Research Progress on Mechanism of Action of DHODH in Progression of Malignant Tumors / 肿瘤防治研究

Dihydroorotate dehydrogenase (DHODH) is a flavin-dependent metabolic enzyme that oxidizes dihydroorotate acid to orotic acid in the de novo synthesis pathway of pyrimidine metabolism. DHODH is located in mitochondria, closely related to cellular oxidative phosphorylation, and an important suppressor of the ferroptosis pathway. This study investigates the influence of DHODH on the progression of malignant tumors, including its important role in the de novo synthesis of pyrimidine, oxidative phosphorylation, and ferroptosis. The objective is to present evidence that DHODH is a potential target for the clinical treatment of tumors.

Morphological Characteristics and the Role of Mitochondria in Ferroptosis / 中国生物化学与分子生物学报

Ferroptosis is a unique mode of iron-dependent cell death driven by lipid peroxidation. It is characterized by morphological changes in mitochondria, including densification of mitochondrial membranes and associated reduction in the volume, rupture of outer membranes and reduction or disappearance of the mitochondrial crest, which is different from that of apoptosis, autophagy and pyroptosis. Mitochondria, as the core of cell metabolism, are important organelles for iron metabolism, lipid metabolism and energy metabolism. However, it remains controversial debates as how mitochondria participate in ferroptosis and the underlying mechanisms during its progression. This review summaries the current understanding of the occurrence and defense mechanisms of ferroptosis, and the role of mitochondria in promoting and inhibiting ferroptosis, which includes the tricarboxylic acid cycle and glycolysis, reactive oxygen species, and lipid metabolism in mitochondria and their roles in driving ferroptosis. Moreover, we also summarize the defense mechanisms against ferroptosis through detoxification of mitochondrial lipid peroxidation by mitochondrial dihydroorotate dehydrogenase, as well as mitochondrial ferritin. Other mitochondrial molecules and their regulation of ferroptosis is stated at the end. This paper reviews the latest research progress of mitochondria in the process of ferroptosis, which aims to further understand the function of mitochondria in ferroptosis and its mechanism in the occurrence and development of ferroptosis, and therefore provides a theoretical foundation for the basic research of cell biology and strategies for investigation of clinical diseases.

Structural characteristics and catalytic cycle of dihydroorotate dehydrogenase-a review / 生物工程学报

Dihydroorotate dehydrogenase is a flavin-dependent mitochondrial enzyme to catalyze the fourth step of the de novo synthesis of pyrimidine and to oxidize dihydroorotate to orotate. By selectively inhibiting dihydroorotate dehydrogenase, thereby inhibiting pyrimidine synthesis, the enzyme has been developed for the treatment of cancer, autoimmune diseases, bacterial or viral infections, parasitic diseases and so on. The development of inhibitory drugs requires a detailed understanding of the structural characteristics and catalytic cycle mechanism of dihydroorotate dehydrogenase. Therefore, this paper reviews these two aspects, and indicates perspectives of these inhibitors in clinical application.

Investigation of the DHODH Genetic Polymorphism (19C ＞ A) Response to Leflunomide Treatment in Rheumatoid Arthritis / 医学研究杂志

Objective Purpose leflunomide (LEF) is a disease-modifying antirheumatic drug used for treating rheumatoid arthritis (RA).It is a prodrug that is rapidly converted in vivo to the active metabolite A77 1726.The mechanism of action of A77 1726 primarily involves inhibition of the enzyme DHODH.Related studies have found that DHOD gene has polymorphism.The aim of this study was to investigate whether genetic polymorphisms in DHODH (19C ＞ A) influence leflunomide pharmacokinetics and treatment response.Methods We studied 105 patients diagnosed with RA and treated with LEF (20 mg daily).Follow-up was 6 months.Clinical improvement was valuated according to the American College of Rheumatology 20％ and 50％ response criteria.The peripheral blood genomic DNA was extracted,and amplified by PCR,analyzed by direct sequencing.The gene detection was performed in our hospital 105 patients with DHODH polymorphism(19C ＞ A).Results After 3 months of therapy,the ACR20 criteria was met by 70.0％ in C allele,51.7％ in A allele.Differences were statistically significant(P =0.012).The ACR50 criteria was met by 38.0％ in C allele,28.3％ in A allele.It did not reach statistically significant(P =0.185).After 6 months of therapy,the ACR20 criteria was met by 84.7％ in C allele,61.7％ in A allele.Differences were statistically significant (P =0.006).The ACR50 criteria was met by 60.7％ in C allele,38.3 ％ in A allele.Differences were statistically significant(P =0.039).Conclusion The remission rate of ACR20 and ACR50 in patients with rheumatoid arthritis was increased with the treatment time.The results of the study suggest that DHODH(19C ＞ A) polymorphism may be associated with LEF treatment outcome in RA patients.Treatment response is better in the patients with the C allele than A allele.This trend is more obvious with the extension of time.

