Human Dihydroorotate Dehydrogenase Research Articles

Novel quinolone derivatives targeting human dihydroorotate dehydrogenase suppress Ebola virus infection in vitro

Ebola virus (EBOV) has emerged as a significant public health concern since the 2013–2016 outbreak in West Africa. Currently, no effective antiviral treatments have been approved for clinical use. Compound 1 RYL-634 is a quinolone-derived compound that can inhibit dihydroorotate dehydrogenase, a rate-limiting enzyme in the de novo pyrimidine synthesis pathway and it exhibited antiviral activity against multiple RNA virus infection. In this study, we evaluated the efficacy of a panel of newly developed compounds based on RYL-634 against EBOV infection. Our data showed that RYL-634 as well as its derivatives are effective against EBOV transcription- and replication-competent virus-like particle (trVLP) infection and authentic EBOV infection in vitro at low nanomolar IC50 values and relatively high CC50. Of note, the new derivative RYL-687 had the lowest IC50 at approximately 7 nM and was almost 6 times more potent than remdesivir (GS-5734). Exogenous addition of different metabolites in the pyrimidine de novo synthesis pathway confirmed DHODH as the target of RYL-687. These data provide evidence that such quinolone-derived compounds are promising therapeutic candidates against EBOV infection.

Abstract 1256: JNJ-74856665, a novel DHODH inhibitor, mediates potent anti-leukemic activity and differentiation in vitro and in vivo

Abstract Acute myeloid leukemia (AML) is a heterogeneous disease characterized by uncontrolled clonal expansion of hematopoietic progenitor cells (myeloid blasts). Disease remission can be achieved with standard induction chemotherapy, but refractory and relapsed disease remains a challenge with a 5-year overall survival rate of approximately 25%. Induction of differentiation is an alternative approach to AML therapy. Dihydroorotate dehydrogenase (DHODH) catalyzes the ubiquinone-mediated oxidation of dihydroorotate (DHO) to orotate in the de novo pyrimidine synthesis pathway. Pre-clinical findings demonstrated that DHODH is a metabolic vulnerability in AML and a new target for differentiation therapy. Given the genetic complexity of myeloid malignancies, future use of DHODH inhibitors in combination with selected antineoplastic agents (e.g., azacitidine) with nonoverlapping mechanisms of action may address specific aspects of the complex pathogenesis of AML and MDS (myelodysplastic syndrome), ultimately improving patients' outcomes.JNJ74856665 is an orally available, potent and selective DHODH inhibitor. JNJ-74856665 inhibited the biochemical activity of human DHODH with a 50% inhibitory concentration (IC50) in the subnanomolar range and in vitro inhibited proliferation of MOLM-13, OCI-AML3, HL60 and THP-1 AML cell lines with low nanomolar IC50 values. Mechanism of action studies using these four cell lines demonstrated induction of CD11b and CD14 differentiation marker mRNA levels. Differentiation was accompanied by cell cycle arrest and apoptosis induction. Importantly, addition of excess uridine to the tissue culture conditions abrogated the anti-proliferative, differentiation and apoptosis inducing activity of JNJ-74856665, indicating on target activity. In vivo, JNJ-74856665 mediated significant activity in decreasing leukemic burden and increasing life span across subcutaneous (MOLM-13 and OCI-AML3) and disseminated (MOLM-13) xenografts, respectively. Pharmacodynamic investigations demonstrated target engagement as evidenced by upregulation of the DHODH substrate dihydroorotate in plasma and induction of differentiation markers in tumor. Combination of JNJ-74856665 with azacitidine in vivo demonstrated lack of antagonism in the anti-leukemic activity.The results described herein demonstrate that JNJ-74856665 exerts potent anti-leukemic activity in vitro and in vivo and warrant further investigation. To that end, a phase 1 clinical trial assessing the safety and efficacy of JNJ-74856665 in patients with AML and MDS is planned to initiate shortly. Citation Format: Christine Pietsch, Scott Kuduk, Xiaochun Zhang, Tammy Bush, Faraz Kazmi, Edgar Jacoby, Zhuming Zhang, Lindsey DeRatt, Weixue Wang, Aaron Patrick, Andrew Steele, Friedericke Pastore, Tony Greway, Nikki Daskalakis, Kathryn Bradford, Kathryn Packman, Gilles Bignan, James Edwards, Ricardo Attar, Yusri Elsayed, Jacqueline Bussolari, Ulrike Philippar. JNJ-74856665, a novel DHODH inhibitor, mediates potent anti-leukemic activity and differentiation in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1256.

