Dihydroorotate Dehydrogenase Inhibitors Research Articles

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

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.

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.

A mesoporous superparamagnetic iron oxide nanoparticle as a generic drug delivery system for tumor ferroptosis therapy

As a famous drug delivery system (DDS), mesoporous organosilica nanoparticles (MON) are degraded slowly in vivo and the degraded components are not useful for cell nutrition or cancer theranostics, and superparamagnetic iron oxide nanoparticles (SPION) are not mesoporous with low drug loading content (DLC). To overcome the problems of MON and SPION, we developed mesoporous SPIONs (MSPIONs) with an average diameter of 70 nm and pore size of 3.9 nm. Sorafenib (SFN) and/or brequinar (BQR) were loaded into the mesopores of MSPION, generating SFN@MSPION, BQR@MSPION and SFN/BQR@MSPION with high DLC of 11.5% (SFN), 10.1% (BQR) and 10.0% (SNF + BQR), demonstrating that our MSPION is a generic DDS. SFN/BQR@MSPION can be used for high performance ferroptosis therapy of tumors because: (1) the released Fe2+/3+ in tumor microenvironment (TME) can produce •OH via Fenton reaction; (2) the released SFN in TME can inhibit the cystine/glutamate reverse transporter, decrease the intracellular glutathione (GSH) and GSH peroxidase 4 levels, and thus enhance reactive oxygen species and lipid peroxide levels; (3) the released BQR in TME can further enhance the intracellular oxidative stress via dihydroorotate dehydrogenase inhibition. The ferroptosis therapeutic mechanism, efficacy and biosafety of MSPION-based DDS were verified on tumor cells and tumor-bearing mice.

Pyrimidine depletion enhances targeted and immune therapy combinations in acute myeloid leukemia.

Acute myeloid leukemia (AML) is a fatal disease characterized by the accumulation of undifferentiated myeloblasts, and agents that promote differentiation have been effective in this disease but are not curative. Dihydroorotate dehydrogenase inhibitors (DHODHi) have the ability to promote AML differentiation and target aberrant malignant myelopoiesis. We introduce HOSU-53, a DHODHi with significant monotherapy activity, which is further enhanced when combined with other standard-of-care therapeutics. We further discovered that DHODHi modulated surface expression of CD38 and CD47, prompting the evaluation of HOSU-53 combined with anti-CD38 and anti-CD47 therapies, where we identified a compelling curative potential in an aggressive AML model with CD47 targeting. Finally, we explored using plasma dihydroorotate (DHO) levels to monitor HOSU-53 safety and found that the level of DHO accumulation could predict HOSU-53 intolerability, suggesting the clinical use of plasma DHO to determine safe DHODHi doses. Collectively, our data support the clinical translation of HOSU-53 in AML, particularly to augment immune therapies. Potent DHODHi to date have been limited by their therapeutic index; however, we introduce pharmacodynamic monitoring to predict tolerability while preserving antitumor activity. We additionally suggest that DHODHi is effective at lower doses with select immune therapies, widening the therapeutic index.

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.

Abstract SY21-02: Coenzyme Q-dependent metabolism: Lessons from structure-function studies of DHODH

