DHODH Inhibitors Research Articles

Identification of isoquinolinone DHODH inhibitor isosteres

DHODH inhibition represents an attractive approach to overcome differentiation blockade for the treatment of AML. In a previous communication, we described our efforts leading to the discovery of compound 3 (JNJ-74856665), an orally bioavailable, potent, and selective DHODH inhibitor for clinical development. Guided by the co-crystal structures bound to human DHODH, other fused six-membered constructs were explored as isosteric replacements of the isoquinolinone central core. The correct positioning of the nitrogen in these core systems proved to be essential in modulating potency. Herein is described the synthesis of these complexly functionalized cores and their profiling, leading to DHODH inhibitors that possess favorable properties suitable for further development.

51223 A novel human disease model of alopecia areata to evaluate benefit of the DHODH inhibitor farudodstat

51223 A novel human disease model of alopecia areata to evaluate benefit of the DHODH inhibitor farudodstat

A Patent Review of Human Dihydroorotate Dehydrogenase (hDHODH) Inhibitors as Anticancer Agents and their Other Therapeutic Applications (1999-2022).

Highly proliferating cells, such as cancer cells, are in high demand of pyrimidine nucleotides for their proliferation, accomplished by de novo pyrimidine biosynthesis. The human dihydroorotate dehydrogenase (hDHODH) enzyme plays a vital role in the rate-limiting step of de novo pyrimidine biosynthesis. As a recognised therapeutic target, hDHODH plays a significant role in cancer and other illness. In the past two decades, small molecules as inhibitors hDHODH enzyme have drawn much attention as anticancer agents, and their role in rheumatoid arthritis (RA), and multiple sclerosis (MS). In this patent review, we have compiled patented hDHODH inhibitors published between 1999 and 2022 and discussed the development of hDHODH inhibitors as anticancer agents. Therapeutic potential of small molecules as hDHODH inhibitors for the treatment of various diseases, such as cancer, is very well recognised. Human DHODH inhibitors can rapidly cause intracellular uridine monophosphate (UMP) depletion to produce starvation of pyrimidine bases. Normal cells can better endure a brief period of starvation without the side effects of conventional cytotoxic medication and resume synthesis of nucleic acid and other cellular functions after inhibition of de novo pathway using an alternative salvage pathway. Highly proliferative cells such as cancer cells do not endure starvation because they are in high demand of nucleotides for cell differentiation, which is fulfilled by de novo pyrimidine biosynthesis. In addition, hDHODH inhibitors produce their desired activity at lower doses rather than a cytotoxic dose of other anticancer agents. Thus, inhibition of de novo pyrimidine biosynthesis will create new prospects for the development of novel targeted anticancer agents, which ongoing preclinical and clinical experiments define. Our work brings together a comprehensive patent review of the role of hDHODH in cancer, as well as various patents related to the hDHODH inhibitors and their anticancer and other therapeutic potential. This compiled work on patented DHODH inhibitors will guide researchers in pursuing the most promising drug discovery strategies against the hDHODH enzyme as anticancer agents.

Abstract 5787: Structure-function mapping of DHODH shows deviations in protein structure