Low cytotoxic quinoline-4-carboxylic acids derived from vanillin precursors as potential human dihydroorotate dehydrogenase inhibitors

Twenty novel 2-substituted quinoline-4-carboxylic acids bearing amide moiety were designed and synthesized by Doebner reaction. Human dihydroorotate dehydrogenase (hDHODH) was recognized as a biological target and all compounds were screened as potential hDHODH inhibitors in an enzyme inhibition assay. The prepared heterocycles were also evaluated for their cytotoxic effects on the healthy HaCaT cell line while lipophilic properties were considered on the basis of experimentally determined logD values at physiological pH. The most promising compound 5j, with chlorine at para-position of terminal phenyl ring, showed good hDHODH inhibitory activity, low cytotoxicity, and optimal lipophilicity. The bioactive conformation of 5j on the hDHODH, determined by means of molecular docking, revealed the compound’s pharmacology and provide guidelines for further lead optimization.

Combination of consensus and ensemble docking strategies for the discovery of human dihydroorotate dehydrogenase inhibitors

The inconsistencies in the performance of the virtual screening (VS) process, depending on the used software and structural conformation of the protein, is a challenging issue in the drug design and discovery field. Varying performance, especially in terms of early recognition of the potential hit compounds, negatively affects the whole process and leads to unnecessary waste of the time and resources. Appropriate application of the ensemble docking and consensus-scoring approaches can significantly increase reliability of the VS results. Dihydroorotate dehydrogenase (DHODH) is a key enzyme in the pyrimidine biosynthesis pathway. It is considered as a valuable therapeutic target in cancer, autoimmune and viral diseases. Based on the conducted benchmark study and analysis of the effect of different combinations of the applied methods and approaches, here we suggested a structure-based virtual screening (SBVS) workflow that can be used to increase the reliability of VS.

Chemical Constituents from Chloranthus elatior and Their Inhibitory Effect on Human Dihydroorotate Dehydrogenase.

A new sesquiterpene, chlorantholide G (1: ), a new sesquiterpene dimer, elatiolactone (2: ), and 2 new diterpenes, elatiorlabdane B (3: ) and elatiorlabdane C (4: ), together with 51known compounds, were isolated from the aerial parts of Chloranthus elatior. The new structures including their absolute configurations were mainly established by mass spectrometric, NMR, and electronic circular dichroism experiments. All isolated compounds were tested for their anti-hDHODH activity. (4S,6R)-4-hydroxy-6-isopropyl-3-methylcyclohex-2-enone (5: ) and (4S,5R,9S,10R)-8(17),12,14-labdatrien-18-oic acid (29: ) were the most active compounds with IC50 values of 18.7 and 30.7 µM, respectively.

Targeting Acute Myelogenous Leukemia Using Potent Human Dihydroorotate Dehydrogenase Inhibitors Based on the 2-Hydroxypyrazolo[1,5-a]pyridine Scaffold: SAR of the Biphenyl Moiety.

The connection with acute myelogenous leukemia (AML) of dihydroorotate dehydrogenase (hDHODH), a key enzyme in pyrimidine biosynthesis, has attracted significant interest from pharma as a possible AML therapeutic target. We recently discovered compound 1, a potent hDHODH inhibitor (IC50 = 1.2 nM), able to induce myeloid differentiation in AML cell lines (THP1) in the low nM range (EC50 = 32.8 nM) superior to brequinar’s phase I/II clinical trial (EC50 = 265 nM). Herein, we investigate the 1 drug-like properties observing good metabolic stability and no toxic profile when administered at doses of 10 and 25 mg/kg every 3 days for 5 weeks (Balb/c mice). Moreover, in order to identify a backup compound, we investigate the SAR of this class of compounds. Inside the series, 17 is characterized by higher potency in inducing myeloid differentiation (EC50 = 17.3 nM), strong proapoptotic properties (EC50 = 20.2 nM), and low cytotoxicity toward non-AML cells (EC30(Jurkat) > 100 μM).

Targeting Chikungunya Virus Replication by Benzoannulene Inhibitors.

