Rezatapopt (PC14586): A First-in-Class Small Molecule p53 Y220C Mutant Protein Stabilizer in Clinical Trials

Abstract

The p53 protein, an important transcription factor, plays a critical role in regulating cell responses to stress. (1,2) Coded by the TP53 gene, p53 is a well-recognized tumor suppressorprotein. Dysregulation of p53 function is closely associated with the initiation and development of various cancers. Although restoration of p53 function appears to be a promising therapeutic approach, p53 is considered to be a traditionally “undruggable” target owing to the lack of active binding pockets or frequent mutations. (3) Mutations in the TP53 gene can assist tumor cells to evade p53 suppressive effects, promoting cell proliferation, migration, and invasion, thereby aggravating tumorigenesis. TP53 genetic mutations present in more than 50% of human cancers, most of which are missense mutations occurring in the DNA-binding domain of p53 (amino acids 94–292). Y220C is the ninth most prevalent mutation among all TP53 mutations observed across various tumor types, accounting for 1.8%, and presents in ∼1% of human cancers. (4) Y220C mutation leads to tyrosine-to-cysteine substitution and creates a pocket in the p53 Y220C mutant protein. p53 Y220C protein is structurally unstable at physiological temperatures and can be ubiquitinated and delivered to proteasomal for degradation. Therefore, targeting p53 Y220C-mutant stabilization may be a viable therapeutic strategy for various associated cancers. Small molecule compounds targeting p53 Y220C stabilization are in active development for potential cancer therapeutics. (5) Rezatapopt (PC14586), discovered by PMV Pharmaceuticals, Inc., is a first-in-class small molecule stabilizer and reactivator of p53 Y220C currently in the Phase II clinical trial (NCT04585750) for treating various locally advanced or metastatic solid tumors harboring a p53 Y220C mutation. (6) In addition, rezatapopt is in a Phase Ib clinical trial (NCT06616636) in combination with azacitidine for patients with TP53 Y220C mutant myeloid malignancies (acute myeloid leukemia or myelodysplastic syndrome). Rezatapopt was designed to tightly occupy the pocket created by mutation-caused amino acid tyrosine-to-cysteine substitution in the p53 Y220C mutant protein. Rezatapopt was obtained based on structure-based drug design (SBDD) and systematical structural optimization, starting from hit compound 1 (Figure 1). The substrate concentration required to increase DNA binding by 1.5-fold (SC₁₅₀) was determined by a time-resolved fluorescence resonance energy transfer (FRET) assay to assess the in vitro potency of the p53 Y220C reactivators. Hit compound 1 was obtained by scaffold combination of hit compounds PhiKan83 (SC₁₅₀ = 37.2 μM) with a carbazole scaffold (7) and PK1596 (SC₁₅₀ = 1.6 μM) with an iodophenol scaffold. (8) Compound 1 contains a widely recognized “privileged” indole scaffold in drug development, which exists in numerous natural products and bioactive molecules. Compound 1 has three anchors attached to the indole scaffold, including a hydrophobic ethyl group, a polar alkylamine extending to the solvent region, and a substituted acetylene occupying the adjacent subsite. Compound 1 (SC₁₅₀ = 13.6 μM) showed inadequate potency (SC₁₅₀ > 100 nM) and poor metabolic stability in human liver microsome (T_1/2 = 4 min). SBDD efforts around 1 (SC₁₅₀ = 13.6 μM) resulted in the lead compound 2 (PC-9859) with significantly improved DNA binding activity (SC₁₅₀ = 54 nM). The cocrystal structure (PDB code: 9BR4) of p53 Y220C mutant protein in complex with 2, revealed that 2 locates at the pocket created by tyrosine-to-cysteine substitution in p53 Y220C mutant protein, with indole scaffold occupying this space (Figure 1). Trifluoromethyl group inserts deeply into the hydrophobic pocket, the 4-aminopiperidine ring extends toward the solvent-exposed region, and the pyridine ring is connected to indole core via an alkyne occupies the adjacent subsite. The pyridine ring forms a favorable CH-π stacking interaction with Pro153. Compound 2 forms two key hydrogen bonds with p53 Y220C mutant protein. One interaction is formed between the amino group on pyridine ring and the carbonyl of Cys220 while the other is formed between the amino group on 1-methylpiperidine ring and the carbonyl of Thr150 on the side chain. Further drug design and structural optimization around lead compound 2 has successfully led to the discovery of rezatapopt with a fluorine atom substitution on the piperidine ring and a polar amide group on the phenyl ring projected toward the solvent region. Figure 1. Drug discovery and development of clinical compound rezatapopt (PC14586), a first-in-class small molecule reactivator of p53 Y220C mutant protein. Cocrystal structure of lead compound 2 (PC-9859) complexed with p53 Y220C mutant protein (PDB code: 9BR4) is shown. The p53 Y220C mutant protein is shown as a cartoon. Hydrogen bonds formed between 2, and the key residues within p53 Y220C mutant protein and water molecules are highlighted by red dashed lines. 