Abstract Ubiquinone or coenzyme Q (CoQ) is perhaps best known as a key component of the mitochondrial electron transport chain and as an endogenous antioxidant. However, CoQ has another critical function in cells, acting as an essential cosubstrate for a number of important metabolic enzymes that are associated with the inner mitochondrial membrane, including dihydroorotate dehydrogenase (DHODH), choline dehydrogenase (CHDH), proline dehydrogenase 1 and 2 (PRODH1, PRODH2), and glycerol-3-phosphate dehydrogenase 2 (GPD2). These FAD-containing mitochondrial enzymes are all hypothesized to utilize a two-step ‘ping-pong’ mechanism of catalysis. In this kinetic model, the baseline conformation of the enzyme has specific affinity for its first cosubstrate (dihydroorotate, choline, proline, glycerol-3-phosphate) but not its second cosubstrate (CoQ). Binding of the first cosubstrate to the enzyme results in oxidation of the cosubstrate and reduction of FAD to FADH2. This reduced ‘intermediate’ form of the enzyme releases the byproduct of the first reaction and then binds CoQ. Upon binding of CoQ, FADH2 is oxidized back to FAD and CoQ is reduced to CoQH2. The oxidized form of the enzyme has a low affinity for CoQH2 and CoQH2 is therefore released, restoring the enzyme to its baseline ‘empty’ conformation. Although there is considerable biochemical evidence to support this ‘double-displacement’ model of CoQ-dependent catalysis, it has been challenging to study the structures of these enzymes in their catalytically-active states due to the profound insolubility of CoQ and due to technical challenges associated with producing highly purified recombinant forms of membrane-associated proteins. Perhaps the most well-studied CoQ-dependent metabolic enzyme is DHODH. DHODH catalyzes the fourth and rate-limiting step in the de novo pyrimidine biosynthesis pathway, oxidizing dihydroorotate to orotate, which is then converted to the nucleotide precursor UMP. Due to the critical requirement of rapidly dividing cells for pyrimidines, DHODH has been proposed as a therapeutic target in cancer, and a number of highly potent and highly specific DHODH inhibitors have been developed, all of which act as competitive inhibitors of CoQ. By binding and stabilizing the protein, these inhibitors have made it possible to solve the x-ray crystal structure of DHODH. These structural studies have revealed that the inhibitors (and presumably CoQ) bind in a narrow amphipathic tunnel that spans from the membrane-embedded surface of the protein to its FAD-containing catalytic core. These structural studies have also found that DHO or orotate can bind DHODH when the CoQ tunnel is occupied by drug. Given that the ‘ping-pong’ model of catalysis is predicated on the assumption that cosubstrates and byproducts cannot simultaneously bind enzyme, it would appear that either the ping-pong model does not accurately reflect the catalytic mechanism of DHODH or, alternatively, that the structure of DHODH bound to drug is not an accurate representation of the structure of DHODH bound to CoQ. In order to gain a greater understanding of the structure of the DHODH-CoQ complex, we undertook to solve the x-ray crystal structure of DHODH in complex with decylubiquinone (dUb), a CoQ analog that can act as an electron acceptor for DHODH in vitro. These studies revealed that the x-ray crystal structure of DHODH in its catalytically-active cofactor-bound state deviates in subtle but important ways from the known structure of drug-bound DHODH, providing novel insights into the catalytic mechanism of CoQ-dependent enzymes. Citation Format: Julie-Aurore Losman. Coenzyme Q-dependent metabolism: Lessons from structure-function studies of DHODH [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr SY21-02.

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

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. 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. 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. These findings highlight DHODH as a potential therapeutic target for treating ATL.

Abstract 4456: De novo pyrimidine synthesis is a druggable vulnerability for chemotherapy resistant nasopharyngeal carcinoma with wild type IDH1