Abstract Dihydroorotate dehydrogenase (DHODH) is a CoQ-dependent enzyme that converts dihydroorotate to orotate in the de novo pyrimidine biosynthesis pathway. Multiple structures of DHODH in complex with various small molecule inhibitors have been solved that indicate that DHODH inhibitors bind in the CoQ binding tunnel of DHODH and act as competitive inhibitors of CoQ. We performed saturating mutagenesis screens to identify mutations in DHODH that confer resistance to brequinar and BAY2402234, two highly potent DHODH inhibitors. As expected, many of the resistance mutations localized to the drug/CoQ-binding tunnel of the enzyme. Among the top-scoring drug-binding-domain mutants in both the brequinar and BAY2402234 screens were substitutions A58H and A58T. In silico analysis predicted that A58H should impair DHODH binding to both brequinar and BAY2402234. However, A58T was predicted to only impair binding of DHODH to brequinar. We expressed the mutants in a DHODH inhibitor sensitive cell line and confirmed that they both confer resistance to brequinar and BAY2402234, and we then assessed the ability of the mutants to bind drug by cellular thermal shift assay (CETSA). We found that, as predicted, DHODH A58H did not bind brequinar or BAY2402234. However, to our surprise, we detected binding of both brequinar and BAY2402234 to DHODH A58T. We generated recombinant DHODH A58T and confirmed that the purified protein was enzymatically active and strongly drug-resistant by DCIP assay and then assessed binding of the mutant protein to brequinar and BAY2402234 by thermal shift assay (TSA). Consistent with our CETSA results, recombinant DHODH A58T bound both brequinar and BAY2402234 in vitro. These results suggested that DHODH A58T can simultaneously bind drug and CoQ. However, previously reported DHODH structures indicated that the drug/CoQ binding tunnel is quite narrow and rigid. To determine whether the structure of catalytically-active DHODH is more flexible than inhibitor-bound catalytically-inactive DHODH, we co-crystallized wild-type DHODH with decylubiquinone (DCU), a CoQ analog that can act as a DHODH co-substrate in vitro. We found that the structure of DCU-bound DHODH differs from previously published DHODH structures in two important ways: (1) there appears to be a second point of ingress into the active site of DHODH next to the CoQ tunnel that may be able to accommodate a highly non-polar molecule such as CoQ/DCU and (2) the ‘flap’ covering the DHO/orotate ingress/egress region of DHODH, which is ‘closed’ in inhibitor-bound DHODH, is ‘open’ in DCU-bound DHODH. We are currently working to solve the crystal structure of DHODH A58T bound to inhibitor and DCU to ask if DCU can access the active site of DHODH through an alternative hydrophobic tunnel and if DHODH A58T remains in the ‘open’ configuration when bound to drug. Citation Format: Ruchika Pokhriyal, Laura Evans, Hyuk-Soo Seo, Jillian K. O'Neil, Betty Rouaisnel, Atanas Kamburov, Judith Guenther, Stefan Gradl, Haribabu Arthanari, Sven Christian, Xiaoping Yang, Sirano Dhe-Paganon, Julie-Aurore Losman. Structure-function mapping of DHODH shows deviations in protein structure [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 5787.

Menaquinone-4 attenuates ferroptosis by upregulating DHODH through activation of SIRT1 after subarachnoid hemorrhage

Background: Menaquinone-4(MK-4), the isoform of vitamin K2 in the brain, exerts neuroprotective effects against a variety of central nervous system disorders. This study aimed to demonstrate the anti-ferroptosis effects of MK-4 in neurons after SAH. Methods: A subarachnoid hemorrhage (SAH) model was prepared by endovascular perforation in mice. In vitro hemoglobin stimulation of primary cortical neurons mimicked SAH. MK-4, Brequinar (BQR, DHODH inhibitor), and Selisistat (SEL, SIRT1 inhibitor) were administered, respectively. Subsequently, WB, immunofluorescence was used to determine protein expression and localization, and transmission electron microscopy was used to observe neuronal mitochondrial structure while other indicators of ferroptosis were measured. Results: MK-4 treatment significantly upregulated the protein levels of DHODH; decreased GSH, PTGS2, NOX1, ROS, and restored mitochondrial membrane potential. Meanwhile, MK-4 upregulated the expression of SIRT1 and promoted its entry into the nucleus. BQR or SEL partially abolished the protective effect of MK-4 on, neurologic function, and ferroptosis. Conclusions: Taken together, our results suggest that MK-4 attenuates ferroptosis after SAH by upregulating DHODH through the activation of SIRT1.

90 Exploring the therapeutic potential of DHODH inhibitor farudodstat for alopecia areata treatment in a novel ex vivo model of human hair follicle immune privilege collapse

90 Exploring the therapeutic potential of DHODH inhibitor farudodstat for alopecia areata treatment in a novel ex vivo model of human hair follicle immune privilege collapse

Blockade of de novo pyrimidine biosynthesis triggers autophagic degradation of oncoprotein FLT3-ITD in acute myeloid leukemia.

