Targeting of Hematologic Malignancies with PTC299, A Novel Potent Inhibitor of Dihydroorotate Dehydrogenase with Favorable Pharmaceutical Properties

Abstract

PTC299 was identified as an inhibitor of VEGFA mRNA translation in a phenotypic screen and evaluated in the clinic for treatment of solid tumors. To guide precision cancer treatment, we performed extensive biological characterization of the activity of PTC299 and demonstrated that inhibition of VEGF production and cell proliferation by PTC299 is linked to a decrease in uridine nucleotides by targeting dihydroorotate dehydrogenase (DHODH), a rate limiting enzyme for de novo pyrimidine nucleotide synthesis. Unlike previously reported DHODH inhibitors that were identified using in vitro enzyme assays, PTC299 is a more potent inhibitor of DHODH in isolated mitochondria suggesting that mitochondrial membrane lipid engagement in the DHODH conformation in situ is required for its optimal activity. PTC299 has broad and potent activity against hematological cancer cells in preclinical models, reflecting a reduced pyrimidine nucleotide salvage pathway in leukemia cells. Archived serum samples from patients treated with PTC299 demonstrated increased levels of dihydroorotate, the substrate of DHODH, indicating target engagement in patients. PTC299 has advantages over previously reported DHODH inhibitors, including greater potency, good oral bioavailability and lack of off-target kinase inhibition and myelosuppression, and thus may be useful for the targeted treatment of hematologic malignancies.

The DHODH Inhibitor PTC299 Arrests SARS-CoV-2 Replication and Suppresses Induction of Inflammatory Cytokines

“…Similar findings were documented in PTC299-treated cancer patients, where administration of PTC299 resulted in increased blood levels of DHO, indicating successful inhibition of DHODH in these patients (Cao et al, 2019). These results are consistent with increased DHO levels observed in Miller syndrome patients who carry mutations in the DHODH gene that reduce DHODH activity (Duley et al, 2016) and with studies showing that PTC299 not only inhibits vascular endothelial growth factor (VEGF) levels in cultured cells, but also normalized VEGF levels in cancer patients (Cao et al, 2019). VEGF is a stress-regulated cytokine the levels of which are increased in these patients (Plotkin et al, 2009).…”

“…PTC299 is an orally bioavailable potent inhibitor of DHODH (Cao et al, 2019). Treatment of cultured cells with PTC299 results in inhibition of DHODH activity, leading to increased levels of DHO, the substrate for the DHODH enzyme (Cao et al, 2019). Similar findings were documented in PTC299-treated cancer patients, where administration of PTC299 resulted in increased blood levels of DHO, indicating successful inhibition of DHODH in these patients (Cao et al, 2019).…”

“…The salvage pathway recycles pre-existing nucleotides from food or other extracellular sources and does not appear to be sufficient to support viral RNA replication. The percentages in red denote the respective extents to which the relative levels of specific nucleotides are reduced during PTC299 treatment (100 nM) of cultured HT1080 fibrosarcoma cells for 8 hours (Cao et al, 2019) Abbreviations: CAD, complex of the following three enzymes: carbamoyl-phosphate synthetase 2, aspartate carbamoyl transferase, and dihydroorotase; CDA, cytidine deaminase; CMP, cytidine monophosphate; CTP, cytidine triphosphate; DHO, dihydroorotate; UCK1/2, Uridine-cytidine kinase 1 and Uridine-cytidine kinase 2; UDP, uridine diphosphate; UMP, uridine monophosphate; UPP, uridine phosphorylase; UPRT, uracil phosphoribosyl transferase; UTP, uridine triphosphate.…”

“…PTC299 was originally identified as a compound that modulates expression of stressregulated proteins like VEGF and was only subsequently found to be a DHODH inhibitor (Cao et al, 2019;Weetall et al, 2016). DHODH inhibitors have been shown to suppress virallyinduced inflammatory cytokine production (Xiong et al, 2020).…”

“…PTC299 is an orally bioavailable potent inhibitor of DHODH (Cao et al, 2019). Treatment of cultured cells with PTC299 results in inhibition of DHODH activity, leading to increased levels of DHO, the substrate for the DHODH enzyme (Cao et al, 2019).…”