Synthesis, stability and activity of the prodrugs of dihydroorotate dehydrogenase inhibitors / 药学学报

This study was conducted to improve structural instability of a highly active DHODH inhibitor A found in our group. Twelve prodrugs were synthesized by modifying the carboxyl group. The enzyme activity test of 12 prodrugs A1−A12 demonstrated that A1−A5 displayed weak inhibitory activity, and A6−A12 displayed no activity, which met the action mechanism of designed prodrug. The structural stability of A1−A12 in methanol and pH 2.0, 9.0 buffers were tested, and the results showed that A12 could avoid intramolecular ring-formation in CH3OH, A1−A8 were easily hydrolyzed under acidic conditions, and A9−A12 were inclined to hydrolyze under alkaline conditions. The cell proliferation inhibitory activity of 12 prodrugs were evaluated, in which compound A12 displayed excellent activity (IC50=0.63 μmol·L−1) similar to brequinar. These results laid a good foundation for conducting further vivo studies.

Dihydroorotate dehydrogenase mutations lead to changes of protein and function in Miller syndrome / 重庆医学

Objective To observe the changes of corresponding proteins and function based on known clinical dihydroorotate dehydrogenase(DHODH) mutation types,i.e.,G202A,R346W and R135C in the patients with Miller syndrome.Methods HeLacell lines stably expressing Miller syndrome pathogenic mutation types G202A,R346W and R135C were established.Then the mitochondrial localization function,protein stability and enzyme activity of corresponding proteins were studied by the immunohistochemistry and mitochondrial layered positioning.Results The mitochondrial localization function of 3 kinds of DHODH mutation were not affected,which existed in the mitochondrial inner membrane after expression.The mutant G202A and R346W protein stability was reduced;the mutant R135C protein was stable,but base induced enzyme activity injury caused the deficiency of corresponding enzymatic activity.Conclusion The DHODH function injury may be related with the symptoms in Miller syndrome.

3D-QSAR studies on thiazole derivatives as potent inhibitors of dihydroorotate dehydrogenase / 药物评价研究

Objective The three-dimensional quantitative structure activity relationship (3D-QSAR) method was applied to study thiazole derivatives as potent inhibitors ofdihydroorotate dehydrogenase,which provided useful guidance for more discovery of potent inhibitors of dihydroorotate dehydrogenase.Methods Molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) were applied to systematicly investigate 3D-QSAR of 38 hiazole derivatives as potent inhibitors of dihydroorotate dehydrogenase.Established models of CoMFA and CoMSIA and the predictive ability of models were validated.Three dimensional map was applied to analyzing the relationship between structure and activity of thiazole derivatives.Results The coefficients of cross validation q2 and non-cross validation r2 for CoMFA model were 0.796 and 0.978,and for CoMSIA model were 0.721 and 0.976 respectively.The prediction of activity of compound was close to the actual value of the two models.Effect of compound structure on its activity could be analyzed comprehensively and intuitively by three dimensional map.Conclusion The model reveals the relationship between the structure characteristics and the inhibitory activity,and has good predictive capability and stability to lay a good foundation for further development and research.

Study on effects of DHODH deficiency on mitochondrial function and differentiation and maturation in osteoblast cells / 重庆医学

Objective To observe the changes of the skeletal development related cells after dihydroorotate dehydrogenase (DHODH) deficiency.Methods The DHODH expression in MC3T3-E1 cells derived from mouse calvaria osteoblast precursor cells was inhibited by specific small interfering RNAs (siRNAs),and cell proliferation,ATP production and expression levels of bone-related genes were investigated in these cells.Results After reducing the DHODH expression by using specific siRNAs,cell proliferation was inhibited and cell cycle was arrested at G1/S stage.In addition,the ATP production was reduced in whole cells,especially in mitochondria.Furthermore,the expression levels of Runt-related transcription factor 2 (Runx2) and osteocalcin (Ocn) mRNAs in the DHODH inhibition group were decreased compared with the control group.Conclusion Inhibiting DHODH protein affects the differentiation and maturation of osteoblasts.The mitochondrial dysfunction in osteoblasts may be one of causes leading to the abnormal bone formation in Miller syndrome.

Design, synthesis and structure-activity relationship studies of human dihydroorotate dehydrogenase inhibitors / 药学学报

In this study, 1-(3-(4-chlorophenyl)-5-methylthio-1H-1,2,4-triazol-1-yl)-butan-1-one discovered previously in our lab was selected as a inhibitor of human dihydroorotate dehydrogenase (HsDHODH) for structural optimization. The co-crystal of HsDHODH with the hit was obtained and analyzed for guiding the subsequent structural optimization. As a result, a series of novel triazole derivatives were designed and synthesized as potent HsDHODH inhibitors. Among them, compound (3-(4-chlorophenyl)-5-ethylthio-1H-1,2,4-triazol-1-yl)-furan-2-yl-methanone displayed high potency in the inhibition of HsDHODH with an IC50 value of 1.50 μmol·L-1. Meanwhile, the structure-activity relationships were analyzed based on the biological data and the co-crystal structure. These results provide a valuable reference for optimization of 1H-1,2,4-triazole derivatives as HsDHODH inhibitors in the future.