The probability of cardiovascular events has been reported lower in rheumatoid arthritis (RA) patients treated with leflunomide. However, the anti-atherosclerotic and cardiovascular protective effects and metabolism of leflunomide are not explored. In this study, we assessed the potential benefits of leflunomide on atherosclerosis and revealed the underlying mechanism. ApoE-/- mice were fed a western diet (WD) alone or supplemented with leflunomide (20 mg/kg, oral gavage, once per day) for 12 weeks. Samples of the aorta, heart, liver, serum, and macrophages were collected. We found that leflunomide significantly reduced lesion size in both en-face aortas and aortic root in WD-fed ApoE-/- mice. Leflunomide also obviously improved dyslipidemia, reduced hepatic lipid content, and improved disorders of glucose and lipid metabolism in vivo. RNA-Seq results showed that leflunomide effectively regulated the genes\' expression involved in the lipid metabolism pathway. Importantly, leflunomide significantly increased the phosphorylation levels of AMPKα and acetyl-CoA carboxylase (ACC) in vivo. Furthermore, leflunomide and its active metabolite teriflunomide suppressed lipid accumulation in free fatty acid (FFA)-induced AML12 cells and improved endothelial dysfunction in palmitic acid (PA)-induced HUVECs through activating AMPK signaling and inhibiting dihydroorotate dehydrogenase (DHODH) signaling pathway. We present evidence that leflunomide and teriflunomide ameliorate atherosclerosis by regulating lipid metabolism and endothelial dysfunction. Our findings suggest a promising use of antirheumatic small-molecule drugs leflunomide and teriflunomide for the treatment of atherosclerosis and related cardiovascular diseases (CVDs).

BACKGROUND: Heart failure (HF) is a clinical syndrome that seriously endangers human health and quality of life as the terminal stage of cardiovascular diseases. Ferroptosis as a new iron-dependent programmed cell death mode that is closely related to the occurrence and development of cardiovascular diseases. Dihydroorotate dehydrogenase (DHODH) has been found to play a crucial role in inhibiting ferroptosis and improving mitochondrial function, and its expression can be upregulated by estradiol (E2). Recent studies have found that DHODH can inhibit ferroptosis by reducing coenzyme Q (CoQ) to CoQH2. Therefore, this study aims to explore the effect of up-regulation of DHODH on the pathological hypertrophy and fibrosis of heart failure and its mechanisms.
METHODS: The mouse heart failure model was established by transverse aortic constriction (TAC), surgery in mice. Two days after the operation, a subcutaneous injection of E2 or the same volume of sesame oil was given for 8 weeks. Then, the left ventricular systolic function related indicators of mice were measured by echocardiography, and the degree of myocardial fibrosis of mice was detected by histological analysis; the expression levels of heart failure markers were detected by quantitative polymerase chain reaction (q-PCR) and western blot (WB) analysis; the morphological changes of mitochondria in cardiac cells of mice were observed by transmission electron microscopy. Cell model were established by stimulating with phenylephrine for 96 hours. Ferroptosis markers were detected by kits and WB analysis. Mitochondrial function was verified by a JC-1 fluorescent probe, and 2\',7\'-Dichlorodihydrofluorescein diacetate (DCFH-DA) staining. The knockdown results were detected by WB analysis after transfection of small interfering RNA (siRNA) of CoQ. Fer-1 was added as a positive control to verify the ferroptosis-related changes of myocardial cells.
RESULTS: In the animal model, we found that E2 treatment alleviates TAC-induced cardiac hypertrophy and fibrosis and suppresses cardiomyocyte ferroptosis by promotes DHODH upregulation in murine cardiomyocytes. In the cell model, DHODH upregulation protects against phenylephrine-induced cardiomyocytes with failure. However, the effect on up-regulating DHODH was inhibited by transfection to down-regulate CoQ expression.
CONCLUSIONS: The up-regulation of DHODH could effectively ameliorate the manifestations of heart failure such as myocardial hypertrophy and fibrosis in mice after TAC surgery, inhibit ferroptosis of cardiac myocytes, and ameliorate mitochondrial function. The mechanism involves CoQ-related biological processes.

