Targeted inhibition of DHODH is synergistic with BCL2 blockade in HGBCL with concurrent MYC and BCL2 rearrangement.

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.

Harnessing Redox Polymer Dynamics for Enhanced Glucose-Oxygen Coupling in Dual Biosensing and Therapeutic Applications.

The burgeoning field of continuous glucose monitoring (CGM) for diabetes management faces significant challenges, particularly in achieving precise and stable biosensor performance under changing environmental conditions such as varying glucose concentrations and O2 levels. To address this, we present a novel biosensor based on the electroless coupling of glucose oxidation catalyzed by flavin-dependent glucose dehydrogenase (FAD-GDH) and O2 reduction catalyzed by bilirubin oxidase (BOD) via a redox polymer, dimethylferrocene-modified linear poly(ethylenimine), FcMe2-LPEI. Initial cyclic voltammetry tests confirm the colocalization of both enzymatic reactions within the potential range of the polymer, indicating an effective electron shuttle mechanism. As a result, we created a hybrid biosensor that operates at open-circuit potential (OCP). It can detect glucose concentrations of up to 100 mM under various O2 conditions, including ambient air. This resulted from optimizing the enzyme ratio to 120 ± 10 mUBOD·UFAD-GDH-1·atmO2-1. This biosensor is highly sensitive, a crucial feature for CGM applications. This distinguishes it from FAD-GDH traditional biosensors, which require a potential to be applied to measure glucose concentrations up to 30 mM. In addition, this biosensor demonstrates the ability to function as a noninvasive, external device that can adapt to changing glucose levels, paving the way for its use in diabetes care and, potentially, personalized healthcare devices. Furthermore, by leveraging the altered metabolic pathways in tumor cells, this system architecture opened up new avenues for targeted glucose scavenging and O2 reduction in cancer therapy.

Loss-of-function of PGL10 impairs photosynthesis and tolerance to high-temperature stress in rice.

High temperature (HT) affects the production of chlorophyll (Chl) pigment and inhibits cellular processes that impair photosynthesis, and growth and development in plants. However, the molecular mechanisms underlying heat stress in rice are not fully understood yet. In this study, we identified two mutants varying in leaf color from the ethylmethanesulfonate mutant library of indica rice cv. Zhongjiazao-17, which showed pale-green leaf color and variegated leaf phenotype under HT conditions. Mut-map revealed that both mutants were allelic, and their phenotype was controlled by a single recessive gene PALE GREEN LEAF 10 (PGL10) that encodes NADPH:protochlorophyllide oxidoreductase B, which is required for the reduction of protochlorophyllide into chlorophyllide in light-dependent tetrapyrrole biosynthetic pathway-based Chl synthesis. Overexpression-based complementation and CRISPR/Cas9-based knockout analyses confirmed the results of Mut-map. Moreover, qRT-PCR-based expression analysis of PGL10 showed that it expresses in almost all plant parts with the lowest expression in root, followed by seed, third leaf, and then other green tissues in both mutants, pgl10a and pgl10b. Its protein localizes in chloroplasts, and the first 17 amino acids from N-terminus are responsible for signals in chloroplasts. Moreover, transcriptome analysis performed under HT conditions revealed that the genes involved in the Chl biosynthesis and degradation, photosynthesis, and reactive oxygen species detoxification were differentially expressed in mutants compared to WT. Thus, these results indicate that PGL10 is required for maintaining chloroplast function and plays an important role in rice adaptation to HT stress conditions by controlling photosynthetic activity.

Biliverdin Reductase-A integrates insulin signaling with mitochondrial metabolism through phosphorylation of GSK3ß.