The internal tandem duplication of the FMS-like tyrosine kinase 3 (FLT3-ITD) is one of the most frequent genetic alterations in acute myeloid leukemia (AML). Limited and transient clinical benefit of FLT3 kinase inhibitors (FLT3i) emphasizes the need for alternative therapeutic options for this subset of myeloid malignancies. Herein, we showed that FLT3-ITD mutant (FLT3-ITD+) AML cells were susceptible toward inhibitors of DHODH, a rate-limiting enzyme of de novo pyrimidine biosynthesis. Genetic and pharmacological blockade of DHODH triggered downregulation of FLT3-ITD protein, subsequently suppressed activation of downstream ERK and STAT5, and promoted cell death of FLT3-ITD+ AML cells. Mechanistically, DHODH blockade triggered autophagy-mediated FLT3-ITD degradation via inactivating mTOR, a potent autophagy repressor. Notably, blockade of DHODH synergized with an FDA-approved FLT3i quizartinib in significantly impairing the growth of FLT3-ITD+ AML cells and improving tumor-bearing mice survival. We further demonstrated that DHODH blockade exhibited profound anti-proliferation effect on quizartinib-resistant cells in vitro and in vivo. In summary, this study demonstrates that the induction of degradation of FLT3-ITD protein by DHODH blockade may offer a promising therapeutic strategy for AML patients harboring FLT3-ITD mutation.

LB1777 A novel ex vivo model of human hair follicle immune privilege collapse reveals the potential of farudodstat, a DHODH inhibitor, as a therapeutic for alopecia areata treatment

LB1777 A novel ex vivo model of human hair follicle immune privilege collapse reveals the potential of farudodstat, a DHODH inhibitor, as a therapeutic for alopecia areata treatment

Design and Synthesis of Isobavachalcone Derivatives as Potential Dihydroorotate Dehydrogenase Inhibitors

AbstractDihydroorotate dehydrogenase (DHODH) inhibitors have been reported to exhibit multi‐therapeutic potential due to their various pharmacological effects. As a result, the design and development of novel DHODH inhibitors is both necessary and an intriguing topic. Previous studies have demonstrated that Isobavachalcone, a natural compound isolated from Psoralea corylifolia, can efficiently inhibit the DHODH activity. Three primary series, designated A, B and C, were designed and synthesized based on the lead compound Isobavachalcone. The inhibitory activities of these compounds were evaluated using the Orotate Production Assay method, and the corresponding structure‐activity relationship (SAR) were thoroughly examined. Additionally, the interactions between the best active compounds and the DHODH protein were analyzed through a molecular docking study. These findings suggest that these derivatives have the potential to be promising candidates for the development of a new family of DHODH inhibitors.

Identification of Potent hDHODH Inhibitors for Lung Cancer via Virtual Screening of a Rationally Designed Small Combinatorial Library.

Cancer is characterized by altered cellular metabolism, and metabolic enzymes are considered as a promising target for anticancer therapy. Pyrimidine metabolism dysregulation is associated with various types of cancer, particularly lung cancer, which is one of the leading causes of cancer-related mortality worldwide. Recent studies have shown that small-cell lung cancer cells are particularly reliant on the pyrimidine biosynthesis pathway and are sensitive to its disruption. DHODH, the rate-limiting enzyme of the de novo pyrimidine production pathway, is essential in the production of RNA and DNA and is overexpressed in malignancies such as AML, skin cancer, breast cancer, and lung cancer, thereby highlighting DHODH as a viable target for developing drugs to combat lung cancer. Herein, rational drug design and computational techniques were used to discover novel DHODH inhibitors. A small combinatorial library was generated, and the top hits were synthesized and tested for anticancer activity against three lung cancer cell lines. Among the tested compounds, compound 5c possessed a stronger cytotoxicity (TC50 of 11 μM) compared to the standard FDA-approved drug (Regorafenib, TC50 of 13 μM) on the A549 cell line. Furthermore, compound 5c demonstrated potent inhibitory activity against hDHODH at a nanomolar level of 421 nM. DFT, molecular docking, molecular dynamic simulations, and free energy calculations were also carried out to understand the inhibitory mechanisms of the synthesized scaffolds. These in silico studies identified key mechanisms and structural features that will be crucial for future studies.

