Computer-aided discovery of novel SmDHODH inhibitors for schistosomiasis therapy: Ligand-based drug design, molecular docking, molecular dynamic simulations, drug-likeness, and ADMET studies.

Schistosomiasis, also known as bilharzia or snail fever, is a tropical parasitic disease resulting from flatworms of the Schistosoma genus. This often overlooked disease has significant impacts in affected regions, causing enduring morbidity, hindering child development, reducing productivity, and creating economic burdens. Praziquantel (PZQ) is currently the only treatment option for schistosomiasis. Given the potential rise of drug resistance and the limited treatment choices available, there is a need to develop more effective inhibitors for this neglected tropical disease (NTD). In view of this, quantitative structure-activity relationship studies (QSAR), molecular docking, molecular dynamics simulations, drug-likeness, and ADMET predictions were applied to 31 inhibitors of Schistosoma mansoni Dihydroorotate dehydrogenase (SmDHODH). The designed QSAR model demonstrated robust statistical parameters including an R2 of 0.911, R2adj of 0.890, Q2cv of 0.686, R2pred of 0.807, and cR2p of 0.825, confirming its robustness. Compound 26, identified as the most active derivative, emerged as a lead candidate for new potential inhibitors through ligand-based drug design. Subsequently, 12 novel compounds (26A-26L) were designed with enhanced inhibition activity and binding affinity. Molecular docking studies revealed strong and stable interactions, including hydrogen bonding and hydrophobic interactions, between the designed compounds and the target receptor. Molecular dynamics simulations over 100 nanoseconds and MM-PBSA free binding energy (ΔGbind) calculations validated the stability of the two best-designed molecules (26A and 26L). Furthermore, drug-likeness and ADMET prediction analyses affirmed the potential of these designed compounds, suggesting their promise as innovative agents for treating schistosomiasis.

Metabolic Engineering of Escherichia coli for Production of a Bioactive Metabolite of Bilirubin.

Bilirubin (BR) is an important ingredient of a valuable Chinese medicine, Calculus bovis. Over recent decades, increasing evidence has confirmed that BR offers health benefits in cardiovascular health, stroke, diabetes, and metabolic syndrome. However, BR is mainly produced by extraction from pig bile. In this study, we assembled an efficient pathway for BR production by metabolic engineering of Escherichia coli. First, heme oxygenase (HO1) and biliverdin reductase were co-expressed in E. coli. HPLC and LC-MS confirmed the accumulation of BR in the recombinant E. coli cells. To improve BR production, the catalytic abilities of HO1 from different species were investigated. In addition, the outermembrane-bound heme receptor (ChuA) and the enzymes involved in heme biosynthesis were overexpressed among which ChuA, 5-aminolevulinic acid dehydratase (HemB), protoporphyrin oxidase (HemG), and ferrochelatase (HemH) were found to enhance BR accumulation in E. coli. In addition, expression of ferredoxin (Fd) was shown to contribute to efficient conversion of heme to BR in E. coli. To increase supply of NADPH, isocitrate dehydrogenase (IDH), NAD kinase (nadK), NADP-specific glutamate dehydrogenase (gdhA), and glucose-6-phosphate 1-dehydrogenase (ZWF) were overexpressed and were found to enhance BR accumulation when these proteins were expressed with a low-copy plasmid pACYCduet-1. Modular optimization of the committed genes led to a titer of 17.2 mg/L in strain M1BHG. Finally, fed-batch fermentation was performed for the strains M1BHG and M1, resulting in accumulation of 75.5 mg/L and 25.8 mg/L of BR, respectively. This is the first report on biosynthesis of BR through metabolic engineering in a heterologous host.

Antirheumatic drug leflunomide attenuates atherosclerosis by regulating lipid metabolism and endothelial dysfunction via DHODH/AMPK signaling pathway.

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).

Molecular cloning and characterization of heat-responsive LcOPR1, a gene encoding oxophytodienoic acid reductase in lentil.

Improving crop plants using biotechnological implications is a promising and modern approach compared to traditional methods. High-temperature exposure to the reproductive stage induces flower abortion and declines grain filling performance, leading to smaller grain production and low yield in lentil and other legumes. Thus, cloning effective candidate genes and their implication in temperature stress tolerance in lentil (Lens culinaris Medik.) using biotechnological tools is highly demandable. The 12-oxophytodienoic acid reductases (OPRs) are flavin mononucleotide-dependent oxidoreductases with vital roles in plants. They are members of the old yellow enzyme (OYE) family. These enzymes are involved in the octadecanoid pathway, which contributes to jasmonic acid biosynthesis and is essential in plant stress responses. Lentil is one of the vital legume crops affected by the temperature fluctuations caused by global warming. Therefore, in this study, the LcOPR1 gene was successfully cloned and isolated from lentils using RT-PCR to evaluate its functional responses in lentil under heat stress. The bioinformatics analysis revealed that the full-length cDNA of LcOPR1 was 1303 bp, containing an 1134 bp open reading frames (ORFs), encoding 377 amino acids with a predicted molecular weight of 41.63 and a theoretical isoelectric point of 5.61. Bioinformatics analyses revealed that the deduced LcOPR1 possesses considerable homology with other plant 12-oxophytodienoic acid reductases (OPRs). Phylogenetic tree analysis showed that LcOPR1 has an evolutionary relationship with other OPRs in different plant species of subgroup I, containing enzymes that are not required for jasmonic acid biosynthesis. The expression analysis of LcOPR1 indicated that this gene is upregulated in response to the heat-stress condition and during recovery in lentil. This study finding might be helpful to plant breeders and biotechnologists in LcOPR1 engineering and/or plant breeding programs in revealing the biological functions of LcOPR1 in lentils and the possibility of enhancing heat stress tolerance by overexpressing LcOPR1 in lentil and other legume plants under high temperature.

