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

Abstract

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 pancaspase inhibitor Z-VAD-FMK. Additional solubility and in vitro metabolic stability profiling revealed compound 51 as a favorable candidate for preclinical efficacy studies.

DHODH inhibition modulates glucose metabolism and circulating GDF15, and improves metabolic balance

“…This demonstrates that DHODH inhibitors increase the synthesis and/or secretion of GDF15 by blocking pyrimidine ribonucleotide synthesis. As mentioned earlier, transcription of GDF15 is known to be induced by p53 (Li et al, 2000), DHODH inhibitors activate p53, and this is prevented in the presence of 100 mM uridine (Ladds et al, 2018;Popova et al, 2020). In line with these observations and as shown in Figures 2A, 2B, and S3, both DHODH inhibitors increased intracellular and secreted GDF15 levels in wild-type p53-expressing MCF7 cultures but not in MCF7 p53 knockout cells.…”

“…MCF7 or MCF7 p53KO cells (previously described ( Zhu et al., 2020 )) were seeded in 6-well plates at a density of 20 × 10 4 cells per well. Next day, cells were treated with the indicated compounds for 48 h. Cell pellets were lysed and processed as described ( Popova et al., 2020 ). Primary antibodies used are as follows: GDF15 mouse monoclonal antibody (Sigma-Aldrich, AMAB90687), p53 mouse monoclonal antibody DO1 (Abcam, ab1101), Rabbit monoclonal antibody to ATF4 (Abcam, ab184909), Rabbit polyclonal antibody to Histone H3 (Abcam, ab1791), MDM2 mouse monoclonal antibody IF2 (Calbiochem, OP46).…”

“…Given that DHODH participates in mitochondrial respiration, that GDF15 expression is induced by the tumor suppressor p53 ( Li et al., 2000 ), that DHODH inhibitors increase p53 synthesis ( Ladds et al., 2018 ; Popova et al., 2020 ), and that an extra TP53 allele can delay aging in mice ( Matheu et al., 2007 ), here we tested the effects of DHODH inhibitors on metabolic balance and on the production of GDF15 by cells and in db/db mice as a model for obesity-induced type 2 diabetes.…”

Structure-Aided Design, Synthesis, and Biological Evaluation of Potent and Selective Non-Nucleoside Inhibitors Targeting Protein Arginine Methyltransferase 5

“…In an effort to further improve the potency and microsomal stability, the next moiety for the SAR study was the central phenyl group. As fluorine atom installation has been extensively utilized to block metabolism, − we introduced a fluorine atom at the C2 position of the central phenyl group of compounds 39–44 , resulting in compounds 45–50 . As shown in Table , consistent with above results, the fluorine-substituted S -enantiomer 47 and 50 exhibited more potent inhibitory activity than their respective racemates ( 45 and 48 ) and R -enantiomers ( 46 and 49 ).…”

Dietary Flavonoids in p53—Mediated Immune Dysfunctions Linking to Cancer Prevention

“…HZ analogs have been shown as promising agents against cancer progression. The cell-based reporter showed that HZ functions by activating the p53-dependent transcription activity [20]. The autosomal dominant polycystic kidney disease (ADPKD) is mainly caused by the mutations in the PKD1 or PKD2 genes.…”