Brain insulin resistance links the failure of energy metabolism with cognitive decline in both type 2 Diabetes Mellitus (T2D) and Alzheimer's disease (AD), although the molecular changes preceding overt brain insulin resistance remain unexplored. Abnormal biliverdin reductase-A (BVR-A) levels were observed in both T2D and AD and were associated with insulin resistance. Here, we demonstrate that reduced BVR-A levels alter insulin signaling and mitochondrial bioenergetics in the brain. Loss of BVR-A leads to IRS1 hyper-activation but dysregulates Akt-GSK3ß complex in response to insulin, hindering the accumulation of pGSK3ßS9 into the mitochondria. This event impairs oxidative phosphorylation and fosters the activation of the mitochondrial Unfolded Protein Response (UPRmt). Remarkably, we unveil that BVR-A is required to shuttle pGSK3ßS9 into the mitochondria. Our data sheds light on the intricate interplay between insulin signaling and mitochondrial metabolism in the brain unraveling potential targets for mitigating the development of brain insulin resistance and neurodegeneration.

DHCR24 in Tumor Diagnosis and Treatment: A Comprehensive Review.

As an important nutrient in the human body, cholesterol can not only provide structural components for the body's cells, but also can be transformed into a variety of active substances to regulate cell signaling pathways. As an important cholesterol synthase, DHCR24 participates in important regulatory processes in the body. The application of DHCR24 in tumor clinical diagnosis and treatment also attracts much attention. This article reviews the structure and regulatory characteristics of DHCR24, and the research of DHCR24 on tumor progression. We summarize the possible mechanisms of DHCR24 promoting tumor progression through reactive oxygen species (ROS), p53, Ras and PI3K-AKT pathways. Through our review, we hope to provide more research ideas and reference value for the application of DHCR24 in tumor prevention and treatment.

Redistribution of defective mitochondria-mediated dihydroorotate dehydrogenase imparts 5-fluorouracil resistance in colorectal cancer.

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.

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

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

Classical swine fever virus non-structural protein 4A recruits dihydroorotate dehydrogenase to facilitate viral replication.

Mitochondria are energy producers in cells, which can affect viral replication by regulating the host innate immune signaling pathways, and the changes in their biological functions are inextricably linked the viral life cycle. In this study, we screened a library of 382 mitochondria-targeted compounds and identified the antiviral inhibitors of dihydroorotate dehydrogenase (DHODH), the rate-limiting enzyme in the de novo synthesis pathway of pyrimidine ribonucleotides, against classical swine fever virus (CSFV). Our data showed that the inhibitors interfered with viral RNA synthesis in a dose-dependent manner, with half-maximal effective concentrations (EC50) ranging from 0.975 to 26.635 nM. Remarkably, DHODH inhibitors obstructed CSFV replication by enhancing the innate immune response including the TBK1-IRF3-STAT1 and NF-κB signaling pathways. Furthermore, the data from a series of compound addition and supplementation trials indicated that DHODH inhibitors also inhibited CSFV replication by blocking the de novo pyrimidine synthesis. Remarkably, DHODH knockdown demonstrated that it was essential for CSFV replication. Mechanistically, confocal microscopy and immunoprecipitation assays showed that the non-structural protein 4A (NS4A) recruited and interacted with DHODH in the perinuclear. Notably, NS4A enhanced the DHODH activity and promoted the generation of UMP for efficient viral replication. Structurally, the amino acids 65-229 of DHODH and the amino acids 25-40 of NS4A were pivotal for this interaction. Taken together, our findings highlight the critical role of DHODH in the CSFV life cycle and offer a potential antiviral target for the development of novel therapeutics against CSF. IMPORTANCE: Classical swine fever remains one of the most economically important viral diseases of domestic pigs and wild boar worldwide. dihydroorotate dehydrogenase (DHODH) inhibitors have been shown to suppress the replication of several viruses in vitro and in vivo, but the effects on Pestivirus remain unknown. In this study, three specific DHODH inhibitors, including DHODH-IN-16, BAY-2402234, and Brequinar were found to strongly suppress classical swine fever virus (CSFV) replication. These inhibitors target the host DHODH, depleting the pyrimidine nucleotide pool to exert their antiviral effects. Intriguingly, we observed that the non-structural protein 4A of CSFV induced DHODH to accumulate around the nucleus in conjunction with mitochondria. Moreover, NS4A exhibited a strong interaction with DHODH, enhancing its activity to promote efficient CSFV replication. In conclusion, our findings enhance the understanding of the pyrimidine synthesis in CSFV infection and expand the novel functions of CSFV NS4A in viral replication, providing a reference for further exploration of antiviral targets against CSFV.