Abstract Chemotherapy resistance is a major obstacle to treat nasopharyngeal carcinoma (NPC), and strategies to overcome this resistance are required. Using proteomic analysis, we found that NPC patients resistant to gemcitabine plus cisplatin (GP) induction chemotherapy were characterized by abundant metabolic pathways. NPC with high expression of wild-type isocitrate dehydrogenase 1 (wtIDH1) was hyperdependent on de novo pyrimidine nucleotide synthesis and resistant to GP. Knockdown of IDH1 augmented the inhibition of de novo pyrimidine nucleotide synthesis induced by gemcitabine and cisplatin but reversed the suppression of purine nucleotide synthesis, which leading to DNA damage caused by nucleotide pool imbalance. IDH1 facilitated transcription of dihydroorotate dehydrogenase (DHODH), a rate-limiting enzyme for de novo pyrimidine nucleotide synthesis, through promoting chromatin accessibility by ALKBH5 in an α-ketoglutaric acid dependent manner. DHODH inhibitor, BAY2402234 significantly sensitized NPC cells to gemcitabine plus cisplatin chemotherapy. Collectively, these results reveal that wtIDH1-induced increase in de novo pyrimidine nucleotide represents a metabolic vulnerability that can be exploited pharmacologically, highlighting BAY2402234 as a promising strategy to enhance the efficacy of chemotherapy against NPC. Citation Format: Yingqin Li, Yuheng Zhao, Wuqi Zhang, Chunxian Ou, Jiaxi Shen, Jun Ma. De novo pyrimidine synthesis is a druggable vulnerability for chemotherapy resistant nasopharyngeal carcinoma with wild type IDH1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4456.

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.

Mitochondria-Targeted Prodrug Nanoassemblies for Efficient Ferroptosis-Based Therapy via Devastating Ferroptosis Defense Systems.

Ferroptosis is a form of regulated cell death accompanied by lipid reactive oxygen species (ROS) accumulation in an iron-dependent manner. However, the efficiency of tumorous ferroptosis was seriously restricted by intracellular ferroptosis defense systems, the glutathione peroxidase 4 (GPX4) system, and the ubiquinol (CoQH2) system. Inspired by the crucial role of mitochondria in the ferroptosis process, we reported a prodrug nanoassembly capable of unleashing potent mitochondrial lipid peroxidation and ferroptotic cell death. Dihydroorotate dehydrogenase (DHODH) inhibitor (QA) was combined with triphenylphosphonium moiety through a disulfide-containing linker to engineer well-defined nanoassemblies (QSSP) within a single-molecular framework. After being trapped in cancer cells, the acidic condition provoked the structural disassembly of QSSP to liberate free prodrug molecules. The mitochondrial membrane-potential-driven accumulation of the lipophilic cation prodrug was delivered explicitly into the mitochondria. Afterward, the thiol-disulfide exchange would occur accompanied by downregulation of reduced glutathione levels, thus resulting in mitochondria-localized GPX4 inactivation for ferroptosis. Simultaneously, the released QA from the hydrolysis reaction of the adjacent ester bond could further devastate mitochondrial defense and evoke robust ferroptosis via the DHODH-CoQH2 system. This subcellular targeted nanoassembly provides a reference for designing ferroptosis-based strategy for efficient cancer therapy through interfering antiferroptosis systems.

Discovery of Thiadiazoles as Human Dihydroorotate Dehydrogenase (hDHODH) Inhibitors by Combined Structure‐Based Modelling Methods

AbstractThis study involves the design of novel thiadiazole derivatives as hDHODH inhibitors using various structure‐based approaches such as pharmacophore modelling, high‐throughput virtual screening (HTVS), prediction of ADMET parameters, docking and molecular dynamics (MD) studies. We used the validated co‐crystal structure of hDHODH to create the six‐feature pharmacophore model ADHRRR, which was validated by the Güner‐Henry scoring (GH score=0.81) approach, enrichment calculations (enrichment factor=51), area under the curve (0.93), receiving operating characteristic (ROC) curve (0.89), and ROC′s Boltzmann‐enhanced discrimination, known as BEDROC (α=8) (0.859), (α=20) (0.828), (α=160) (0.912). Pharmacophore model as a 3D search query was used for the screening of zinc database followed by docking investigations and energy calculation (Prime/MMGBSA). For the design of novel compounds, we selected the thiadiazole moiety as the central scaffold and various functional groups based on pharmacophoric characters and binding interactions. In docking study, designed compounds showed favourable interactions with Gln47 and Arg136 at the active site of the hDHODH enzyme, and optimal drug likeness properties for ADMET prediction study. Stability of enzyme‐ligand complex was validated by MD simulation study with RMSD, RMSF and RoG calculations. These findings suggested that novel thiadiazoles are potential candidates as hDHODH inhibitors and anticancer agents.