Analysis of homodimer formation in 12-oxophytodienoate reductase 3 in solutio and crystallo challenges the physiological role of the dimer.

12-oxophytodienoate reductase 3 (OPR3) is a key enzyme in the biosynthesis of jasmonoyl-L-isoleucine, the receptor-active form of jasmonic acid and crucial signaling molecule in plant defense. OPR3 was initially crystallized as a self-inhibitory dimer, implying that homodimerization regulates enzymatic activity in response to biotic and abiotic stresses. Since a sulfate ion is bound to Y364, mimicking a phosphorylated tyrosine, it was suggested that dimer formation might be controlled by reversible phosphorylation of Y364 in vivo. To investigate OPR3 homodimerization and its potential physiological role in more detail, we performed analytical gel filtration and dynamic light scattering on wild-type OPR3 and three variants (R283D, R283E, and Y364P). The experiments revealed a rapid and highly sensitive monomer-dimer equilibrium for all OPR3 constructs. We crystallized all constructs with and without sulfate to examine its effect on the dimerization process and whether reversible phosphorylation of Y364 triggers homodimerization in vivo. All OPR3 constructs crystallized in their monomeric and dimeric forms independent of the presence of sulfate. Even variant Y364P, lacking the putative phosphorylation site, was crystallized as a self-inhibitory homodimer, indicating that Y364 is not required for dimerization. Generally, the homodimer is relatively weak, and our results raise doubts about its physiological role in regulating jasmonate biosynthesis.

Efficient production of 22(R)-hydroxycholesterol via combination optimization of Saccharomyces cerevisiae.

22(R)-hydroxycholesterol (22(R)-HCHO) is a crucial precursor of steroids biosynthesis with various biological functions. However, the production of 22(R)-HCHO is expensive and unsustainable due to chemical synthesis and extraction from plants or animals. This study aimed to construct a microbial cell factory to efficiently produce 22(R)-HCHO through systems metabolic engineering. First, we tested 7-dehydrocholesterol reductase (Dhcr7s) and cholesterol C22-hydroxylases from different sources in Saccharomyces cerevisiae, and the titer of 22(R)-HCHO reached 128.30 mg L-1 in the engineered strain expressing Dhcr7 from Columba livia (ClDhcr7) and cholesterol 22-hydroxylase from Veratrum californicum (VcCyp90b27). Subsequently, the 22(R)-HCHO titer was significantly increased to 427.78 mg L-1 by optimizing the critical genes involved in 22(R)-HCHO biosynthesis. Furthermore, hybrid diploids were constructed to balance cell growth and 22(R)-HCHO production and to improve stress tolerance. Finally, the engineered strain produced 2.03 g L-1 of 22(R)-HCHO in a 5-L fermenter, representing the highest 22(R)-HCHO titer reported to date in engineered microbial cell factories. The results of this study provide a foundation for further applications of 22(R)-HCHO in various industrially valuable steroids.

Bilirubin oxidase expression and activity enhancement from Myrothecium verrucaria in Aspergillus species.

We constructed a new Aspergillus expression vector (pSENSU2512nid) under the control of the enolase promoter with 12 tandem repeats of cis-acting elements (region III) and the heat shock protein 12 (Hsp12) 5' untranslated region (UTR). Bilirubin oxidase (EC: 1.3.3.5) from Myrothecium verrucaria, which catalyzes the oxidation of bilirubin to biliverdin, was overexpressed in Aspergillus oryzae and A. niger. The productivity was estimated to be approximately 1.2 g/L in the culture broth, which was approximately 6-fold higher than that of recombinant bilirubin oxidase (BOD) expressed in Pichia pastoris (Komagataella phaffii). BOD was purified using hydrophobic interaction chromatography, followed by ion exchange chromatography. The specific activity of the purified BOD against 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) substrate was 57.6 U/mg and 66.4 U/mg for A. oryzae and A. niger, respectively. l-Ascorbic acid (4 mM) addition and storage under deoxygenated conditions for 3-7 d increased the specific activity of these Aspergillus-expressed BODs approximately 2.3-fold (154.1 U/mg). The BOD specific activity was enhanced by incubation at higher temperature (30-50 °C). Further characterization of the enzyme catalytic efficiency revealed that the Km value remained unchanged, whereas the kcat value improved 3-fold. In conclusion, this high-level of BOD expression meets the requirements for industrial-level production. Additionally, we identified an effective method to enhance the low specific activity during expression, making it advantageous for industrial applications.