Depletion of SOD2 enhances nasopharyngeal carcinoma cell radiosensitivity via ferroptosis induction modulated by DHODH inhibition

BackgroundRecurrence due to the development of radioresistance remains a major challenge in the clinical management of nasopharyngeal carcinoma. The objective of this study was to increase the sensitivity of nasopharyngeal carcinoma cells to ionizing radiation by enhancing oxidative stress and ferroptosis caused by disrupting the mitochondrial anti-oxidant enzyme system.MethodsOxidative stress cell model was constructed by SOD2 knockdown using shRNA. The expression and activity of DHODH was suppressed by siRNA and brequinar in SOD2 depleted cells. Protein levels were determined by western blotting and ferroptosis was assessed by C11 BODIPY and malondialdehyde assay. Cell viability was evaluated using CCK-8 assay while radiotoxicity was assessed by colony formation assay. Cellular ATP level was determined by ATP assay kits, ROS was determined by DCFD and DHE, while mitochondrial oxygen consumption was determined by seahorse assay. Data were analyzed by two-tailed independent t-test.ResultsRadiation upregulated SOD2 expression and SOD2 depletion increased cellular O2.−, malondialdehyde, and the fluorescence intensity of oxidized C11 BODIPY. It also resulted in mitochondrial damage. Its depletion decreased colony formation both under ionizing and non-ionizing radiation conditions. The ferroptosis inhibitor, deferoxamine, rescued cell viability and colony formation in SOD2 depleted cells. Cellular level of malondialdehyde, fluorescence intensity of oxidized C11 BODIPY, O2.− level, ATP, and mitochondrial oxygen consumption decreased following DHODH inhibition in SOD2 depleted cells. Cell viability and colony formation was rescued by DHODH inhibition in SOD2 depleted cells.ConclusionInducing oxidative stress by SOD2 inhibition sensitized nasopharyngeal carcinoma cells to ionizing radiation via ferroptosis induction. This was found to be dependent on DHODH activity. This suggests that DHODH inhibitors should be used with caution during radiotherapy in nasopharyngeal carcinoma patients.

Preclinical Development of Hosu-53, a Novel Potent Dihydroorotate Dehydrogenase Inhibitor with Impressive Single Agent Efficacy and Combination Approaches in Hematological Malignancy

Common FDA approved strategies for hematological malignancies include chemotherapy, targeted therapies, immune therapies, hypomethylating agents, and cell-based therapeutics. However, recurrence often occurs resulting in poor prognosis particularly in acute myeloid leukemia (AML) with first relapse and multiple myeloma (MM) following several relapses based upon more therapies being available. The emergence of dihydroorotate dehydrogenase as an appealing mechanistic target in AML has propelled the development of several clinical candidate DHODH inhibitors (DHODHi) such as brequinar. Here we describe HOSU-53, a novel DHODHi with favorable oral bioavailability (>50% in mouse, rat, and dog) and a pharmacodynamic (PD) biomarker for target engagement, enabling clinical in vivo monitoring of potency and on-target anti-tumor activity. Here we describe the development of a series of DHODHi through a collaboration between Hendrix College and The Ohio State University (OSU). Our initial screen of 18 novel compounds synthesized at Hendrix College identified HOSU-3 as a potent DHODHi with efficacy equivalent to brequinar, however in vivo efficacy in a MOLM-13 AML xenograft model revealed further optimization would be required to compete with leading clinical candidates. Further structure activity relationship studies and lead optimization performed in collaboration with the Drug Development Institute at OSU led to screening over 200 additional analogs and the development of three additional compounds (HOSU-28, -49 and -53). HOSU-53 has subnanomolar biochemical activity against human DHODH in a cell-free enzyme assay, with a cellular IC50 ranging from 2 - 45 nM across a panel of AML cell lines, and 12-42 nM in MM cell lines. While HOSU-53 is highly plasma protein bound, it exhibits a moderate serum shift in assays using human plasma proteins with an IC50 of 457 nM, well below the in vivo Cmax and AUC achieved in rodent and canine pharmacokinetic (PK) studies (Cmax = 79.4-210 μM and AUC = 997-7961 μM-h). Additional absorption, distribution, metabolic and excretion (ADME) and safety studies revealed that HOSU-53 displays very favorable drug-like properties sufficient for development as a pre-clinical candidate. A MOLM-13 xenograft study verified that HOSU-53 was a potential new best in class DHODHi, with efficacy superior to the Bayer clinical candidate. We performed a follow-up study to determine the efficacy of dosing daily at 4, 10, and 20 mg/kg versus 30 mg/kg twice a week. Correlative PK/PD studies were also conducted to measure plasma HOSU-53 and DHO levels at day 1 and 14. We observed a dose linear relationship between HOSU-53 plasma concentration and DHO accumulation. Collectively, our efficacy and PK/PD studies indicate that 10 mg/kg is a highly efficacious and tolerable daily regimen, while higher doses can be safely administered intermittently, and DHO accumulation serves as an important plasma biomarker to identify in vivo therapeutic responses and toxicity. Confirmatory studies in rat subcutaneous MOLM-13 and MV4-11 xenograft models further demonstrated the efficacy of HOSU-53. Our group has previously described synergy between HOSU-53 and decitabine in AML, as well as monoclonal antibodies directed to CD38 in AML and MM. Furthermore, a novel combination between HOSU-53 and CD47 antibody is described in a separate abstract at this current meeting. We expanded our combination studies by testing HOSU-53 with another standard of care, the FLT3 inhibitor gilteritinib, using the MOLM-13 FLT3-ITD mutant xenograft model. NCG mice were enrolled ten days post engraftment to receive daily 4 mg/kg HOSU-53, 30 mg/kg gilteritinib or the combination. Single agent HOSU-53 and gilteritinib prolonged survival (median survival of 37 and 47 days, respectively) while the combination significantly improved survival with 9/10 mice in the combination group remaining on study at day 80. Collectively, we provide a thorough preclinical assessment for HOSU-53 for treatment in hematological malignancy both as a monotherapy and combined with other effective approved therapies including decitabine, monoclonal antibodies and gilteritinib. We have initiated IND enabling studies including GLP toxicity studies planned for late 2022 and have initiated human dose projection models based on our multi-species PK/PD analysis. We anticipate filing an IND application in 2023 for a phase 1 clinical trial in AML and MM.