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

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

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

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

Bioinformatics analysis and experimental verification of the cancer-promoting effect of DHODH in clear cell renal cell carcinoma.

Clear cell renal cell carcinoma (ccRCC) is a malignant tumor of the urinary system. To explore the potential mechanisms of DHODH in ccRCC, we analyzed its molecular characteristics using public databases. TCGA pan-cancer dataset was used to analyze DHODH expression in different cancer types and TCGA ccRCC dataset was used to assess differential expression, prognosis correlation, immune infiltration, single-gene, and functional enrichment due to DHODH. The GSCALite and CellMiner databases were employed to explore drugs and perform molecular docking analysis with DHODH. Protein-protein interaction networks and ceRNA regulatory networks of DHODH were constructed using multiple databases. The effect of DHODH on ccRCC was confirmed in vitro. DHODH was highly expressed in ccRCC. Immune infiltration analysis revealed that DHODH may be involved in regulating the infiltration of immunosuppressive cells such as Tregs. Notably, DHODH influenced ccRCC progression by forming regulatory networks with molecules, such as hsa-miR-26b-5p and UMPS and significantly enhanced the malignant characteristics of ccRCC cells. Several drugs, such as lapatinib, silmitasertib, itraconazole, and dasatinib, were sensitive to DHODH expression and exhibited strong molecular binding with it. Thus, DHODH may promote ccRCC progression and is a candidate effective therapeutic target for ccRCC.

Elucidation of the Catalytic Sequence of Dihydroorotate Dehydrogenase B from Lactoccocus lactis: Evidence for Accumulation of a Flavin Bisemiquinone State in Catalysis.

The physiological role of dihydroorotate dehydrogenase (DHOD) enzymes is to catalyze the oxidation of dihydroorotate to orotate in pyrimidine biosynthesis. DHOD enzymes are structurally diverse existing as both soluble and membrane-associated forms. The Family 1 enzymes are soluble and act either as conventional single subunit flavin-dependent dehydrogenases known as Class 1A (DHODA) or as unusual heterodimeric enzymes known as Class 1B (DHODB). DHODBs possess two active sites separated by ∼20 Å, each with a noncovalently bound flavin cofactor. NAD is thought to interact at the FAD containing site, and the pyrimidine substrate is known to bind at the FMN containing site. At the approximate center of the protein is a single Fe2S2 center that is assumed to act as a conduit, facilitating one-electron transfers between the flavins. We present anaerobic transient state analysis of a DHODB enzyme from Lactoccocus lactis. The data presented primarily report the exothermic reaction that reduces orotate to dihydroorotate. The reductive half reaction reveals rapid two-electron reduction that is followed by the accumulation of a four-electron reduced state when NADH is added in excess, suggesting that the initial two electrons acquired reside on the FMN cofactor. Concomitant with the first reduction is the accumulation of a long-wavelength absorption feature consistent with the blue form of a flavin semiquinone. Spectral deconvolution and fitting to a model that includes reversibility for the second electron transfer reveals equilibrium accumulation of a flavin bisemiquinone state that has features of both red and blue semiquinones. Single turnover reactions with limiting NADH and excess orotate reveal that the flavin bisemiquinone accumulates with reduction of the enzyme by NADH and decays with reduction of the pyrimidine substrate, establishing the bisemiquinone as a fractional state of the two-electron reduced intermediate observed.

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

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

DHCR7 links cholesterol synthesis with neuronal development and axonal integrity.

