Activating p53Y220C with a Mutant-Specific Small Molecule

Abstract

TP53 is the most commonly mutated gene in cancer, but it remains recalcitrant to clinically meaningful therapeutic reactivation. We present here the discovery and characterization of a small molecule chemical inducer of proximity that activates mutant p53. We named this compound TRanscriptional Activator of p53 (TRAP-1) due to its ability to engage mutant p53 and BRD4 in a ternary complex, which potently activates mutant p53 and triggers robust p53 target gene transcription. Treatment of p53^Y220C expressing pancreatic cell lines with TRAP-1 results in rapid upregulation of p21 and other p53 target genes and inhibits the growth of p53^Y220C-expressing cell lines. Negative control compounds that are unable to form a ternary complex do not have these effects, demonstrating the necessity of chemically induced proximity for the observed pharmacology. This approach to activating mutant p53 highlights how chemically induced proximity can be used to restore the functions of tumor suppressor proteins that have been inactivated by mutation in cancer.

Figure 1:. Chemical Targeting of p53 in Cancer Cells.

A. Schematic of the traditional occupancy-based approach to develop MDM2 inhibitors that interrupt the MDM2-p53^WT interaction (left) and p53^Y220C correctors that bind to p53^Y220C and increase its thermal stability (right). B. Schematic of the event-driven transcriptional activator of p53 (TRAP) targeting p53^Y220C mutant through the recruitment of BRD4.

Figure 2:. Development of B-1-based bivalent TRAPs.

A. The structures of the B-1-based bivalent TRAP library. B. TR-FRET. The ternary complex formation measured by TR-FRET between purified p53^Y220C and BRD4_BD1. The data is shown as means ± s.d. of n=2 independent experiments. C. Luciferase reporter. Activation of p53-regulated transcription in BxPC-3 (p53^Y220C) cells after compound treatment for 24 h. The data is shown as means ± s.d. of n=2 independent experiments.

Figure 3:. Ternary complex formation is required TRAP-mediated transcription activation.

A. Structures of negative controls containing minor chemical modifications to remove BRD4 binding (TRAP-1-Neg1) or p53^Y220C binding (TRAP-1-Neg2). B. TR-FRET. The ternary complex formation of TRAPs and their negative controls measured by TR-FRET between p53^Y220C and BRD4_BD1. The data is shown as means ± s.d. of n=2 independent experiments. C. Luciferase reporter. The p53-regulated transcriptional activation of TRAPs and their negative controls at 24 h in BxPC-3 cells. The data is shown as means ± s.d. of n=2 independent experiments. D. The co-immunoprecipitation of TRAPs compared with the cotreatment of p53^Y220C binder (B-1 linker) and BRD4 binder (JQ1) in HEK293T cells after 4 h of compound treatment.

Figure 4:. Activation of p53 target gene transcription.

A. RT-qPCR. Changes of gene expression after 3 μM TRAP-1 incubation for 8 h in BxPC-3 cells compared with the controls. A representative data is shown with mean ± s.d. of n=3 replicates. B. Changes in protein level after TRAPs treatment at 16 h in BxPC-3 cells compared with the controls. C. p21 protein level changes after TRAPs treatment at 2 h in BxPC-3 cells compared with the controls. D. Washout experiment. BxPC-3 cells were incubated with 2 μM TRAPs and controls for 2 h followed by a washout and measurement of cell proliferation at 74 h. A representative data is shown with mean ± s.d. of n=48 replicates.

Figure 5:. The antiproliferative activity of TRAPs in different cell lines.

A. The antiproliferative activity of TRAPs compared with weaker or inactive compounds at 72 h in BxPC-3 cells. The data is shown as means ± s.d. of n=2 independent experiments. B. Heat map showing the antiproliferative potency of TRAP-1 compared with controls in BxPC-3 (p53^Y220C), A549 (p53^WT), and CCD 841 CoN (p53^WT). The data is shown as means of n=2 independent experiments.