Gene Editing and Multi-Omics Reveal Novel Metabolic Vulnerabilities in t(4;14)-Associated Multiple Myeloma

Background: Chromosomal translocation t(4;14) is a recurrent genetic lesion in multiple myeloma (MM) which drives aberrant overexpression of the histone methyltransferase NSD2 and is associated with a more guarded prognosis. Despite its oncogenic outcomes, NSD2 overexpression in MM cells could simultaneously confer cellular vulnerabilities that can be therapeutically exploited. Aim: To define potential therapeutic susceptibilities driven by NSD2 overexpression in t(4;14) MM. Methods: We performed genome-wide knockout screening in isogenic NSD2 high and low KMS11 cells using the Brunello CRISPR library, followed by validation of key target genes in several different t(4;14) MM cell lines. Results: These screens revealed increased reliance of NSD2-overexpressing cells on multiple cellular processes including mitochondrial respiration, transcriptional elongation and proteasomal degradation. One of the top preferential dependencies in NSD2-high MM cells is the mitochondrial adenine nucleotide regulator adenylate kinase 2 (AK2). AK2 catalyzes the reversible interconversion of ADP to ATP and AMP. Cancer Dependency Map (depmap) analysis indicated that AK2 loss is detrimental to most MM cell lines but it is not a dependency in most solid tumor cells. Reliance on AK2 was modest in t(4;14) MM cells engineered to express low levels of NSD2 but the detrimental effect of AK2 depletion by shRNA or CRISPR was exacerbated upon enforced overexpression of NSD2. Gene expression studies showed that AK2 suppression leads to induction of genes involved in oxidative stress response. Metabolomic profiling of NSD2 high and low isogenic MM cells demonstrated striking reduction of NAD+ and elevation of nicotinamide (NAM) levels in NSD2 overexpressing cells, which correlates with a 10-fold induction of expression of the NAD+ consuming enzyme CD38 by NSD2. NAD+ and its metabolite NADP+ are essential co-factors of multiple antioxidant enzymes and its depletion can exacerbate oxidative damage. Therefore, NAD+ depletion likely accounts for the increased susceptibility of NSD2 high MM cells to AK2 loss. In vitro cell proliferation/viability assays demonstrated increased sensitivity of NSD2 high MM cells to PI exposure suggesting that NSD2 overexpression drives a higher load of unfolded proteins, correlating with loss of NAD+ and redox system activity. AK2 suppression in MM cells also impairs activation of the adaptive unfolded protein response through the IRE1a-XBP1 axis leading to decreased protein folding capacity. The combination of high unfolded protein load and poor ATP shuttling to the endoplasmic reticulum (ER) secondary to AK2 loss may represent an additional mechanism of selective lethality. Furthermore, AK2 deficient MM cells displayed increased sensitivity to PIs. Transcriptomic analyses in NSD2 high and low MM cells revealed that NSD2 overexpression, besides driving an oncogenic transcriptional program, alters the expression of several genes involved in nucleotide metabolism. Perturbed nucleotide metabolism was further documented by untargeted metabolite profiling which showed a notable depletion of several pyrimidine metabolites when NSD2 is overexpressed. Intriguingly, induced reduction of pyrimidine synthesis by treatment with DHODH inhibitors appeared to be more detrimental in NSD2 overexpressing MM cells. Conclusions: NSD2 overexpression in MM cells alters cellular metabolism and confers higher susceptibility to oxidative stress and unfolded protein loads. By inducing the expression of NAD+ consuming enzymes, NSD2 overexpression results in NAD+ depletion which likely underlies the described vulnerabilities. Loss of the mitochondrial adenine nucleotide regulator AK2 is more detrimental to NSD2 overexpressing MM cells as it diminishes efficient ATP utilization in the ER perturbing the ability of MM cells to handle high loads of unfolded immunoglobulins and mitigate oxidative stress. Furthermore, integrated analyses of the different omics datasets highlighted other NSD2-driven liabilities, including mitochondrial respiration, transcriptional elongation and nucleotide metabolism, that can be targeted to improve prognosis of MM.