2 is shown as green sticks. Key amino acid residues Cys220, Thr150, and Pro153 in p53 Y220C mutant protein are shown as yellow sticks. Water molecules are shown as red balls. As the optimal p53 Y220C reactivator, rezatapopt binds to p53 Y220C mutant in a similar mode to that of 2 and stabilizes its structure in the wide-type (WT) conformation, thereby restoring its tumor suppressive functions. The introduction of fluorine atom to the piperidine ring within rezatapopt further strengthened the interaction with p53 Y220C mutant protein, including increased hydrogen bond interaction with Thr150, enhanced conformational rigidity of piperidine ring, and reduced basicity. Moreover, the chiral conformation of fluorine-substituted piperidine ring showed obvious effect on protein binding and 3S,4R configuration is the optimal. Furthermore, the introduction of polar amide group to the phenyl ring remarkably improved the pharmacokinetic (PK) profiles. Rezatapopt significantly increases DNA binding to p53 Y220C (SC₁₅₀ = 9 nM). Furthermore, it potently suppresses the growth of NUGC-3 cells (a gastric cancer cell line with a TP53 Y220C mutation) with an IC₅₀ value of 504 nM, determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Moreover, rezatapopt (po, 50 mg/kg) displays desirable PK profiles in mouse (C_max = 16600 ng/mL and AUC_0-last = 163342 ng.h/mL). Excitingly, rezatapopt (po, QD) at doses of both 25 mg/kg and 50 mg/kg, robustly suppresses the tumor growth in a NUGC-3 tumor xenograft mouse model (TGI = 33% and 71%, respectively). Moreover, rezatapopt causes a significant tumor growth suppression at a higher dose of 100 mg/kg, resulting in an impressive 80% tumor regression. Notably, in toxicological studies, rezatapopt presents a favorable safety profile. Given its potent in vitro and in vivo efficacy as well as favorable PK safety profiles, rezatapopt was advanced into human clinical trials. In a Phase I clinical trial (NCT04585750), rezatapopt displayed a favorable safety profile across all efficacious doses. (9) Moreover, rezatapopt, as a monotherapy, produced robust clinical efficacy in heavily pretreated patients with multiple solid tumor types harboring the TP53 Y220C mutation. (9) A Phase II clinical trial of rezatapopt (NCT04585750, https://clinicaltrials.gov) is currently recruiting, serving as a registrational study to investigate its safety, tolerability and efficacy as a monotherapy in participants with locally advanced or metastatic solid tumors harboring a TP53 Y220C mutation. In some cases, protein destabilization is the root cause of several classes of diseases, including tumor suppressive proteins (e.g., p53, p21, p57, FOXO3A, IRF3 and BAX), and mutated and misfolded proteins, such as ΔF508-cystic fibrosis transmembrane conductance regulator (ΔF508-CFTR) in cystic fibrosis, glucokinase in pancreatic cells in maturity-onset diabetes of the young type 2 (MODY2), and transthyretin (TTR) in cardiac amyloidosis (ATTR-CM). (10) Therefore, in such cases, target protein stabilization (TPS) rather than target protein degradation (TPD) would be therapeutically beneficial. TPS is attracting increasing attention of researchers from both academic and industrial settings and several molecule protein stabilizers have been developed. Inspiringly, two TTR stabilizers, including tafamidis developed by researchers at Pfizer and acoramidis developed by researchers at Stanford University, had been approved by the U.S. FDA for treating ATTR-CM in 2019 and 2024 respectively. (11) Notably, emergence of rezatapopt, the first-in-class stabilizer of p53 Y220C mutant in Phase II clinical trial, represents a great breakthrough in the field of drug discovery targeting p53, a traditionally considered “undruggable protein. Moreover, it also provides the basis for developing p53-based chemical inducers of proximity (CIPs). Besides monovalent stabilizers, several heterobifunctional stabilizers (CIPs) have been developed, including deubiquitinase-targeting chimeras (DUBTACs), (12) RESTORACs, and enhancement-targeting chimeras (ENTACs). (10) Biotech companies Vicinitas, Stablix, and Entact Bio are centering on the development of DUBTACs, RESTORACs and ENTACs, respectively. (13) With continued efforts, more and more protein stabilizers are anticipated to be developed as clinical trial drug candidates, conferring therapeutic benefits. In summary, the discovery of rezatapopt is an exciting breakthrough for drug discovery targeting p53 function restoration. Like drugs targeting KRAS G12C, rezatapopt targeting p53 Y220C shows potential to convert p53 from an “undruggable” to “druggable” target. Rezatapopt is currently in a phase II clinical trial (NCT04585750), benefiting patients with locally advanced or metastatic solid tumors harboring a TP53 Y220C mutation. More clinical results of rezatapopt will be achieved and released in the near future. This work was partially supported by R01CA226001 and R01CA231150 grants from the National Institutes of Health, Breast Cancer Research Program (BCRP) Breakthrough Awards W81XWH-17-1-0071 and W81XWH-17-1-0072 from the Department of Defense (DoD), the John D. Stobo, M.D. Distinguished Chair Endowment, and the Edith & Robert Zinn Chair Endowment in Drug Discovery. This article references 13 other publications. This article has not yet been cited by other publications.