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.
Intracellular lipid accumulation leads to lipid peroxidation overload, while mitochondrial DHODH deficiency weakens the ferroptosis defense system. This dual vulnerability makes 5-FU-resistant CRC cells susceptible to ferroptosis. Moreover, the redistribution of mitochondrial DHODH to the cytosol increases intracellular pyrimidine pools, impairing the effectiveness of 5-FU through molecular competition. Targeting ferroptosis or DHODH has emerged as a novel therapeutic avenue for treating patients with CRC exhibiting resistance to 5-FU therapies. Display omitted
•The susceptibility of 5-FU-resistant CRC to ferroptosis is attributed to the combination of lipid accumulation and mitochondrial DHODH deficiency.•Cytosolic DHODH, like its mitochondrial counterpart, catalyzes dihydroorotate and is involved in pyrimidine biosynthesis.•Ferroptosis and DHODH could be therapeutic targets for 5-FU-resistant CRC patients.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Mycobacterium tuberculosis (MTB) is the etiologic agent of tuberculosis (TB), an ancient disease which causes 1.5 million deaths worldwide. Dihydroorotate dehydrogenase (DHODH) is a key enzyme of the ...MTB de novo pyrimidine biosynthesis pathway, and it is essential for MTB growth in vitro, hence representing a promising drug target. We present: (i) the biochemical characterization of the full‐length MTB DHODH, including the analysis of the kinetic parameters, and (ii) the previously unreleased crystal structure of the protein that allowed us to rationally screen our in‐house chemical library and identify the first selective inhibitor of mycobacterial DHODH. The inhibitor has fluorescence properties, potentially instrumental to in cellulo imaging studies, and exhibits an IC50 value of 43 μm, paving the way to hit‐to‐lead process.
Dihydroorotate dehydrogenase (DHODH) is a key enzyme of the pyrimidine biosynthesis pathway in Mycobacterium tuberculosis. Annotated as an essential gene for mycobacterial growth, it represents a potential drug target. Here, we present the crystal structure of the full‐length DHODH along with its biochemical characterization. Structural analysis has driven the screening for the identification of the first inhibitor of mycobacterial DHODH.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Ferroptosis is a form of programmed cell death and plays an important role in many diseases. Dihydroorotate dehydrogenase (DHODH) and glutathione peroxidase 4 (GPX4) play major roles in cell ...resistance to ferroptosis. Therefore, inactivation of these proteins provides an excellent opportunity for efficient ferroptosis-based synergistic cancer therapy. In this study, a multifunctional nanoagent (BPN
) containing a GPX4 targeting boron dipyrromethene (Bodipy) probe (BP) and a DHODH targeting proteolysis targeting chimera (PROTAC) is reported. BPN
is prepared using a nanoprecipitation method in the presence of a thermoresponsive liposome, where BP is encapsulated inside and the cathepsin B (CatB)-cleavable PROTAC peptide (DPCP) is modified on the outer surface. In the presence of near-infrared (NIR) photoirradiation, BPN
is melted and BP is released in tumor cells. Subsequently, BP inhibits the activity of GPX4 by covalently bonding with the selenocysteine at the enzyme active site. In addition, DPCP achieves sustained degradation of DHODH upon activation by CatB overexpressed in the tumor. The synergistic deactivation of GPX4 and DHODH induces extensive ferroptosis and subsequent cell death. In vivo and in vitro studies clearly show that the proposed ferroptosis therapy provides excellent antitumor effect.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Fourteen novel quinoline‐4‐carboxylic acid‐chalcone hybrids were obtained via Claisen–Schmidt condensation and evaluated as potential human dihydroorotate dehydrogenase (hDHODH) inhibitors. The ...ketone precursor 2 was synthesized by the Pfitzinger reaction and used for further derivatization at position 3 of the quinoline ring for the first time. Six compounds showed better hDHODH inhibitory activity than the reference drug leflunomide, with IC50 values ranging from 0.12 to 0.58 μM. The bioactive conformations of the compounds within hDHODH were resolved by means of molecular docking, revealing their tendency to occupy the narrow tunnel of hDHODH within the N‐terminus and to prevent ubiquinone as the second cofactor from easily approaching the flavin mononucleotide as a cofactor for the redox reaction within the redox site. The results of the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay revealed that 4d and 4h demonstrated the highest cytotoxic activity against the A375 cell line, with IC50 values of 5.0 and 6.8 µM, respectively. The lipophilicity of the synthesized hybrids was obtained experimentally and expressed as logD7.4 values at physiologicalpH while the solubility assay was conducted to define physicochemical characteristics influencing the ADMET properties.