Structure-Based Prototyping of Allosteric Inhibitors of Human Uridine/Cytidine Kinase 2 (UCK2).

Pyrimidine nucleotide biosynthesis in humans is a promising chemotherapeutic target for infectious diseases caused by RNA viruses. Because mammalian cells derive pyrimidine ribonucleotides through a combination of de novo biosynthesis and salvage, combined inhibition of dihydroorotate dehydrogenase (DHODH; the first committed step in de novo pyrimidine nucleotide biosynthesis) and uridine/cytidine kinase 2 (UCK2; the first step in salvage of exogenous nucleosides) strongly attenuates viral replication in infected cells. However, while several pharmacologically promising inhibitors of human DHODH are known, to date there are no reports of medicinally viable leads against UCK2. Here, we use structure-based drug prototyping to identify two classes of promising leads that noncompetitively inhibit UCK2 activity. In the process, we have identified a hitherto unknown allosteric site at the intersubunit interface of this homotetrameric enzyme. By reducing the kcat of human UCK2 without altering its KM, these new inhibitors have the potential to enable systematic dialing of the fractional inhibition of pyrimidine salvage to achieve the desired antiviral effect with minimal host toxicity.

A druggable addiction to de novo pyrimidine biosynthesis in diffuse midline glioma.

Diffuse midline glioma (DMG) is a uniformly fatal pediatric cancer driven by oncohistones that do not readily lend themselves to drug development. To identify druggable targets for DMG, we conducted a genome-wide CRISPR screen that reveals a DMG selective dependency on the de novo pathway for pyrimidine biosynthesis. This metabolic vulnerability reflects an elevated rate of uridine/uracil degradation that depletes DMG cells of substrates for the alternate salvage pyrimidine biosynthesis pathway. A clinical stage inhibitor of DHODH (rate-limiting enzyme in the de novo pathway) diminishes uridine-5'-phosphate (UMP) pools, generates DNA damage, and induces apoptosis through suppression of replication forks-an "on-target" effect, as shown by uridine rescue. Matrix-assisted laser desorption/ionization (MALDI) mass spectroscopy imaging demonstrates that this DHODH inhibitor (BAY2402234) accumulates in the brain at therapeutically relevant concentrations, suppresses de novo pyrimidine biosynthesis invivo, and prolongs survival of mice bearing intracranial DMG xenografts, highlighting BAY2402234 as a promising therapy against DMGs.

Caffeic Acid Phenethyl Ester as a DHODH Inhibitor and Its Synergistic Anticancer Properties in Combination with 5-Fluorouracil in a Breast Cancer Cell Line.