Safety and Tolerability of KIO-101 Eye Drops in Healthy Volunteers and Patients with Ocular Surface Disease-A Phase I Study.

Inhibitors of dihydroorotate dehydrogenase (DHODH) have been found to be potent anti-inflammatory agents. Recently, a topical formulation (KIO-101 eye drops) of a DHODH inhibitor has been developed. The aim of the present study was to evaluate the safety and tolerability of KIO-101 eye drops in Healthy Volunteers (HVs) and patients with conjunctival hyperemia. The study was carried out in a double-masked, placebo-controlled, randomized, parallel-group design with two parts. In part I, HVs received single and multiple instillations (four times daily for 12 consecutive days) of KIO-101 eye drops in ascending doses of 0.05%, 0.15%, and 0.30%, respectively. Part II was conducted in patients with conjunctival hyperemia who received 0.15% KIO-101 eye drops twice daily for 12 consecutive days. Ophthalmic and systemic safety examinations were performed on all participants. In part II, ocular hyperemia grading and an ocular surface disease index (OSDI) questionnaire were performed. 24 HVs participated in part I and 21 patients in part II. KIO-101 eye drops were well tolerated in all subjects. No serious adverse events (SAEs) occurred, and all AEs that were reported were transient and considered mild to moderate. In the highest dose cohort (0.30%), epistaxis occurred in two subjects after multiple instillations. In part II, after 12 days treatment with 0.15% KIO-101, conjunctival hyperemia decreased by -1.1 ± 0.27 points in the treatment and -0.6 ± 0.79 points in the placebo group (p = 0.0385). OSDI decreased from 47.9 ± 18.7 to 27.6 ± 19.13 points in the treatment group, while in the placebo group, a change from 41.3 ± 12.08 to 27.3 ± 18.63 points occurred. A 12-day treatment regimen with topical KIO-101 eye drops at low and mid doses was safe and well tolerated in both HVs and patients with conjunctival hyperemia. The obtained results point towards an early sign of reduction in conjunctival hyperemia.

A novel mitochondria-targeting DHODH inhibitor induces robust ferroptosis and alleviates immune suppression

Dihydroorotate dehydrogenase (DHODH)-mediated ferroptosis defense is a targetable vulnerability in cancer. Currently, only a few DHODH inhibitors have been utilized in clinical practice. To further enhance DHODH targeting, we introduced the mitochondrial targeting group triphenylphosphine (TPP) to brequinar (BRQ), a robust DHODH inhibitor, resulting in the creation of active molecule B2. This compound exhibits heightened anticancer activity, effectively inhibiting proliferation in various cancer cells, and restraining tumor growth in melanoma xenografts in mice. B2 achieves these effects by targeting DHODH, triggering the formation of reactive oxygen species (ROS), promoting mitochondrial lipid peroxidation, and inducing ferroptosis in B16F10 and A375 cells. Surprisingly, B2 significantly downregulates PD-L1 and alleviates immune suppression. Importantly, B2 exhibits no apparent adverse effects in mice. Collectively, these findings highlight that enhancing the mitochondrial targeting capability of the DHODH inhibitor is a promising therapeutic approach for melanoma treatment.

Discovery of Alternative Binding Poses through Fragment-Based Identification of DHODH Inhibitors.

Dihydroorotate dehydrogenase (DHODH) is a mitochondrial enzyme that affects many aspects essential to cell proliferation and survival. Recently, DHODH has been identified as a potential target for acute myeloid leukemia therapy. Herein, we describe the identification of potent DHODH inhibitors through a scaffold hopping approach emanating from a fragment screen followed by structure-based drug design to further improve the overall profile and reveal an unexpected novel binding mode. Additionally, these compounds had low P-gp efflux ratios, allowing for applications where exposure to the brain would be required.