Biofuel cell-based self-powered immunosensor for detection of 17ß-estradiol by integrating the target-induced biofuel release and biogate immunoassay.

A novel biofuel cell (BFC)-based self-powered electrochemical immunosensing platform was developed by integrating the target-induced biofuel release and biogate immunoassay for ultrasensitive 17ß-estradiol (E2) detection. The carbon nanocages/gold nanoparticle composite was employed in the BFCs device as the electrode material, through which bilirubin oxidase and glucose oxidase were wired to form the biocathode and bioanode, respectively. Positively charged mesoporous silica nanoparticles (PMSN) were encapsulated with glucose molecules as biofuel and subsequently coated by the negatively charged AuNPs-labelled anti-E2 antibody (AuNPs-Ab) serving as a biogate. The biogate could be opened efficiently and the trapped glucose released once the target E2 was recognized and captured by AuNPs-Ab due to the decreased adhesion between the antigen-antibody complex and PMSN. Then, glucose oxidase oxidized the glucose to produce a large number of electrons, resulting in significantly increased open-circuit voltage (EOCV). Promisingly, the proposed BFC-based self-powered immunosensor demonstrated exceptional sensitivity for the detection of E2 in the concentration range from 1.0 pg mL-1 to 10.0 ng mL -1, with a detection limit of 0.32 pg mL-1 (S/N = 3). Furthermore, the prepared BFC-based self-powered homogeneous immunosensor showed significant potential for implementation as a viable prototype for a mobile and an on-site bioassay system in food and environmental safety applications.

DHODH Alleviates Heart Failure via the Modulation of CoQ-Related Ferroptotic Inhibition.

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.

DHODH inhibition enhances the efficacy of immune checkpoint blockade by increasing cancer cell antigen presentation.

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.

Molecular insights into the differences between cyanobacterial and plant LPORs and prolamellar body formation: In vitro studies.

Light-dependent protochlorophyllide oxidoreductase (LPOR) has captivated the interest of the research community for decades. One reason is the photocatalytic nature of the reaction catalyzed by the enzyme, and the other is the involvement of LPOR in the formation of a paracrystalline lattice called a prolamellar body (PLB) that disintegrates upon illumination, initiating a process of photosynthetic membrane formation. In this paper, we have integrated three traditional methods previously employed to study the properties of the enzyme: molecular biology, spectroscopy, and electron microscopy. We found that for cyanobacterial LPOR, substrates binding appears to be independent of lipids, with membrane interaction primarily affecting the enzyme post-reaction, with MGDG and PG having opposite effects on SynPOR. In contrast, plant isoforms exhibit sequence alterations, rendering the enzyme effective in substrate binding mainly in the presence of anionic lipids, depending on residues at positions 122, 312, and 318. Moreover, we demonstrated that the interaction with MGDG could initially serve as enhancement of the substrate specificity towards monovinyl-protochlorophyllide (Pchlide). We have shown that the second LPOR isoforms of eudicots and monocots accumulated mutations that made these variants less and more dependent on anionic lipids, respectively. Finally, we have shown that in the presence of Pchlide, NADP+, and the lipids, plant but not cyanobacterial LPOR homolog remodel membranes into the cubic phase. The cubic phase is preserved if samples supplemented with NADP + are enriched with NADPH. The results are discussed in the evolutionary context, and the model of PLB formation is presented.

Exploration of dietary interventions to treat mitochondrial fatty acid disorders in a mouse model.

Mitochondrial fatty acids synthesis (mtFAS) is a conserved metabolic pathway essential for mitochondrial respiration. The best characterized mtFAS product is the medium-chain fatty acid octanoate (C8) used as a substrate in the synthesis of lipoic acid (LA), a cofactor required by several mitochondrial enzyme complexes. In humans, mutations in the mtFAS component enoyl reductase MECR cause childhood-onset neurodegenerative disorder MEPAN. A complete deletion of Mecr in mice is embryonically lethal, while selective deletion of Mecr in cerebellar Purkinje cells causes neurodegeneration in these cells. A fundamental question in the research of mtFAS deficiency is if the defect is amenable to treatment by supplementation with known mtFAS products. Here we used the Purkinje-cell specific mtFAS deficiency neurodegeneration model mice to study if feeding the mice with a medium-chain triacylglycerol-rich formula supplemented with LA could slow down or prevent the neurodegeneration in Purkinje cell-specific Mecr KO mice. Feeding started at the age of 4 weeks and continued until the age of 9 months. The neurological status on the mice was assessed at the age of 3, 6, and 9 months with behavioral tests and the state of the Purkinje cell deterioration in the cerebellum was studied histologically. We showed that feeding the mice with medium chain triacylglycerols and LA affected fatty acid profiles in the cerebellum and plasma but did not prevent the development of neurodegeneration in these mice. Our results indicate that dietary supplementation with medium chain fatty acids and LA alone is not an efficient way to treat mtFAS disorders.

Discovery of JNJ-74856665: A Novel Isoquinolinone DHODH Inhibitor for the Treatment of AML.

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).

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.

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.

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.

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.

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.

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.