolism, making it a multi-functional node linking pyrimidine synthesis to ferroptosis defense, immune evasion, and differentiation.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"DHODH is a flavin-dependent mitochondrial inner-membrane enzyme that catalyzes the oxidation of dihydroorotate to orotate in de novo pyrimidine biosynthesis, coupling this reaction to the reduction of ubiquinone to ubiquinol; its activity functions as a metabolic node linking nucleotide supply to redox defense, differentiation, and immune signaling [#0, #5]. Beyond supplying pyrimidines, the ubiquinol generated by DHODH acts as a radical-trapping antioxidant that suppresses mitochondrial lipid peroxidation and ferroptosis in parallel with mitochondrial GPX4 but independently of cytosolic GPX4 or FSP1 [#0]. Inhibitors bind competitively at the ubiquinone site, and high-resolution structures define their binding modes and chiral specificity [#2, #3, #4]. Biallelic loss-of-function mutations that reduce DHOdehase activity cause Miller syndrome, consistent with strong embryonic expression in pharyngeal arch and limb bud [#5]. DHODH activity is tuned post-translationally: HERC4-mediated ubiquitination at K306 (antagonized by PRR11) and USP24-mediated deubiquitination control DHODH stability and ferroptosis sensitivity, while SIRT3-regulated acetylation modulates its function under stress [#14, #16, #15]. Pyrimidine depletion downstream of DHODH inhibition arrests cells in S phase and drives differentiation, p53 induction, and antitumor responses through ATR/Chk1 and MYC-dependent programs, and is also exploited as an antiviral mechanism against SARS-CoV-2 and other RNA viruses [#3, #19, #20, #7]. DHODH additionally influences transcriptional elongation and antigen presentation via P-TEFb, supports tumor macropinocytosis through NRP1 O-GlcNAcylation, and binds \\u03b2-catenin to regulate Wnt signaling independently of its catalytic activity [#1, #18, #17, #11].\"\n,\n  \"teleology\": [\n    {\n      \"year\": 2006,\n      \"claim\": \"Establishing where inhibitors act on DHODH was needed for rational drug design; structures showed inhibitors occupy the ubiquinone binding pocket.\",\n      \"evidence\": \"High-resolution X-ray crystallography of DHODH-inhibitor complexes with enzymatic correlation\",\n      \"pmids\": [\"16480261\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Does not address non-catalytic functions\", \"Single-lab structural study\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Whether human DHODH function depends on mitochondrial targeting was unresolved; complementation showed a functional mitochondrial targeting signal and conserved enzymatic role.\",\n      \"evidence\": \"Genetic complementation of auxotrophic U. maydis pyr4 mutants with humanized DHODH and brequinar sensitivity\",\n      \"pmids\": [\"17369345\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Heterologous system\", \"Does not address regulation in human cells\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"It was unknown whether DHODH had functions beyond pyrimidine synthesis; inhibition was shown to suppress transcriptional elongation of neural crest and melanoma genes.\",\n      \"evidence\": \"Chemical genetic screen in zebrafish, mammalian stem cell assays, melanoma xenografts\",\n      \"pmids\": [\"21430780\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular link between DHODH and elongation machinery not fully defined\", \"Catalytic vs non-catalytic contribution unclear\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"The cause of Miller syndrome was unknown; biallelic DHODH loss-of-function mutations reducing enzymatic activity were shown to be causative.\",\n      \"evidence\": \"Yeast complementation, in vitro enzymatic assays of 11 missense mutations, urine metabolomics, mouse embryo in situ hybridization\",\n      \"pmids\": [\"22692683\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Tissue-specific basis of craniofacial/limb defects not mechanistically resolved\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Whether DHODH inhibition could overcome AML differentiation blockade was untested; ML390 directly inhibited DHODH and induced differentiation in HoxA9 AML.\",\n      \"evidence\": \"Phenotypic HTS, genetic resistance mapping, X-ray crystallography of DHODH-ML390\",\n      \"pmids\": [\"27994748\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Downstream differentiation signaling not detailed in this study\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"How DHODH inhibition triggers tumor suppression was incompletely understood; (R)-HZ05 was shown to cause S-phase accumulation, pyrimidine depletion, and increased p53 synthesis.\",\n      \"evidence\": \"Target deconvolution, crystal structure, cell cycle and p53 synthesis assays, in vivo tumor models\",\n      \"pmids\": [\"29549331\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Generality across p53-mutant contexts not established\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Whether DHODH-driven pyrimidine supply is required for RNA virus replication and inflammatory cytokine production was tested; inhibition blocked SARS-CoV-2 replication DHODH-dependently and suppressed cytokines.\",\n      \"evidence\": \"DHODH inhibitors, knockout cells, uridine rescue, cytokine ELISA, in vivo antiviral efficacy\",\n      \"pmids\": [\"32754890\", \"33249060\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Host-vs-viral target specificity in vivo limited\", \"Cytokine effects mechanistically indirect\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Whether DHODH has catalysis-independent signaling roles was unknown; DHODH was shown to bind \\u03b2-catenin and stabilize it by blocking GSK3\\u03b2 interaction, independent of enzymatic activity.