Small Molecule Targeting Mutant p53-R273H Restores DNA Binding and Activity

Although mutations in TP53 are the most frequent genetic alteration in human cancers, approaches that target mutant p53 to restore p53-wild type (WT) tumor suppressor functions have had limited success and there are currently no FDA-approved therapeutics targeting mutant p53. We report a membrane-permeant small molecule, Compound K, that selectively increases tumor suppressor functions of p53-R273H, one of the two most common p53 mutants in cancers. Key to understanding the loss of tumor suppressor functions of p53-R273H is deprotonation of His273 at the higher intracellular pH (pHi) of cancer cells, which attenuates binding to the negatively charged phosphate backbone of DNA. We identified Compound K in a computational screen of 3.5 million small molecules for targeting deprotonated His273 and imparting a positive charge to restore electrostatic binding to DNA, like positively charged WT p53-Arg273. We find that Compound K selectively increases DNA binding by p53-R273H at the higher pH of cancer cells with no effect on DNA binding by p53-R273H at the lower pH of untransformed cells, or by p53-WT, p53-R175H, and other pH-regulated transcription factors at lower and higher pH values. Compound K also restores p53-WT functions in human PANC-1 pancreatic cancer cells that are homozygous for p53-R273H, with limited effects in human HPDE untransformed pancreatic epithelial cells, including >4200 DEGs in PANC-1 and <200 DEGs in HPDE, as determined by RNA-seq. In PANC-1 cells Compound K increases expression of loss-of-function, anti-tumorigenic genes, including GADD45A, KLF6, and IFIT2, and decreases expression of gain-of-function pro-tumorigenic genes, including CDC20, PLK1, AURKA, and for TGF-β signaling, as determined by RNA-seq and immunoblotting cell lysates. Compound K also selectively decreases viability of PANC-1 cells with no effect on HPDE cells, and tumorigenicity, determined by 80% reduced colony formation in soft agar. Our findings are an important step toward the unmet need of developing therapeutics targeting mutant p53 to limit cancer progression. Additionally, beyond p53-R273H, Arg>His mutations are enriched in many cancers, most notably medulloblastoma, acute myeloid leukemia, and pancreatic and prostate cancers, which makes our approach of targeting an Arg>His substitution to restore a positive charge have broad cancer therapeutic promise. Citation Format: Anne Kingsland, Kyle P. Kisor, Emil Alexov, Diane L. Barber. Small molecule restoring mutant p53R273H DNA binding and activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 4581.