Fourteen novel quinoline‐4‐carboxylic acid‐chalcone hybrids were synthesized and screened as potential human dihydroorotate dehydrogenase (hDHODH) inhibitors. Six compounds showed better hDHODH inhibitory activity than the reference drug, leflunomide. Compound 4d demonstrated the highest cytotoxic activity against A375 cells. Bioactive conformations within hDHODH were resolved by molecular docking.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
The last new herbicidal modes-of-action with commercial significance were introduced to the marketplace multiple decades ago. Serious weed resistance to most herbicidal classes have since emerged ...with widespread use. Aryl pyrrolidinone anilides represent an entirely new mode-of-action class of herbicides that interfere with
pyrimidine biosynthesis in plants via inhibition of dihydroorotate dehydrogenase. The chemical lead for this new herbicide class discovery was identified from high-volume sourced greenhouse screening that required structural reassignment of the hit molecule followed by an extensive synthetic optimization effort. With excellent grass weed control and pronounced safety on rice, the selected commercial development candidate has a proposed common name of tetflupyrolimet and represents the first member of the new HRAC (Herbicide Resistance Action Committee) Group 28. This paper describes the discovery path to tetflupyrolimet with an added focus on the bioisosteric modifications pursued in optimization, including replacements of the lactam core itself.
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IJS, KILJ, NUK, PNG, UL, UM, UPUK
Metabolic reprogramming is an integral part of the growth-promoting program driven by the MYC family of oncogenes. However, this reprogramming also imposes metabolic dependencies that could be ...exploited therapeutically. Here we report that the pyrimidine biosynthetic enzyme dihydroorotate dehydrogenase (DHODH) is an attractive therapeutic target for MYCN-amplified neuroblastoma, a childhood cancer with poor prognosis. Gene expression profiling and metabolomic analysis reveal that MYCN promotes pyrimidine nucleotide production by transcriptional upregulation of DHODH and other enzymes of the pyrimidine-synthesis pathway. Genetic and pharmacological inhibition of DHODH suppresses the proliferation and tumorigenicity of MYCN-amplified neuroblastoma cell lines. Furthermore, we obtain evidence suggesting that serum uridine is a key factor in determining the efficacy of therapeutic agents that target DHODH. In the presence of physiological concentrations of uridine, neuroblastoma cell lines are highly resistant to DHODH inhibition. This uridine-dependent resistance to DHODH inhibitors can be abrogated by dipyridamole, an FDA-approved drug that blocks nucleoside transport. Importantly, dipyridamole synergizes with DHODH inhibition to suppress neuroblastoma growth in animal models. These findings suggest that a combination of targeting DHODH and nucleoside transport is a promising strategy to overcome intrinsic resistance to DHODH-based cancer therapeutics.
Leflunomide, an anti-inflammatory agent, has been shown to be effective in multiple myeloma (MM) treatment; however, the mechanism of this phenomenon has not been fully elucidated. The aim of the ...study was to assess the role of mitochondria and dihydroorotate dehydrogenase (DHODH) inhibition in the cytotoxicity of leflunomide in relation to the MM cell line RPMI 8226. The cytotoxic effect of teriflunomide—an active metabolite of leflunomide—was determined using MTT assay, apoptosis detection, and cell cycle analysis. To evaluate DHODH-dependent toxicity, the cultures treated with teriflunomide were supplemented with uridine. Additionally, the level of cellular thiols as oxidative stress symptom was measured as well as mitochondrial membrane potential and protein tyrosine kinases (PTK) activity. The localization of the compound in cell compartments was examined using HPLC method. Teriflunomide cytotoxicity was not abolished in uridine presence. Observed apoptosis occurred in a mitochondria-independent manner, there was also no decrease in cellular thiols level. Teriflunomide arrested cell cycle in the G2/M phase which is not typical for DHODH deficiency. PTK activity was decreased only at the highest drug concentration. Interestingly, teriflunomide was not detected in the mitochondria. The aforementioned results indicate DHODH- and mitochondria-independent mechanism of leflunomide toxicity against RPMI 8226 cell line.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Abstract
The inconsistencies in the performance of the virtual screening (VS) process, depending on the used software and structural conformation of the protein, is a challenging issue in the drug ...design and discovery field. Varying performance, especially in terms of early recognition of the potential hit compounds, negatively affects the whole process and leads to unnecessary waste of the time and resources. Appropriate application of the ensemble docking and consensus-scoring approaches can significantly increase reliability of the VS results. Dihydroorotate dehydrogenase (DHODH) is a key enzyme in the pyrimidine biosynthesis pathway. It is considered as a valuable therapeutic target in cancer, autoimmune and viral diseases. Based on the conducted benchmark study and analysis of the effect of different combinations of the applied methods and approaches, here we suggested a structure-based virtual screening (SBVS) workflow that can be used to increase the reliability of VS.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Inhibition of
pyrimidine synthesis in proliferating T and B lymphocytes by teriflunomide, a pharmacological inhibitor of dihydroorotate dehydrogenase (DHODH), has been shown to be an effective ...therapy to treat patients with MS in placebo-controlled phase 3 trials. Nevertheless, the underlying mechanism contributing to the efficacy of DHODH inhibition has been only partially elucidated. Here, we aimed to determine the impact of teriflunomide on the immune compartment in a longitudinal high-dimensional follow-up of patients with relapse-remitting MS (RRMS) treated with teriflunomide.