Abstract B085: K17-induced pyrimidine biosynthesis drives chemoresistance in PDAC

Abstract Background: The 5-year survival rate of pancreatic cancer is extremely low compared with other cancers, although it has increased to 12% in 2023 in the United States. 80% of pancreatic ductal adenocarcinoma (PDAC) patients are not eligible for surgical procedure, and unfortunately, PDAC is refractory to standard-of-care chemotherapy. Thus, developing novel and more effective treatment strategies for PDAC is urgently needed. We previously reported keratin 17 (K17) as a novel negative prognostic and predictive biomarker, whose overexpression confers the resistance to chemotherapies. Here, we investigated the mechanisms of chemoresistance and tumor-specific vulnerabilities that can be exploited for K17-mediated targeted therapies. Methods: Survival analyses, immunohistochemistry (IHC), and matrix assisted laser desorption/ionization mass spectrometry (MALDI MSI) metabolite imaging were applied in patient derived samples. The K17 stable expression cell line models (KPC and PANC-1) and K17 loss-of-function cell line model (L3.6) were generated, where the analyses were applied including western blots, qRT-PCR, mass spectrometric metabolomics analysis, immunofluorescence imaging, cell viability and proliferation assays. KPC cells with or without K17 expression were orthotopically implanted into the head of the pancreas of c57B6J mice for tumor growth and treatment study. Results: Unbiased metabolomic studies in isogenic PDAC models identified several key metabolic pathways that are upregulated in the presence of K17. We demonstrate that K17 increases pyrimidine biosynthesis, a pathway that drives chemoresistance. Patient dataset analysis revealed that K17 expression and enzymes involved in pyrimidine, but not purine, de novo biosynthesis is associated with shorter patient survival. Furthermore, K17-expressing cells were more sensitive to Brequinar, a specific inhibitor of dihydroorotate dehydrogenase (DHODH), the rate-limiting enzyme in de novo pyrimidine biosynthetic pathway. Targeting DHODH by small interfering RNA or by Brequinar with Gemcitabine synergistically inhibited the viability of K17- positive cells. Importantly, the combination of Gemcitabine and Brequinar significantly inhibited the growth of K17-expressing tumors and extended survival of mice bearing K17-positive tumors. Mechanistically, K17 increased mitochondrial DHODH translocation which made these cells more sensitive to Brequinar. We also found that the coil 2 domain of K17 was crucial for triggering metabolic reprogramming and promoting tumor aggression. Conclusions: Overall, we define a novel connection between K17, nucleotide metabolism, and chemoresistance in PDAC. We report for the first time that K17, a negative biomarker of the most aggressive PDAC subtype, drives chemoresistance by up-regulating de novo pyrimidine biosynthesis, which provides a tumor-specific vulnerability: DHODH. Further study could lead to the development of a DHODH targeted therapy for K17-expressing PDAC. Citation Format: Yinghuan Lyu, Kenneth Shroyer, Chun-Hao Pan, Robert Tseng, Md Afjalus Siraj, Girish Halemirle Rajacharya, Bo Chen, Katie L Donnelly, Michael Horowitz, Carlos Mauricio Mejia Arbelaez, Sumedha Chowdhury, Lyanne Oblein, Natalia Marchenko, Luisa Escobar-Hoyos, Pankaj K. Singh. K17-induced pyrimidine biosynthesis drives chemoresistance in PDAC [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B085.

Discovery of Nanomolar Inhibitors for Human Dihydroorotate Dehydrogenase Using Structure-Based Drug Discovery Methods.