A benzo[6]annulene, 4-(tert-butyl)-N-(3-methoxy-5,6,7,8-tetrahydronaphthalen-2-yl) benzamide (1a), was identified as an inhibitor against Chikungunya virus (CHIKV) with antiviral activity EC90 = 1.45 μM and viral titer reduction (VTR) of 2.5 log at 10 μM with no observed cytotoxicity (CC50 = 169 μM) in normal human dermal fibroblast cells. Chemistry efforts to improve potency, efficacy, and drug-like properties of 1a resulted in a novel lead compound 8q, which possessed excellent cellular antiviral activity (EC90 = 270 nM and VTR of 4.5 log at 10 μM) and improved liver microsomal stability. CHIKV resistance to an analog of 1a, compound 1c, tracked to a mutation in the nsP3 macrodomain. Further mechanism of action studies showed compounds working through inhibition of human dihydroorotate dehydrogenase in addition to CHIKV nsP3 macrodomain. Moderate efficacy was observed in an in vivo CHIKV challenge mouse model for compound 8q as viral replication was rescued from the pyrimidine salvage pathway.

IMU-838, a Developmental DHODH Inhibitor in Phase II for Autoimmune Disease, Shows Anti-SARS-CoV-2 and Broad-Spectrum Antiviral Efficacy In Vitro.

The ongoing pandemic spread of the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) demands skillful strategies for novel drug development, drug repurposing and cotreatments, in particular focusing on existing candidates of host-directed antivirals (HDAs). The developmental drug IMU-838, currently being investigated in a phase 2b trial in patients suffering from autoimmune diseases, represents an inhibitor of human dihydroorotate dehydrogenase (DHODH) with a recently proven antiviral activity in vitro and in vivo. Here, we established an analysis system for assessing the antiviral potency of IMU-838 and DHODH-directed back-up drugs in cultured cell-based infection models. By the use of SARS-CoV-2-specific immunofluorescence, Western blot, in-cell ELISA, viral yield reduction and RT-qPCR methods, we demonstrated the following: (i) IMU-838 and back-ups show anti-SARS-CoV-2 activity at several levels of viral replication, i.e., protein production, double-strand RNA synthesis, and release of infectious virus; (ii) antiviral efficacy in Vero cells was demonstrated in a micromolar range (IMU-838 half-maximal effective concentration, EC50, of 7.6 ± 5.8 µM); (iii) anti-SARS-CoV-2 activity was distinct from cytotoxic effects (half-cytotoxic concentration, CC50, >100 µM); (iv) the drug in vitro potency was confirmed using several Vero lineages and human cells; (v) combination with remdesivir showed enhanced anti-SARS-CoV-2 activity; (vi) vidofludimus, the active determinant of IMU-838, exerted a broad-spectrum activity against a selection of major human pathogenic viruses. These findings strongly suggest that developmental DHODH inhibitors represent promising candidates for use as anti-SARS-CoV-2 therapeutics.

Experimental approach, theoretical investigation and molecular docking of 2- chloro-5-fluoro phenol antibacterial compound

The molecular structural dimerization of biologically potent 2-chloro-5-fluoro phenol (2C5FP) is optimized. A combined experimental and theoretical characteristics of vibrational spectral determinations (NMR, FT-IR and Raman) on 2-chloro-5-fluoro phenol (2C5FP) were used at DFT-B3LYP/6–31++G (d,p) level of computation. A close coherence is achieved when experimentally observed wave numbers are compared with calculated wave numbers by refinement of the scale factors. Calculated values of global chemical descriptors of the present molecule reveal significant molecular stability and chemical reactivity. Non-Linear optical (NLO) property of the present molecule is investigated by determining the second order non linear parameter of first hyperpolarizability β. Moreover, hydrogen bond and thermodynamic parameters at various temperatures are determined and discussed. Investigated compound 2C5FP possesses a better antibacterial activity against Echerichia coli, Streptococcus aureus, Pseudomonas aureus,and Staphylococcus aureus, respectively. The title molecule is subjected to molecular docking studies with two different proteins, namely Staphylococcus aureus Tyrosyl-tRNA synthetase (PDB ID: 1JIL) and human dihydroorotate dehydrogenase (hDHODH) (PDB ID: 6CJF). The results of molecular docking analysis support the antibacterial activity and demonstrate a strong interaction with the DHODH inhibitor.