High-dimensional spectral flow cytometry was used to analyze the phenotype and the function of innate and adaptive immune system of patients with RRMS before and 12 months after teriflunomide treatment. In addition, we assessed the impact of teriflunomide on the migration of memory CD8 T cells in patients with RRMS, and we defined patient immune metabolic profiles.
We found that 12 months of treatment with teriflunomide in patients with RRMS does not affect the B cell or CD4 T cell compartments, including regulatory T
follicular helper T
cell and helper T
cell subsets. In contrast, we observed a specific impact of teriflunomide on the CD8 T cell compartment, which was characterized by decreased homeostatic proliferation and reduced production of TNFα and IFNγ. Furthermore, we showed that DHODH inhibition also had a negative impact on the migratory velocity of memory CD8 T cells in patients with RRMS. Finally, we showed that the susceptibility of memory CD8 T cells to DHODH inhibition was not related to impaired metabolism.
Overall, these findings demonstrate that the clinical efficacy of teriflunomide results partially in the specific susceptibility of memory CD8 T cells to DHODH inhibition in patients with RRMS and strengthens active roles for these T cells in the pathophysiological process of MS.
Coenzyme Q (CoQ, ubiquinone) is the electron‐carrying lipid in the mitochondrial electron transport system (ETS). In mammals, it serves as the electron acceptor for nine mitochondrial inner membrane ...dehydrogenases. These include the NADH dehydrogenase (complex I, CI) and succinate dehydrogenase (complex II, CII) but also several others that are often omitted in the context of respiratory enzymes: dihydroorotate dehydrogenase, choline dehydrogenase, electron‐transferring flavoprotein dehydrogenase, mitochondrial glycerol‐3‐phosphate dehydrogenase, proline dehydrogenases 1 and 2, and sulfide:quinone oxidoreductase. The metabolic pathways these enzymes are involved in range from amino acid and fatty acid oxidation to nucleotide biosynthesis, methylation, and hydrogen sulfide detoxification, among many others. The CoQ‐linked metabolism depends on CoQ reoxidation by the mitochondrial complex III (cytochrome bc1 complex, CIII). However, the literature is surprisingly limited as for the role of the CoQ‐linked metabolism in the pathogenesis of human diseases of oxidative phosphorylation (OXPHOS), in which the CoQ homeostasis is directly or indirectly affected. In this review, we give an introduction to CIII function, and an overview of the pathological consequences of CIII dysfunction in humans and mice and of the CoQ‐dependent metabolic processes potentially affected in these pathological states. Finally, we discuss some experimental tools to dissect the various aspects of compromised CoQ oxidation.
Coenzyme Q (CoQ) is the electron‐carrying lipid in the mitochondrial electron transport system. In mammals, it is the electron acceptor for mitochondrial dehydrogenases involved in metabolic pathways ranging from ATP synthesis, amino acid, and fatty acid oxidation to nucleotide biosynthesis, methylation, and hydrogen sulfide detoxification. The CoQ‐linked metabolism depends on CoQ reoxidation by the mitochondrial complex III.
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BFBNIB, DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, SIK, UILJ, UKNU, UL, UM, UPUK
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