This study aimed to repurpose Drug Bank Compounds against P. falciparum Dihydroorotate dehydrogenase (Pf-DHODH)a potential molecular target for antimalarial drug development due to its vital role in P. falciparum survival. Initially, the MATGEN server was used to screen drugs against Pf-DHODH (PDB ID 6GJG), followed by revalidating the results through docking by Autodock Vina through PyRx. Based on the docking results, three drugs namely, Talnifumate, Sulfaphenazole, and (3S)-N-[(2S)-1-[2-(1H-indol-3-yl)ethylamino]-1-oxopropan-2-yl]-1-(4-methoxyphenyl)-5-oxopyrrolidine-3-carboxamide-were subjected to molecular dynamics simulation for 100 ns. Molecular dynamics simulation results indicate that (3S)-N-[(2S)-1-[2-(1H-indol-3-yl)ethylamino]-1-oxopropan-2-yl]-1-(4-methoxyphenyl)-5-oxopyrrolidine-3-carboxamide- and Sulfaphenazole may target Pf-DHODH by forming a stable protein-ligand complex as they showed better free binding energy -130.58 kJ/mol, and -79.84 kJ/mol, respectively as compared to the free binding energy 116.255 kJ/mol of the reference compound; 3,6-dimethyl- ~ {N}-[4-(trifluoromethyl)phenyl]-[1,2]oxazolo[5,4-d]pyrimidin-4-amine. Although the studied compounds are drugs, still we applied Lipinski\'s rules and ADMET analysis that reconfirmed that these drugs have favorable drug-like properties. In conclusion, the results of the study show that Talniflumate and Sulfaphenazole may be potential antimalarial drug candidates.The derivatives of these drugs could be designed and tested to develop better drugs against Plasmodium species.
UNASSIGNED:

Pyrimidine nucleotide biosynthesis is a druggable metabolic dependency of cancer cells, and chemotherapy agents targeting pyrimidine metabolism are the backbone of treatment for many cancers. Dihydroorotate dehydrogenase (DHODH) is an essential enzyme in the de novo pyrimidine biosynthesis pathway that can be targeted by clinically approved inhibitors. However, despite robust preclinical anticancer efficacy, DHODH inhibitors have shown limited single-agent activity in phase 1 and 2 clinical trials. Therefore, novel combination therapy strategies are necessary to realize the potential of these drugs. To search for therapeutic vulnerabilities induced by DHODH inhibition, we examined gene expression changes in cancer cells treated with the potent and selective DHODH inhibitor brequinar (BQ). This revealed that BQ treatment causes upregulation of antigen presentation pathway genes and cell surface MHC class I expression. Mechanistic studies showed that this effect is (1) strictly dependent on pyrimidine nucleotide depletion, (2) independent of canonical antigen presentation pathway transcriptional regulators, and (3) mediated by RNA polymerase II elongation control by positive transcription elongation factor B (P-TEFb). Furthermore, BQ showed impressive single-agent efficacy in the immunocompetent B16F10 melanoma model, and combination treatment with BQ and dual immune checkpoint blockade (anti-CTLA-4 plus anti-PD-1) significantly prolonged mouse survival compared to either therapy alone. Our results have important implications for the clinical development of DHODH inhibitors and provide a rationale for combination therapy with BQ and immune checkpoint blockade.

Inhibition of human dihydroorotate dehydrogenase (hDHODH) represents a promising strategy for suppressing the proliferation of cancer cells. To identify novel and potent hDHODH inhibitors, a total of 28 piperine derivatives were designed and synthesized. Their cytotoxicities against three human cancer cell lines (NCI-H226, HCT-116, and MDA-MB-231) and hDHODH inhibitory activities were also evaluated. Among them, compound H19, exhibited the strongest inhibitory activities (NCI-H226 IC50 = 0.95 µM, hDHODH IC50 = 0.21 µM). Further pharmacological investigations revealed that H19 exerted anticancer effects by inducing ferroptosis in NCI-H226 cells, with its cytotoxicity being reversed by ferroptosis inhibitors. This was supported by the intracellular growth or decline of ferroptosis markers, including lipid peroxidation, Fe2+, GSH, and 4-HNE. Overall, H19 emerges as a promising hDHODH inhibitor with potential anticancer properties warranting development.