Design, synthesis, molecular modeling, and biological evaluation of acrylamide derivatives as potent inhibitors of human dihydroorotate dehydrogenase for the treatment of rheumatoid arthritis

Human dihydroorotate dehydrogenase (DHODH) is a viable target for the development of therapeutics to treat cancer and immunological diseases, such as rheumatoid arthritis (RA), psoriasis and multiple sclerosis (MS). Herein, a series of acrylamide-based novel DHODH inhibitors as potential RA treatment agents were designed and synthesized. 2-Acrylamidobenzoic acid analog 11 was identified as the lead compound for structure–activity relationship (SAR) studies. The replacement of the phenyl group with naphthyl moieties improved inhibitory activity significantly to double-digit nanomolar range. Further structure optimization revealed that an acrylamide with small hydrophobic groups (Me, Cl or Br) at the 2-position was preferred. Moreover, adding a fluoro atom at the 5-position of the benzoic acid enhanced the potency. The optimization efforts led to potent compounds 42 and 53‒55 with IC50 values of 41, 44, 32, and 42 nmol/L, respectively. The most potent compound 54 also displayed favorable pharmacokinetic (PK) profiles and encouraging in vivo anti-arthritic effects in a dose-dependent manner.

Potent human dihydroorotate dehydrogenase inhibitory activity of new quinoline-4-carboxylic acids derived from phenolic aldehydes: Synthesis, cytotoxicity, lipophilicity and molecular docking studies

A series of novel 2-substituted quinoline-4-carboxylic acids was synthesized by Doebner reaction starting from freely available protocatechuic aldehyde and vanillin precursors. Human dihydroorotate dehydrogenase (hDHODH) was recognised as a clear molecular target for these heterocycles. All compounds were also tested for their antiproliferative potential against three cancer cells (MCF-7, A549, A375) and one normal cell line (HaCaT) to evaluate the selective cytotoxicity. Quinoline derivatives 3f and 3g were identified as potent hDHODH inhibitors while 3k and 3l demonstrated high cytotoxic activity against MCF-7 and A375 cells and good selectivity. In addition, the logD7.4 values obtained by the experimental method were found to be in the range from −1.15 to 1.69. The chemical structures of all compounds were confirmed by IR, NMR and elemental analysis. The compounds pharmacology on the molecular level was revealed by means of molecular docking, highlighting the structural differences that distinguish highly active from medium and low active hDHODH inhibitors.

N-phenyl ureidobenzenesulfonates, a novel class of promising human dihydroorotate dehydrogenase inhibitors

N-phenyl ureidobenzenesulfonates (PUB-SOs) is a new class of promising anticancer agents inducing replication stresses and cell cycle arrest in S-phase. However, the pharmacological target of PUB-SOs was still unidentified. Consequently, the objective of the present study was to identify and confirm the pharmacological target of the prototypical PUB-SO named 2-ethylphenyl 4-(3-ethylureido)benzenesulfonate (SFOM-0046) leading to the cell cycle arrest in S-phase. The antiproliferative and the cytotoxic activities of SFOM-0046 were characterized using the NCI-60 screening program and its fingerprint was analyzed by COMPARE algorithm. Then, human dihydroorotate dehydrogenase (hDHODH) colorimetric assay, uridine rescuing cell proliferation and molecular docking in the brequinar-binding site were performed. As a result, SFOM-0046 exhibited a mean antiproliferative activity of 3.5 μM in the NCI-60 screening program and evidenced that leukemia and colon cancer cell panels were more sensitive to SFOM-0046. COMPARE algorithm showed that the SFOM-0046 cytotoxic profile is equivalent to the ones of brequinar and dichloroallyl lawsone, two inhibitors of hDHODH. SFOM-0046 inhibited the hDHODH in the low nanomolar range (IC50 = 72 nM) and uridine rescued the cell proliferation of HT-29, HT-1080, M21 and MCF-7 cancer cell lines in the presence of SFOM-0046. Finally, molecular docking showed a binding pose of SFOM-0046 interacting with Met43 and Phe62 present in the brequinar-binding site. In conclusion, PUB-SOs and notably SFOM-0046 are new small molecules hDHODH inhibitors triggering replication stresses and S-phase arrest.

A Small-Scale Medication of Leflunomide as a Treatment of COVID-19 in an Open-Label Blank-Controlled Clinical Trial.

