Small-molecule correctors and stabilizers to target p53

Abstract

The tumor suppressor p53 is the most frequently mutated protein in human cancer and tops the list of high-value precision oncology targets. p53 prevents initiation and progression of cancer by inducing cell-cycle arrest and various forms of cell death. Tumors have thus evolved ways to inactivate p53, mainly by TP53 mutations or by hyperactive p53 degradation. This review focuses on two types of p53 targeting compounds, MDM2 antagonists and mutant p53 correctors. MDM2 inhibitors prevent p53 protein degradation, while correctors restore tumor suppressor activity of p53 mutants by enhancing thermodynamic stability. Herein we explore both novel and repurposed p53 targeting compounds, discuss their mode of action, and examine the challenges in advancing them to the clinic.

Keywords: MDM2 antagonists; TP53; cancer; mutant reactivation; nutlins; p53; small molecules.

Figure 1.. Types and distribution of p53 alterations in cancer.

(A) A large fraction of tumors show mutations in p53 or amplification of MDM2, which encodes a ubiquitin ligase that triggers p53 degradation by the proteasome. Other pathways that overcome p53 tumor suppression include degradation of p53 by the high-risk human papilloma protein E6 [93], epigenetic silencing of TP53 [94], and diverse mechanisms that block p53 signaling without directly affecting p53. (B) Type of mutations in p53 that contribute to human cancer. (C) Amino acid position distribution and frequency of p53 missense mutations. (D) Frequency of p53 mutations in various tumor types. Graphs are based on data generated by The Cancer Genome Atlas (TCGA) Research Network: www.cancer.gov/tcga. Abbreviations: AD, activation domain; DBD, DNA-binding domain; TD, tetramerization domain. Created with BioRender.com.

Figure 2.. Concepts of wild-type (WT) p53 activation and small-molecule-based mutant p53 reactivation.

(A) Many tumors with WT p53 overexpress the ubiquitin (Ub) ligase MDM2. This leads to rapid degradation of p53 by the proteasome and inactivation of tumor suppression. Nutlins and other small-molecule MDM2 antagonists block the MDM2/p53 interaction resulting in accumulation of p53, assembly of the active p53 tetramer, and induction of genes that trigger cell-cycle arrest and cell death. (B) Small molecules that bind mutant p53 and stabilize a WT-like conformation are known as corrector or reactivation drugs. Several small molecules with different proposed binding modes and mechanisms have been identified. PhiKan0083 and PC14586 bind to a cryptic crevice created by the Y220C mutation. Zinc metallochaperones (ZMCs) act as zinc ionophores to facilitate zinc binding to p53 mutants with reduced zinc binding capacity. Arsenicals (arsenic trioxide, ATO) and antimonials (potassium antimony tartrate, PAT) release arsenic and antimony, respectively, which are complexed by three specific cysteine residues (three blue circles) that comprise the C124–C135–C141 pocket (light blue region). Arsenic and antimony binding to this pocket (ABP) stabilizes an active conformation of p53 mutants. A degradation product of PRIMA-1 covalently attaches to several cysteines and reactivates p53 mutants, and UCI-LC0023 binds noncovalently to a transiently open pocket (L1/S3 pocket, green area) in the same region to restore activity to p53 mutants. Note that C124 is located in the L1/S3 pocket. Created with BioRender.com.

Figure 3.. Cysteine residues in the p53 DNA-binding domain involved in mutant p53 reactivation.

The p53 DNA binding domain bound to DNA (PDB ID: 1TSR) with four cysteine residues involved in p53 reactivation. Cys124 and Cys277 have been linked to PRIMA-1/APR-246-mediated reactivation of p53 mutants [71,72]. Cys124, Cys135, and Cys141 coordinate antimony and arsenic as part of the corrector drug mode of action for antimonials and arsenic trioxide (ATO), respectively [3,4]. Note that arsenic covalently binds to these cysteines, whereas antimony interacts noncovalently. Figure was created in PyMOL.

Figure 4.. Proposed off-target mechanism for thiol-reactive p53 correctors.

Glutathione (GSH) depletion by thiol-reactive compounds such as the PRIMA-1/APR-246 degradation product methylene quinudidinone (MQ) has been suggested as an important off-target activity that contributes to the In vivo mode of action of these compounds. Some p53 mutants gain the ability to bind and inhibit the major antioxidant transcription factor NRF2. This gain-of-function activity reduces expression of the NRF2 target SLC7A11 (a cysteine/glutamate antiporter), resulting in reduced GSH levels specifically in p53 mutants. MQ covalently reacts with the thiol in GSH and further reduces the pool of active antioxidants. Cells with p53 mutants that bind to NRF2 are already sensitized to oxidative stress and cannot tolerate the additional PRIMA-1/APR-246-mediated GSH depletion. TP53 missense mutant cell lines are thus hypersensitive to these compounds. Abbreviation: ROS, reactive oxygen species. Created with BioRender.com.