The DHCR7 enzyme converts 7-DHC into cholesterol. Mutations in DHCR7 can block cholesterol production, leading to abnormal accumulation of 7-DHC and causing Smith-Lemli-Opitz syndrome (SLOS). SLOS is an autosomal recessive disorder characterized by multiple malformations, including microcephaly, intellectual disability, behavior reminiscent of autism, sleep disturbances, and attention-deficit/hyperactivity disorder (ADHD)-like hyperactivity. Although 7-DHC affects neuronal differentiation in ex vivo experiments, the precise mechanism of SLOS remains unclear. We generated Dhcr7 deficient (dhcr7-/-) zebrafish that exhibited key features of SLOS, including microcephaly, decreased neural stem cell pools, and behavioral phenotypes similar to those of ADHD-like hyperactivity. These zebrafish demonstrated compromised myelination, synaptic anomalies, and neurotransmitter imbalances. The axons of the dhcr7-/- zebrafish showed increased lysosomes and attenuated autophagy, suggesting that autophagy-related neuronal homeostasis is disrupted.

Viperin inhibits interferon-γ production to promote Mycobacterium tuberculosis survival by disrupting TBK1-IKKε-IRF3-axis and JAK-STAT signaling.

OBJECTIVES AND DESIGN: As an interferon-inducible protein, Viperin has broad-spectrum antiviral effects and regulation of host immune responses. We aim to investigate how Viperin regulates interferon-γ (IFN-γ) production in macrophages to control Mycobacterium tuberculosis (Mtb) infection. METHODS: We use Viperin deficient bone-marrow-derived macrophage (BMDM) to investigate the effects and machines of Viperin on Mtb infection. RESULTS: Viperin inhibited IFN-γ production in macrophages and in the lung of mice to promote Mtb survival. Further insight into the mechanisms of Viperin-mediated regulation of IFN-γ production revealed the role of TANK-binding kinase 1 (TBK1), the TAK1-dependent inhibition of NF-kappa B kinase-epsilon (IKKε), and interferon regulatory factor 3 (IRF3). Inhibition of the TBK1-IKKε-IRF3 axis restored IFN-γ production reduced by Viperin knockout in BMDM and suppressed intracellular Mtb survival. Moreover, Viperin deficiency activated the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling pathway, which promoted IFN-γ production and inhibited Mtb infection in BMDM. Additionally, a combination of the anti-TB drug INH treatment in the absence of Viperin resulted in further IFN-γ production and anti-TB effect. CONCLUSIONS: This study highlights the involvement of TBK1-IKKε-IRF3 axis and JAK-STAT signaling pathways in Viperin-suppressed IFN-γ production in Mtb infected macrophages, and identifies a novel mechanism of Viperin on negatively regulating host immune response to Mtb infection.

Hypothiocyanous acid reductase is critical for host colonization and infection by Streptococcus pneumoniae.

The major human pathogen Streptococcus pneumoniae encounters the immune-derived oxidant hypothiocyanous acid (HOSCN) at sites of colonization and infection. We recently identified the pneumococcal hypothiocyanous acid reductase (Har), a member of the flavoprotein disulfide reductase enzyme family, and showed that it contributes to the HOSCN tolerance of S. pneumoniae in vitro. Here, we demonstrate in mouse models of pneumococcal infection that Har is critical for colonization and invasion. In a colonization model, bacterial load was attenuated dramatically in the nasopharynx when har was deleted in S. pneumoniae. The Δhar strain was also less virulent compared to wild type in an invasion model as reflected by a significant reduction in bacteria in the lungs and no dissemination to the blood and brain. Kinetic measurements with recombinant Har demonstrated that this enzyme reduced HOSCN with near diffusion-limited catalytic efficiency, using either NADH (kcat/KM = 1.2 × 108 M-1s-1) or NADPH (kcat/KM = 2.5 × 107 M-1s-1) as electron donors. We determined the X-ray crystal structure of Har in complex with the FAD cofactor to 1.50 Å resolution, highlighting the active site architecture characteristic for this class of enzymes. Collectively, our results demonstrate that pneumococcal Har is a highly efficient HOSCN reductase, enabling survival against oxidative host immune defenses. In addition, we provide structural insights that may aid the design of Har inhibitors.