We used a structure-based drug discovery approach to identify novel inhibitors of human dihydroorotate dehydrogenase (DHODH), which is a therapeutic target for treating cancer and autoimmune and inflammatory diseases. In the case of acute myeloid leukemia, no previously discovered DHODH inhibitors have yet succeeded in this clinical application. Thus, there remains a strong need for new inhibitors that could be used as alternatives to the current standard-of-care. Our goal was to identify novel inhibitors of DHODH. We implemented prefiltering steps to omit PAINS and Lipinski violators at the earliest stages of this project. This enriched compounds in the data set that had a higher potential of favorable oral druggability. Guided by Glide SP docking scores, we found 20 structurally unique compounds from the ChemBridge EXPRESS-pick library that inhibited DHODH with IC50,DHODH values between 91 nM and 2.7 μM. Ten of these compounds reduced MOLM-13 cell viability with IC50,MOLM-13 values between 2.3 and 50.6 μM. Compound 16 (IC50,DHODH = 91 nM) inhibited DHODH more potently than the known DHODH inhibitor, teriflunomide (IC50,DHODH = 130 nM), during biochemical characterizations and presented a promising scaffold for future hit-to-lead optimization efforts. Compound 17 (IC50,MOLM-13 = 2.3 μM) was most successful at reducing survival in MOLM-13 cell lines compared with our other hits. The discovered compounds represent excellent starting points for the development and optimization of novel DHODH inhibitors.

In-silico studies of hydroxyxanthone derivatives as potential pfDHFR and pfDHODH inhibitor by molecular docking, molecular dynamics simulation, MM-PBSA calculation and pharmacokinetics prediction

The investigation of hydroxyxanthone derivatives has been conducted, including molecular docking, molecular dynamics simulation MM-PBSA binding energy calculation, and pharmacokinetics prediction of the potential plasmodium falciparum dihydrofolate reductase (pfDHFR) and plasmodium falciparum dihydroorotate dehydrogenase (pfDHODH) inhibitor. The Docking result showed that compound 1,3,6,7-tetrahydroxy-5,8-bis(3-methyl-2-buten-1-yl)-9H-xanthen-9-one (X16) was found to be the best ligand with good inhibitory action against pfDHFR. Meanwhile, the pfDHODH protein was compounded 1,3,6,7-tetrahydroxy-5,8-dinitro-9H-xanthen-9-one (X14). Additionally, the hydroxyxanthone X16 complex showed more excellent stability in the molecular dynamics simulation of the pfDHFR protein than the ligand WR99210 and chloroquine. The MM-PBSA calculation showed that compound X16 had lower binding energy than ligand WR99210. However, 1,3-dihydroxy-8-(3-methyl-2-buten-1-yl)-9H-xanthen-9-one (X4), 1,3,6,7-tetrahydroxy-8-nitro-9H-xanthen-9-one (X10), 1,3,6,7-tetrahydroxy-9-oxo-9H-xanthene-8-sulfonic acid (X11), and 1,3,6,7-tetrahydroxy-5,8-dinitro-9H-xanthen-9-one (X14) complexes were shown to be more stable than chloroquine and to have the same stability when compared to the native ligand A26, according to a molecular dynamics simulation conducted in pfDHODH protein. The MM-PBSA calculation showed that compound X14 had lower binding energy than ligand A26. The hydroxyxanthones X4, X10–11, X14, and X16 fulfill Lipinski's rule parameters in terms of physicochemical and ADMET qualities and parameters related to absorption, distribution, metabolism, excretion, and toxicity tests. To sum up, hydroxyxanthones X4, X10–11, X14, and X16 have the potential to be antimalarial medications, but more in vivo and in vitro testing is needed to confirm this.

Identification of dihydroorotate dehydrogenase inhibitor, vidofludimus, as a potent and novel inhibitor for influenza virus.