We recently reported that inhibitors against human dihydroorotate dehydrogenase (DHODH) have broad-spectrum antiviral activities including their inhibitory efficacies on SARS-CoV-2 replication in infected cells. However, there are limited data from clinical studies to prove the application of DHODH inhibitors in Coronavirus disease 2019 (COVID-19) patients. In the present study, we evaluated Leflunomide, an approved DHODH inhibitor widely used as a modest immune regulator to treat autoimmune diseases, in treating COVID-19 disease with a small-scale of patients. Cases of 10 laboratory-confirmed COVID-19 patients of moderate type with obvious opacity in the lung were included. Five of the patients were treated with Leflunomide, and another five were treated as blank controls without a placebo. All the patients accepted standard supportive treatment for COVID-19. The patients given Leflunomide had a shorter viral shedding time (median of 5 days) than the controls (median of 11 days, P = 0.046). The patients given Leflunomide also showed a significant reduction in C-reactive protein levels, indicating that immunopathological inflammation was well controlled. No obvious adverse effects were observed in Leflunomide-treated patients, and they all discharged from the hospital faster than controls. This preliminary study on a small-scale compassionate use of Leflunomide provides clues for further understanding of Leflunomide as a potential antiviral drug against COVID-19.

Bifunctional Naphtho[2,3-d][1,2,3]triazole-4,9-dione Compounds Exhibit Antitumor Effects In Vitro and In Vivo by Inhibiting Dihydroorotate Dehydrogenase and Inducing Reactive Oxygen Species Production

Human dihydroorotate dehydrogenase (hDHODH) is an attractive target for cancer therapy. Based on its crystal structure, we designed and synthesized a focused compound library containing the structural moiety of 1,4-benzoquinone, which possesses reactive oxygen species (ROS) induction capacity. Compound 3s with a naphtho[2,3-d][1,2,3]triazole-4,9-dione scaffold exhibited inhibitory activity against hDHODH. Further optimization led to compounds 11k and 11l, which inhibited hDHODH activity with IC50 values of 9 and 4.5 nM, respectively. Protein-ligand cocrystal structures clearly depicted hydrogen bond and hydrophobic interactions of 11k and 11l with hDHODH. Compounds 11k and 11l significantly inhibited leukemia cell and solid tumor cell proliferation and induced ROS production, mitochondrial dysfunction, apoptosis, and cell cycle arrest. Nanocrystallization of compound 11l displayed significant in vivo antitumor effects in the Raji xenograft model. Overall, this study provides a novel bifunctional compound 11l with hDHODH inhibition and ROS induction efficacy, which represents a promising anticancer lead worthy of further exploration.

An in silico Workflow that Yields Experimentally Comparable Inhibitors for Human Dihydroorotate Dehydrogenase.

Rheumatoid Arthritis [RA] is an autoimmune disease that can cause chronic inflammation of the joints. Human DiHydroOrotate DeHydrogenase [DHODH] is a clinically validated drug target for the treatment of Rheumatoid Arthritis. DHODH inhibition results in beneficial immunosuppressant and anti-proliferative effects. Leflunomide [LEF] and Brequinar Sodium [BREQ], drugs used in the treatment of RA, suppresses the immune cells responsible for inflammation but has several side-effects, most predominant being symptomatic liver damage and toxicity. An existing scaffold based on structural analogies with LEF and BREQ was used to screen out potent inhibitors of DHODH, in ZINC Database using 2D binary fingerprint. 10 structures similar to the scaffold were shortlisted due to their Tanimoto similarity coefficient. Selected structures were docked using the tools AutoDock, Ligand fit and iGEMDOCK with target human DHODH. High scoring compounds having similar interactions as that of scaffold were checked to evaluate their Drug-Likeliness. The five shortlisted compounds were then subjected to Molecular Dynamics Simulation studies for 50ns using GROMACS. Measures of structural similarity based on 2D Fingerprint Screening and Molecular Dynamics Simulation studies can suggest good leads for drug designing. The novelty of this study is that the workflow used here yields the same results that are at par with the experimental data. This suggests the use of the 2D fingerprint similarity search in various databases, followed by multiple docking algorithms and dynamics as a workflow that will lead to finding novel compounds that a structurally and functionally similar to LEF and BREQ.