High-grade B-cell lymphoma (HGBCL), the subtype of non-Hodgkin lymphoma, to be relapsed or refractory in patients after initial therapy or salvage chemotherapy. Dual dysregulation of MYC and BCL2 is one of the important pathogenic mechanisms. Thus, combined targeting of MYC and BCL2 appears to be a promising strategy. Dihydroorotate dehydrogenase (DHODH) is the fourth rate-limiting enzyme for the de novo biosynthesis of pyrimidine. It has been shown to be a potential therapeutic target for multiple diseases. In this study, the DHODH inhibitor brequinar exhibited growth inhibition, cell cycle blockade, and apoptosis promotion in HGBCL cell lines with MYC and BCL2 rearrangements. The combination of brequinar and BCL2 inhibitors venetoclax had a synergistic inhibitory effect on the survival of DHL cells through different pathways. Venetoclax could upregulate MCL-1 and MYC expression, which has been reported as a resistance mechanism of BCL2 inhibitors. Brequinar downregulated MCL-1 and MYC, which could potentially overcome drug resistance to venetoclax in HGBCL cells. Furthermore, brequinar could downregulate a broad range of genes, including ribosome biosynthesis genes, which might contribute to its anti-tumor effects. In vivo studies demonstrated synergetic tumor growth inhibition in xenograft models with brequinar and venetoclax combination treatment. These results provide preliminary evidence for the rational combination of DHODH and BCL2 blockade in HGBCL with abnormal MYC and BCL2.

Acute myelogenous leukemia (AML), a heterogeneous disease of the blood and bone marrow, is characterized by the inability of myeloblasts to differentiate into mature cell types. Dihydroorotate dehydrogenase (DHODH) is an enzyme well-known in the pyrimidine biosynthesis pathway and preclinical findings demonstrated that DHODH is a metabolic vulnerability in AML as inhibitors can induce differentiation across multiple AML subtypes. As a result of virtual screening and structure-based drug design approaches, a novel series of isoquinolinone DHODH inhibitors was identified. Further lead optimization afforded JNJ-74856665 as an orally bioavailable, potent, and selective DHODH inhibitor with favorable physicochemical properties selected for clinical development in patients with AML and myelodysplastic syndromes (MDS).

Temozolomide (TMZ) is a widely utilized chemotherapy treatment for patients with glioblastoma (GBM), although drug resistance constitutes a major therapeutic hurdle. Emerging evidence suggests that ferroptosis-mediated therapy could offer an appropriate alternative treatment option against cancer cells that are resistant to certain drugs. However, recurrent gliomas display robust ferroptosis resistance, although the precise mechanism of resistance remains elusive. In the present work, we report that proline rich protein 11 (PRR11) depletion significantly sensitizes GBM cells to TMZ by inducing ferroptosis. Mechanistically, PRR11 directly binds to and stabilizes dihydroorotate dehydrogenase (DHODH), which leads to glioma ferroptosis-resistant in a DHODH-dependent manner in vivo and in vitro. Furthermore, PRR11 inhibits HERC4 and DHODH binding, by suppressing the recruitment of E3 ubiquitin ligase HERC4 and polyubiquitination degradation of DHODH at the K306 site, which maintains DHODH protein stability. Importantly, downregulated PRR11 increases lipid peroxidation and alters DHODH-mediated mitochondrial morphology, thereby promoting ferroptosis and increasing TMZ chemotherapy sensitivity. In conclusion, our results reveal a mechanism via which PRR11 drives ferroptosis resistance and identifies ferroptosis induction and TMZ as an attractive combined therapeutic strategy for GBM.

Although 5-fluorouracil (5-FU) is the primary chemotherapy treatment for colorectal cancer (CRC), its efficacy is limited by drug resistance. Ferroptosis activation is a promising treatment for 5-FU-resistant cancer cells; however, potential therapeutic targets remain elusive. This study investigated ferroptosis vulnerability and dihydroorotate dehydrogenase (DHODH) activity using stable, 5-FU-resistant CRC cell lines and xenograft models. Ferroptosis was characterized by measuring malondialdehyde levels, assessing lipid metabolism and peroxidation, and using mitochondrial imaging and assays. DHODH function is investigated through gene knockdown experiments, tumor behavior assays, mitochondrial import reactions, intramitochondrial localization, enzymatic activity analyses, and metabolomics assessments. Intracellular lipid accumulation and mitochondrial DHODH deficiency led to lipid peroxidation overload, weakening the defense system of 5-FU-resistant CRC cells against ferroptosis. DHODH, primarily located within the inner mitochondrial membrane, played a crucial role in driving intracellular pyrimidine biosynthesis and was redistributed to the cytosol in 5-FU-resistant CRC cells. Cytosolic DHODH, like its mitochondrial counterpart, exhibited dihydroorotate catalytic activity and participated in pyrimidine biosynthesis. This amplified intracellular pyrimidine pools, thereby impeding the efficacy of 5-FU treatment through molecular competition. These findings contribute to the understanding of 5-FU resistance mechanisms and suggest that ferroptosis and DHODH are promising therapeutic targets for patients with CRC exhibiting resistance to 5-FU.

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.