IntroductionA combination of chemotherapy agents is the best choice in breast cancer treatment to increase the patient survival rate. 5-fluorouracil (5-FU) is one of the drugs applied in combination with other drugs to control and delay development of cancer cells. Nevertheless, the occurrence of multidrug resistance and dose-limiting cytotoxicity have limited the efficacy of 5-FU treatment. Therefore, the discovery of new anti-breast cancer drugs should be pursued.ObjectiveTo study potency of a promising naturally derived compound, caffeic acid phenethyl ester (CAPE), for breast cancer treatment in single and combination with 5-FU.MethodsCytotoxicity of CAPE, 5-FU, and 5-FU+CAPE was studied by in vitro MTT experiment in MCF-7 cell line, and RT-PCR analysis was used to evaluate the change in gene expression due to the treatment. Moreover, an enzymatic assay and molecular docking analysis were applied to evaluate the possible mechanism of substance-induced apoptosis.ResultsThe study revealed that a single treatment of CAPE showed cytotoxicity with IC50 6.6 ± 1.0 µM and 6.5 ± 2.9 µM at 24 h and 48 h, respectively. Meanwhile, 5-FU showed cytostatic activity. The 5-FU + CAPE has a synergistic effect at 24 h treatment with a CI = 0.5 and an additive effect at 48 h treatment with CI = 1.0. CAPE was also found to enhances the mRNA expression of caspase-8 and BAX within 6 hours in combination with 5-FU compared to 5-FU treatment alone. Our study reveals a new mechanism of CAPE which is related to the inhibition of human dihydroorotate dehydrogenase (HsDHODH) with an IC50 of 120.7 ± 6.8 µM, by bound to the ubiquinone-binding site of the enzyme and could be responsible for inducing extrinsic and intrinsic apoptosis.ConclusionThis study demonstrated the cytotoxicity of CAPE potential to induce apoptosis of breast cancer MCF-7 cell line single and cytotoxic–cytostatic combination with 5-FU. Therefore, further studies to develop CAPE and its derivatives will be required to discover new candidates for breast cancer agents.

P1301: BIOLOGICAL SIGNIFICANCE OF UCK2 IN HTLV-1-INFECTED CELLS IN ATL LEUKEMOGENESIS AND ACQUIRED RESISTANCE TO AZACITIDINE

Background: Adult T-cell leukemia-lymphoma (ATL) is an aggressive hematological malignancy of CD4+ T-cells transformed by human T-cell lymphotropic virus-1 (HTLV-1). We have reported that regional DNA hypermethylation in HTLV-1-infected T-cells reflects the disease status of ATL and the anti-ATL effects of DNA demethylating agents, azacitidine (AZA) and decitabine (Blood 2020, 136: 871-884). We recently generated AZA-resistant (AZA-R) cells from ATL cell lines via long-term drug exposure and found down-regulation of uridine cytidine kinase2 (UCK2) correlated with lower susceptibility to AZA (Int J Cancer 2022, 150: 1184-1197). UCK1 and UCK2 are involved in mono-phosphorylation of uridine and cytidine in pyrimidine nucleotide biosynthesis. Both proteins also catalyze mono-phosphorylation of AZA. Overexpression of UCK2 has been observed in several types of solid tumor tissues. Aims: Here, we aimed to understand the role of UCK2 in HTLV-1-infected cells during multi-step carcinogenesis of ATL and the biological significance of inactivation of UCK2 in AZA-R cells. Methods: All studies using human samples were performed in accordance with the guidelines set out in the Declaration of Helsinki and were approved by the institutional review board at Saga University (2018-03-02). Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood samples from healthy volunteers, HTLV-1 carriers and patients with ATL. ATL cell lines were cultured in RPMI-1640 medium containing 10% FBS. Results: We found that UCK2 gene expression was higher in HTLV-1-infected T-cells than in normal T-cells using the gene expression datasets GSE55851 and GSE33615. UCK2 protein expression was increased in HTLV-1-infected T-cells (CADM1+ cells) isolated from patients with ATL compared with uninfected T-cells (Fig. A). We also found UCK2 gene expression was upregulated via activation of T-cell receptor (TCR) signaling pathway in normal CD4+ T-cells (Fig. B). Somatic alterations and epigenetic modifications activating TCR signaling have been identified in ATL cells. Therefore, we think UCK2 protein is overexpressed through dysregulated TCR signaling in HTLV-1-infected cells during ATL leukemogenesis. We have successfully established AZA-R cell lines, whose UCK2 expression was lost or decreased. Although they proliferated normally in vitro, the knockdown of UCK2 dramatically suppressed cell growth of ATL cell lines (Fig. C). Since UCK2 is involved in pyrimidine nucleotide biosynthesis, we then performed metabolome analysis in AZA-R cells. Although uridine and cytidine were accumulated in AZA-R cells, possibly due to loss of UCK2, the amount of UTP and CTP was almost same with parental cells. On the other hands, dihydroorotic acid (DHO) was decreased and orotate was increased in AZA-R cells (Fig. D). DHODH catalyzes the oxidation of DHO to orotic acid and is a rate-limiting enzyme in de novo pyrimidine nucleotide synthesis. AZA-R cells were more susceptible to BAY2402234, a DHODH inhibitor (Fig. E), indicating the activation of pyrimidine de novo synthesis in AZA-R cells. Image:Summary/Conclusion: Taken together, we think UCK2 supports the vigorous cell proliferation not only in normal TCR-stimulated T-cells but also in HTLV-1-infected cells. Inactivation of UCK2 makes cells resistant to AZA but suppresses the cell growth possibly due to the pyrimidine nucleotide starvation. AZA-R cells with reduced UCK2 expression proliferate normally in vitro by the activation of pyrimidine de novo synthesis.