Vidofludimus calcium, a next generation DHODH inhibitor for the Treatment of relapsing-remitting multiple sclerosis

BackgroundInhibition of dihydroorotate dehydrogenase (DHODH) is an established mechanism for the treatment of relapsing-remitting multiple sclerosis (RRMS). Currently approved treatments have several shortcomings. Consequently, new and effective treatments with improved safety and convenience profiles are sought after by patients. ObjectiveTo explore the overall profile of vidofludimus for the treatment of RRMS. MethodsPreclinical investigations were done exploring the species-dependency of DHODH inhibition of vidofludimus. In addition, the preclinical efficacy in a rat experimental autoimmune encephalomyelitis (EAE) model and the inhibition of cytokine release from activated PBMC were investigated. Pharmacokinetic data were also obtained in a Phase 1 multiple ascending dose trial of the formulation IMU–838 (vidofludimus calcium). ResultsIt was shown that vidofludimus is 2.6 times more potent in inhibiting DHO oxidation by human DHODH compared to teriflunomide. Although both compounds increased cell apoptosis, vidofludimus was more efficacious in the inhibition of T-lymphocyte proliferation compared to teriflunomide. The same was also observed for the secretion of IL–17 and IFN–γ. Interestingly, the potency or vidofludimus to inhibit rat or mouse DHODH is 7.5 and 64.4 time lower than the for the human DHODH, respectively. The rat EAE study clearly exhibited a dose-dependent inhibition of cumulative disease scores by vidofludimus. In the multiple ascending dose Phase 1 clinical trial, the serum half-life of about 30 h provides a favorable profile for once daily dosing of IMU–838, with quick dosing to steady state through levels within 5 days and the ability to wash out drug quickly, if required. ConclusionsThe investigations highlighted that the desired selective immunomodulatory properties can be separated from general antiproliferative effects seen and related adverse events in first-generation DHODH inhibitors. Based on data obtained from a series of pre-clinical as well as phase 1 and phase 2 studies, IMU–838 is a promising next-generation candidate for the oral treatment of RRMS. However, this will need to be confirmed in the currently ongoing Phase 2 study in RRMS patients.

Optimization of Tetrahydroindazoles as Inhibitors of Human Dihydroorotate Dehydrogenase and Evaluation of Their Activity and In Vitro Metabolic Stability.

Human dihydroorotate dehydrogenase (DHODH), an enzyme in the de novo pyrimidine synthesis pathway, is a target for the treatment of rheumatoid arthritis and multiple sclerosis and is re-emerging as an attractive target for cancer therapy. Here we describe the optimization of recently identified tetrahydroindazoles (HZ) as DHODH inhibitors. Several of the HZ analogues synthesized in this study are highly potent inhibitors of DHODH in an enzymatic assay, while also inhibiting cancer cell growth and viability and activating p53-dependent transcription factor activity in a reporter cell assay. Furthermore, we demonstrate the specificity of the compounds toward the de novo pyrimidine synthesis pathway through supplementation with an excess of uridine. We also show that induction of the DNA damage marker γ-H2AX after DHODH inhibition is preventable by cotreatment with the pan-caspase inhibitor Z-VAD-FMK. Additional solubility and in vitro metabolic stability profiling revealed compound 51 as a favorable candidate for preclinical efficacy studies.

Structural Optimization and Structure–Activity Relationship of 4-Thiazolidinone Derivatives as Novel Inhibitors of Human Dihydroorotate Dehydrogenase

Human dihydroorotate dehydrogenase (hDHODH), one of the attractive targets for the development of immunosuppressive drugs, is also a potential target of anticancer drugs and anti-leukemic drugs. The development of promising hDHODH inhibitors is in high demand. Based on the unique binding mode of our previous reported 4-thiazolidinone derivatives, via molecular docking method, three new series 4-thiazolidinone derivatives were designed and synthesized as hDHODH inhibitors. The preliminary structure–activity relationship was investigated. Compound 9 of biphenyl series and compound 37 of amide series displayed IC50 values of 1.32 μM and 1.45 μM, respectively. This research will provide valuable reference for the research of new structures of hDHODH inhibitors.