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

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

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

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

Identification and validation of DHCR7 as a diagnostic biomarker involved in the proliferation and mitochondrial function of breast cancer.

BACKGROUND: Energy metabolism has a complex intersection with pathogenesis and development of breast cancer (BC). This allows for the possibility of identifying energy-metabolism-related genes (EMRGs) as novel prognostic biomarkers for BC. 7-dehydrocholesterol reductase (DHCR7) is a key enzyme of cholesterol biosynthesis involved in many cancers, and in this paper, we investigate the effects of DHCR7 on the proliferation and mitochondrial function of BC. METHODS: EMRGs were identified from the Gene Expression Omnibus (GEO) and MSigDB databases using bioinformatics methods. Key EMRGs of BC were then identified and validated by functional enrichment analysis, interaction analysis, weighted gene co-expression network analysis (WGCNA), least absolute shrinkage and selection operator (LASSO) regression, Cox analysis, and immune infiltration. Western blot, qRT-PCR, immunohistochemistry (IHC), MTT assay, colony formation assay and flow cytometry assay were then used to analyze DHCR7 expression and its biological effects on BC cells. RESULTS: We identified 31 EMRGs in BC. These 31 EMRGs and related transcription factors (TFs), miRNAs, and drugs were enriched in glycerophospholipid metabolism, glycoprotein metabolic process, breast cancer, and cell cycle. Crucially, DHCR7 was a key EMRG in BC identified and validated by WGCNA, LASSO regression and receiver operating characteristic (ROC) curve analysis. High DHCR7 expression was significantly associated with tumor immune infiltration level, pathological M, and poor prognosis in BC. In addition, DHCR7 knockdown inhibited cell proliferation, induced apoptosis and affected mitochondrial function in BC cells. CONCLUSIONS: DHCR7 was found to be a key EMRG up-regulated in BC cells. This study is the first to our knowledge to report that DHCR7 acts as an oncogene in BC, which might become a novel therapeutic target for BC patients.

A comprehensive review of synthetic strategies and SAR studies for the discovery of PfDHODH inhibitors as antimalarial agents. Part 1: triazolopyrimidine, isoxazolopyrimidine and pyrrole-based (DSM) compounds.

One of the deadliest infectious diseases, malaria, still has a significant impact on global morbidity and mortality. Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH) catalyzes the fourth step in de novo pyrimidine nucleotide biosynthesis and has been clinically validated as an innovative and promising target for the development of novel targeted antimalarial drugs. PfDHODH inhibitors have the potential to significantly slow down parasite growth at the blood and liver stages. Several PfDHODH inhibitors based on various scaffolds have been explored over the past two decades. Among them, triazolopyrimidines, isoxazolopyrimidines, and pyrrole-based derivatives known as DSM compounds showed tremendous potential as novel antimalarial agents, and one of the triazolopyrimidine-based compounds (DSM265) was able to reach phase IIa clinical trials. DSM compounds were synthesized as PfDHODH inhibitors with various substitutions based on structure-guided medicinal chemistry approaches and further optimised as well. For the first time, this review provides an overview of all the synthetic approaches used for the synthesis, alternative synthetic routes, and novel strategies involving various catalysts and chemical reagents that have been used to synthesize DSM compounds. We have also summarized SAR study of all these PfDHODH inhibitors. In an attempt to assist readers, scientists, and researchers involved in the development of new PfDHODH inhibitors as antimalarials, this review provides accessibility of all synthetic techniques and SAR studies of the most promising triazolopyrimidines, isoxazolopyrimidines, and pyrrole-based PfDHODH inhibitors.