Identification of a Structural Determinant for Selective Targeting of HDMX

Abstract

p53 is a critical tumor-suppressor protein that guards the human genome against mutations by inducing cellcycle arrest or apoptosis. Cancer cells subvert p53 by deletion, mutation, or overexpression of the negative regulators HDM2 and HDMX. For tumors that retain wild-type p53, its reactivation by pharmacologic targeting of HDM2 and/or HDMX represents a promising strategy, with a series of selective small-molecule HDM2 inhibitors and a dual HDM2/HDMX stapled-peptide inhibitor being evaluated in clinical trials. Because selective HDM2 targeting can cause hematologic toxicity, selective HDMX inhibitors could provide an alternative p53-reactivation strategy, but clinical candidates remain elusive. Here, we applied a mutation-scanning approach to uncover p53-based stapled peptides that are selective for HDMX. Crystal structures of stapled-peptide/HDMX complexes revealed a molecular mechanism for the observed specificity, which was validated by HDMX mutagenesis. Thus, we provide a blueprint for the development of HDMX-selective inhibitors to dissect and target the p53/HDMX interaction.

Recent Progress and Clinical Development of Inhibitors that Block MDM4/p53 Protein–Protein Interactions

“…70 SAH-p53-4 binds to MDM4 and MDM2 with equal potency with K d values of 12.9 and 12.0 nM, respectively, in the FP assay. 21 SAH-p53-8 binds to MDM4 and MDM2 with weaker IC 50 values of 229 and 216 nM, respectively. 71 Cocrystal structure of SAH-p53-8/ MDM4 (zebra fish) complex has been resolved, and the structure was superimposed with the p53 peptide/MDM4 crystal structure as shown in Figure 9A.…”

“…71 Cocrystal structure of SAH-p53-8/ MDM4 (zebra fish) complex has been resolved, and the structure was superimposed with the p53 peptide/MDM4 crystal structure as shown in Figure 9A. 21 The amino acid residues of human and zebra fish MDM4 that constitute the p53 binding pockets are identical and overlap very well three dimensionally (Figure 9A). Four residues, Phe19, Leu22, Trp23, and Leu26, of SAH-p53-8 are involved in MDM4 interactions.…”

“…A crystal structure has also been resolved for MDM4/SAH-p53-4, and the interaction details are similar to that of SAH-p53-8/MDM4 because SAH-p53-4 and SAH-p53-8 only differ by Lys27/Arg27 residues, which are not involved in MDM4 interactions. 21 To improve MDM4 selectivity of SAH-p53-4, alanine-, arginine-, and glutamate-scanning studies were performed and a series of stapled peptides were synthesized. 21 Interestingly, SAH-p53-4-L26E (Figure 5 Glu26 of 13 formed a hydrogen bond with Tyr96, partially explaining why Glu26 mutation is tolerated (Figure .9C).…”

The role of miRNAs in MDMX‐p53 interplay

“…have been developed and shown anticancer activity in vitro and in vivo. 16,47,[80][81][82][83][84][85][86] However, none of these inhibitors has been approved for clinical treatment. 7,87 Almost all of these inhibitors have been designed to reactivate wild-type p53 by inhibiting MDM2 and/or MDMX, and it is speculated that these inhibitors may have little or no inhibitory effect on human cancer cells with p53 mutation or p53 deletion.…”

“…Dozens of MDMX inhibitors and MDMX/MDM2 dual inhibitors have been developed and shown anticancer activity in vitro and in vivo 16,47,80–86 . However, none of these inhibitors has been approved for clinical treatment 7,87 .…”