\",\n      \"evidence\": \"Co-IP, proximity ligation, catalytically inactive mutant, nuclear fractionation, downstream gene expression\",\n      \"pmids\": [\"33060568\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lung/esophageal context\", \"Structural basis of binding undefined\", \"No reciprocal in vivo genetic validation\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"A non-pyrimidine role for DHODH in cell death was unresolved; DHODH was shown to suppress ferroptosis via ubiquinol generation in the mitochondrial inner membrane, parallel to mitochondrial GPX4.\",\n      \"evidence\": \"DHODH KO, brequinar, dihydroorotate/orotate rescue, fractionation, lipid peroxidation assays, in vivo tumor models\",\n      \"pmids\": [\"33981038\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Relative contribution vs GPX4 across tissues\", \"Coupling of catalytic flux to local CoQH2 pool not quantified\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Mechanisms of DHODH inhibition in MDS and metabolic disease were sought; pyrimidine depletion was shown to induce S-phase arrest with MYC suppression and to reprogram metabolism toward glycolysis with p53/GDF15 activation.\",\n      \"evidence\": \"RNA-seq, MYC overexpression rescue, decitabine incorporation, Seahorse, GLUT4 imaging, db/db and xenograft models\",\n      \"pmids\": [\"33496740\", \"34113829\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab studies\", \"Direct vs indirect link to p53/GDF15 not fully dissected\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"How DHODH inhibition drives differentiation and affects nuclear RNA was probed; pyrimidine depletion activated ATR/Chk1-dependent differentiation and caused ATM-dependent nuclear poly(A)+ RNA accumulation.\",\n      \"evidence\": \"Immunoblotting, Chk1 genetic inactivation, FISH for poly(A)+ RNA, ATM inhibition, uridine rescue\",\n      \"pmids\": [\"35790845\", \"36412986\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanistic link between pyrimidine pools and RNA export incomplete\", \"Single-lab findings\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Post-translational control of DHODH stability and its ferroptosis output was clarified; PRR11/HERC4 ubiquitination at K306, SIRT3-dependent acetylation, and CIRBP-dependent expression were shown to govern DHODH-mediated antioxidant defense.\",\n      \"evidence\": \"Co-IP, ubiquitination assays, K306 mutagenesis, SIRT3 KO, CoQH2 measurement, in vivo xenograft/AKI/heart-transplant models\",\n      \"pmids\": [\"38838551\", \"39303219\", \"38690728\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Each regulator validated in a single disease context\", \"Crosstalk between modifications unexplored\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"How DHODH influences immune visibility of tumors was unknown; it was shown to repress MHC-I via P-TEFb-dependent elongation, sustain NRP1 O-GlcNAcylation to drive macropinocytosis and MHC-II repression, and reshape phospholipids to modulate ferroptosis and T-cell killing.\",\n      \"evidence\": \"CRISPR screen, gene expression profiling, P-TEFb inhibition, O-GlcNAcylation and glutarylation assays, lipidomics, CD8+ T-cell co-culture, in vivo immunocompetent models with checkpoint blockade\",\n      \"pmids\": [\"38973593\", \"40816268\", \"40274823\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Distinct mechanistic arms not integrated\", \"Each demonstrated in single model systems\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Upstream control of DHODH abundance in therapy resistance was further defined; USP24 deubiquitinates and stabilizes DHODH to protect against ferroptosis, and an OTUB1\\u2013DDX3X axis stabilizes DHODH mRNA to confer gemcitabine resistance.\",\n      \"evidence\": \"Co-IP, ubiquitination assays, mRNA stability assays, genetic silencing/rescue, in vivo tumoroid and ferroptosis models\",\n      \"pmids\": [\"40715045\", \"41053036\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab studies\", \"Tissue specificity of regulators unclear\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How DHODH's catalytic ubiquinol output is mechanistically partitioned among pyrimidine synthesis, ferroptosis defense, transcriptional elongation, and its catalysis-independent protein-binding roles remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unified model coupling enzymatic flux to non-canonical functions\", \"Structural basis of \\u03b2-catenin and signaling interactions undefined\", \"Tissue-specific weighting of these roles unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016491\", \"supporting_discovery_ids\": [0, 5, 2]},\n      {\"term_id\": \"GO:0016787\", \"supporting_discovery_ids\": [5]},\n      {\"term_id\": \"GO:0016209\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005739\", \"supporting_discovery_ids\": [0, 6]},\n      {\"term_id\": \"GO:0005743\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [0, 5, 9]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [0, 14, 16]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [3, 19, 20]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [17, 18, 13]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [5]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"CTNNB1\", \"PRR11\", \"HERC4\", \"USP24\", \"SIRT3\", \"DDX3X\", \"NRP1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":7,"faith_pct":85.71428571428571}}