Abstract 2: Discovery of BAY 2402234 by phenotypic screening: A human Dihydroorotate Dehydrogenase (DHODH) inhibitor in clinical trials for the treatment of myeloid malignancies

Abstract DHODH is a key enzyme in the biosynthesis of pyrimidines and recent studies have renewed interest in this old anti-cancer target. Here, we disclose the discovery of 4-triazolosalicylamides as inhibitors of DHODH and their structure activity relationship (SAR). The hit cluster was discovered during a phenotypic high throughput screen (HTS) of 2.5 million compounds where proliferation of H460 lung cancer cells was used as read-out. DHODH was successfully identified as the molecular target by comparing the activity profile of the hits in a panel of cell lines to a set of inhibitors with known pharmacological activity. The hit compounds showed good cellular potency but had undesirable DMPK properties. Interestingly, the compounds are non-ionizable in contrast to many other DHODH inhibitors and show no potency shift from biochemical to cellular assays. Structural modifications lead to compounds with sub-nanomolar potency in cellular assays and increased metabolic stability enabling the proof of concept in vivo xenograft experiments. Further optimization guided by lipophilicity efficiency and identification of metabolic hot spots resulted in molecules with low clearance and improved solubility. BAY 2402234 was selected as the clinical candidate after side by side comparison of a number of promising compounds. It shows great oral bioavailability, target engagement in all preclinical species tested, induces differentiation in AML models, and has excellent activity in a variety of leukemia models. A clinical phase I study has been initiated in patients with myeloid malignancies. (NCT03404726) Citation Format: Stefan N. Gradl, Thomas Mueller, Steven Ferrara, Sherif El Sheikh, Andreas Janzer, Han-Jie Zhou, Anders Friberg, Judith Guenther, Martina Schaefer, Timo Stellfeld, Knut Eis, Michael Kroeber, Duy Nguyen, Claudia Merz, Michael Niehues, Detlef Stoeckigt, Sven Christian, Katja Zimmermann, Pascal Lejeune, Michael Bruening, Hanna Meyer, Vera Puetter, David T. Scadden, David B. Sykes, Henrik Seidel, Ashley Eheim, Martin Michels, Andrea Haegebarth, Marcus Bauser. Discovery of BAY 2402234 by phenotypic screening: A human Dihydroorotate Dehydrogenase (DHODH) inhibitor in clinical trials for the treatment of myeloid malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2.

Abstract 3597: BAY 2402234: Preclinical evaluation of a novel, selective dihydroorotate dehydrogenase (DHODH) inhibitor for the treatment of diffuse large B-cell lymphoma (DLBCL)

Abstract DLBCL is the most common type of non-Hodgkin lymphoma. It is an aggressive and fast growing tumor with two major molecular subtypes: germinal center (GCB) and activated B-cell like (ABC). While the majority of patients are 60 years or older, DLBCL can occur at any age. Despite a cure rate of around 50% the need for novel therapies remains high, especially for relapsed/refractory DLBCL patients not eligible for stem cell transplant. DHODH is a key enzyme in the de novo pyrimidine synthesis converting dihydroorotate to orotate. We recently discovered its role in AML differentiation (Sykes et al 2016, Cell) and we are investigating the novel DHODH inhibitor BAY 2402234 in an ongoing phase I study in myeloid malignancies (NCT03404726). Further screening of non-leukemia cancer types identified DLBCL as highly responsive to DHODH inhibition in preclinical studies. Here, we disclose for the first time the functional preclinical characterization of BAY 2402234 in DLBCL. BAY 2402234 is a selective low-nanomolar inhibitor of human DHODH enzymatic activity. In vitro it potently inhibits proliferation of DLBCL cell lines in the sub-nanomolar to low-nanomolar range. The anti-proliferative effects can be rescued by uridine supplementation which bypasses DHODH via the salvage pathway and demonstrates the on-target specificity of the inhibitor. In vivo, BAY 2402234 exhibits strong in vivo anti-tumor efficacy in monotherapy in subcutaneous models derived from patient-derived xenograft (PDX) and cell lines representing various DLBCL subtypes, including GCB and ABC. Dose dependent target engagement and drug exposure of BAY 2402234 could be observed by increases in plasma dihydroorotate levels and unbound plasma drug levels after treatment with the inhibitor. Based on preclinical data presented herein we plan to start clinical investigations of BAY 2402234 in patients with DLBCL in early 2019. Citation Format: Ashley Eheim, Sven Christian, Hanna Meyer, Detlef Stoeckigt, Claudia Merz, Katja Zimmermann, Marcus Bauser, Andrea Haegebarth, Steven Ferrara, David B. Sykes, David T. Scadden, Stefan Gradl, Andreas Janzer. BAY 2402234: Preclinical evaluation of a novel, selective dihydroorotate dehydrogenase (DHODH) inhibitor for the treatment of diffuse large B-cell lymphoma (DLBCL) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3597.