American Association for Cancer Research
15357163mct180863-sup-suppfigs1_s7_supptabs1_s7_meth.pdf (1.15 MB)

Supplementary Figures S1-S7; Supplementary Tables S1-S7; and Supplementary Materials and Methods from Targeting of Hematologic Malignancies with PTC299, A Novel Potent Inhibitor of Dihydroorotate Dehydrogenase with Favorable Pharmaceutical Properties

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journal contribution
posted on 2023-04-03, 16:06 authored by Liangxian Cao, Marla Weetall, Christopher Trotta, Katherine Cintron, Jiyuan Ma, Min Jung Kim, Bansri Furia, Charles Romfo, Jason D. Graci, Wencheng Li, Joshua Du, Josephine Sheedy, Jean Hedrick, Nicole Risher, Shirley Yeh, Hongyan Qi, Tamil Arasu, Seongwoo Hwang, William Lennox, Ronald Kong, Janet Petruska, Young-Choon Moon, John Babiak, Thomas W. Davis, Allan Jacobson, Neil G. Almstead, Art Branstrom, Joseph M. Colacino, Stuart W. Peltz

Supplementary Figures S1-S7: Supplementary Figure S1 shows chemical Synthesis and biological characterization of PTC299. Supplementary Figure S2 shows generation and characterization of PTC299-resistant HT1080 cells. Supplementary Figure 3 shows that PTC299 interacts with and inhibits the activity of DHODH. Supplementary Figure S4 shows that PTC299 inhibits de novo UMP production in both sensitive and insensitive cell lines. Supplementary Figure S5 shows that PTC299 has broad activity against leukemia in vitro and in vivo. Supplementary Figure S6 shows

that PTC299 inhibits production of VEGF protein in tumor cells more potently than does brequinar or teriflunomide. 

Supplementary Figure S7 shows the comparison of GSK983 and PTC299 in pharmacokinetic study in mice; Supplementary Tables S1-S7: Supplementary Table S1 shows the information on the source of cells and when they were obtained. Supplementary Table S2 shows that the metabolism of 15N-glutamine in cells treated with PTC299 is similar to that measured in cells treated with brequinar, a known DHODH inhibitor. Supplementary Table S3 shows the summary of enriched proteins identified in PTC299 pull-down samples. Supplementary Table S4 shows PTC299 species selectivity: inhibition of de novo UMP. Supplementary Table S5 shows the preclinical safety profile of PTC299. Supplementary Table S6 shows the demographic information for patients in the NF-2 clinical trial. Supplementary Table S7 shows the differences in expression of genes related to pyrimidine nucleotide synthesis and salvage pathway in hematopoietic cancers versus solid tumors.





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 hematologic 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.

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