Influenza A virus (IAV) infection causes respiratory disease. Recently, infection of IAV H5N1 among mammals are reported in farmed mink. Therefore, to discover antivirals against IAV, we screened a compound library by using the RNA-dependent RNA polymerase (RdRp) assay system derived from H5N1 IAV including a drug-resistant PA mutant (I38T) and a viral polymerase activity enhancing PB2 mutant (T271A). Upon screening, we found vidofludimus can be served as a potential inhibitor for IAV. Vidofludimus an orally active inhibitor for dihydroorotate dehydrogenase (DHODH), a key enzyme for the cellular de novo pyrimidine biosynthesis pathway. We found that vidofludimus exerted antiviral activity against wild-type and drug-resistant mutant IAV, with effective concentrations (EC50 ) of 2.10 and 2.11 μM, respectively. The anti-IAV activity of vidofludimus was canceled by the treatment of uridine or cytidine through pyrimidine salvage synthesis pathway, or orotic acid through pyrimidine de novo synthesis pathway. This indicated that the main target of vidofludimus is DHODH in IAV RdRp expressing cells. We also produced recombinant seasonal IAV H1N1 virion and influenza B virus (IBV) RdRp assay system and confirmed vidofludimus also carried highly antiviral activity against seasonal IAV and IBV. Vidofludimus is a candidate drug for the future threat of IAV H5N1 infection among humans as well as seasonal influenza virus infection.

PENDEKATAN KOMPUTASI UNTUK MENGATASI MALARIA DARI Coscinium fenestratum

Malaria is a disease with signs of fever and is caused by Plasmodium. One way to inhibit the growth of plasmodium is using Dihydroorotate dehydrogenase inhibitors. The method used in this study was docking with laptop aspire ES 14 equipment and PLANTS software for docking and the Discovery Studio for visualization and the materials used were compounds contained in C. fenestratum. This research was carried out using protein code 6VTY which removed water molecules and ligands that were not linked to the active site, and continued with the docking of the compounds contained in C. fenestratum. The results of this study are the Berberine docking score -82.2408; Berberrubine -84.6866 and Thalifendine -81.1591, the result of ligand interaction with Dihydroorotate dehydrogenase, the three ligands occupy the catalytic site of the enzyme, namely HIS185, PHE227, ARG 265 and CYS 175. The conclusion in this study is Berberrubine with a docking score of -82.488 is compounds that have the potential as Dihydroorotate dehydrogenase inhibitors

Glutathione depletion and dihydroorotate dehydrogenase inhibition actuated ferroptosis-augment to surmount triple-negative breast cancer

Ferroptosis is a promising therapeutic approach for combating malignant cancers, but its effectiveness is limited in clinical due to the adaptability and self-repair abilities of cancer cells. Mitochondria, as the pivotal player in ferroptosis, exhibit tremendous therapeutic potential by targeting the intramitochondrial anti-ferroptotic pathway mediated by dihydroorotate dehydrogenase (DHODH). In this study, an albumin-based nanomedicine was developed to induce augmented ferroptosis in triple-negative breast cancer (TNBC) by depleting glutathione (GSH) and inhibiting DHODH activity. The nanomedicine (ATO/SRF@BSA) was developed by loading sorafenib (SRF) and atovaquone (ATO) into bovine serum albumin (BSA). SRF is an FDA-approved ferroptosis inducer and ATO is the only drug used in clinical that targets mitochondria. By combining the effects of SRF and ATO, ATO/SRF@BSA promoted the accumulation of lipid peroxides within mitochondria by inhibiting the glutathione peroxidase 4 (GPX4)-GSH pathway and downregulating the DHODH-coenzyme Q (CoQH2) defense mechanism, triggers a burst of lipid peroxides. Simultaneously, ATO/SRF@BSA suppressed cancer cell self-repair and enhanced cell death by inhibiting the synthesis of adenosine triphosphate (ATP) and pyrimidine nucleotides. Furthermore, the anti-cancer results showed that ATO/SRF@BSA exhibited tumor-specific killing efficacy, significantly improved the tumor hypoxic microenvironment, and lessened the toxic side effects of SRF. This work presents an efficient and easily achievable strategy for TNBC treatment, which may hold promise for clinical applications.