Identification of clinical candidates against West Nile virus by activity screening in vitro and effect evaluation in vivo.

The West Nile virus (WNV) is a member of the flavivirus and is known to causeencephalitis. There is currently no specific treatment for WNV infection. Repurposing of clinically approved drugs appeared promising for rapidly identifying effective, safe, and readily available candidates for antiviral drugs. Here, we screened the small-molecule compounds with anti-WNV activity from 978 Food Drug Administration-approved drugs. Four compounds, including cilnidipine, mycophenolate mofetil, nitazoxanide, and teriflunomide, were found to efficiently abrogate WNV infection in Vero cells and human neuroblastoma SH-SY5Y cells. The four compounds also exert broad-spectrum antiviral activity against the Zika virus, Japanese encephalitis virus, yellow fever virus, tick-borne encephalitis virus, and chikungunya virus. Furthermore, nitazoxanide (a synthetic benzamide) and teriflunomide (an inhibitor of dihydroorotate dehydrogenase, DHODH) protected 20% and 40% of mice from lethal WNV challenge, respectively. Both drugs, which are orally bioavailable and have been approved clinically for many years, may be promising therapeutics for WNV infection. Moreover, the other two DHODH inhibitors,ML390 and vidofludimus, also displayed potent activity against WNV infection in vitro and in vivo.

A novel and potent dihydroorotate dehydrogenase inhibitor suppresses the proliferation of colorectal cancer by inducing mitochondrial dysfunction and DNA damage

AbstractDihydroorotate dehydrogenase (DHODH) is a quite attractive target in cancer therapy. Nevertheless, the antitumor effects of DHODH inhibitors against colorectal cancer (CRC) and the underlying mechanism have seldom been studied. Here, we explored the anti‐CRC efficacy of M62, a novel and potent DHODH inhibitor. In the study, M62 significantly inhibited the proliferation of CRC cells and induced S phase arrest. The antiproliferative effects caused by M62 were rescued by uridine supplementation. Mechanistically, messenger RNA sequencing results showed that M62‐triggered gene changes related to mitochondrial dysfunction, DNA damage, and DNA damage repair. Further validation showed that M62 treatment induced the generation of reactive oxygen species and decreased ΔΨm and ATP production. Meanwhile, M62 induced the accumulation of γ‐H2AX and longer comet tail moment, which were both markers of DNA damage. The downregulated DNA repair proteins and PI3K/ATK pathway were observed after M62 treatment. Furthermore, M62 significantly inhibited CRC xenograft tumor growth without detectable toxicity. Therefore, we conclude that M62 inhibits CRC growth both in vitro and in vivo by causing mitochondrial dysfunction and DNA damage, suggesting that DHODH inhibitors are potential therapeutic strategies for treating CRC.

Targeting MEN1-deficient tumors with DHODH inhibitor

Targeting MEN1-deficient tumors with DHODH inhibitor