Structural basis for high-affinity peptide inhibition of p53 interactions with MDM2 and MDMX

Abstract

The oncoproteins MDM2 and MDMX negatively regulate the activity and stability of the tumor suppressor protein p53-a cellular process initiated by MDM2 and/or MDMX binding to the Nterminal transactivation domain of p53. MDM2 and MDMX in many tumors confer p53 inactivation and tumor survival, and are important molecular targets for anticancer therapy. We screened a duodecimal peptide phage library against site-specifically biotinylated p53-binding domains of human MDM2 and MDMX chemically synthesized via native chemical ligation, and identified several peptide inhibitors of the p53-MDM2/MDMX interactions. The most potent inhibitor (TSFAEYWNLLSP), termed PMI, bound to MDM2 and MDMX at low nanomolar affinities-approximately 2 orders of magnitude stronger than the wild-type p53 peptide of the same length (ETFSDLWKLLPE). We solved the crystal structures of synthetic MDM2 and MDMX, both in complex with PMI, at 1.6 Å resolution. Comparative structural analysis identified an extensive, tightened intramolecular H-bonding network in bound PMI that contributed to its conformational stability, thus enhanced binding to the 2 oncogenic proteins. Importantly, the C-terminal residue Pro of PMI induced formation of a hydrophobic cleft in MDMX previously unseen in the structures of p53-bound MDM2 or MDMX. Our findings deciphered the structural basis for highaffinity peptide inhibition of p53 interactions with MDM2 and MDMX, shedding new light on structure-based rational design of different classes of p53 activators for potential therapeutic use.

Protein grafting of p53TAD onto a leucine zipper scaffold generates a potent HDM dual inhibitor

“…3d). p53LZ2 strongly bound to HDM2NTD (K D ¼ 18 nM), exceeding a previously reported value of WT p53TAD for HDM2NTD (K D ¼ 61.3 nM, measured in PBS containing 0.1 mM TCEP and 0.01% NaN 3 ) 17 , whereas the p53LZ2 homodimer exhibited a moderately higher affinity for HDMXNTD (K D ¼ 80 nM) in comparison to a previously determined ITC data (K D ¼ 202 nM, measured in PBS containing 0.1 mM TCEP and 0.01% NaN 3 ) 17 . The enthalpy-entropy compensation in both cases was revealed by favourable enthalpy (DH) and unfavourable entropy ( À TDS) changes, which contribute to a high-affinity binding of p53LZ2 to both HDM N-terminal domains.…”

“…These grafted p53TAD mimetics, which are named p53LZ1, p53LZ2 and p53LZ3, contain 3, 5 and 9 mutation points, respectively. p53LZ1 has the simplest aminoacid substitution on the GCN4LZ, whereas p53LZ3, bearing more a complicated substitution, is designed to be more similar to the pMI sequence (TSFAEYWALLS), one of the previously identified high-affinity peptides against the HDM2 and HDMX 17 . Two additional changes onto the p53LZ2, E10G and K15S, in addition to the three key hydrophobic residues, were carefully identified to strengthen the protein-protein interaction between the designed p53LZ and HDM proteins based on the structural analysis by comparative structure modelling.…”

“…We chose to perform the study with human colorectal carcinoma HCT 116 (p53 þ/þ ). Previous in vitro and/or in vivo studies of HCT 116 with either Nutlin-3 or pMI peptide treatment exhibited a relatively low sensitivity 17,39 , encouraging us to tackle this tumour model to assess in vivo potency of TAT-p53LZ2 homodimer, harbouring B2.5-fold higher affinity against HDMX in vitro compared with WT p53TAD as well as a superb in vitro binding to HDM2. An orally administered Nutlin-3 (200 mg per kg twice a day for 3 weeks) strongly suppressed tumour growth (Po0.005 versus control; two-sided Student's t-test), while its intravenous administration was largely ineffective compared with vehicle control (P ¼ 0.37 versus control; two-sided Student's t-test).…”

“…Thus, reactivation of the p53 pathway by displacing the negative regulator HDM2 and/or its homologue HDMX from endogenous wild-type p53 in cancer cells has long been considered an important therapeutic strategy in oncology. Accordingly, numerous innovative efforts from academic laboratories as well as pharmaceutical industries have been focused on developing synthetic compounds, peptidomimetics or miniature protein antagonists of the p53-HDM2 (and HDMX) interaction [14][15][16][17][18][19][20][21][22][23] . Among them, the first reported HDM2-specific inhibitor Nutlin-3, which mimics three essential hydrophobic residues of wild-type p53 (F19, W23 and L26; human p53 numbering) required for HDM2 recognition, demonstrated that this small compound disrupting the p53-HDM2 interaction recovered p53 function and resensitized cancer cells to apoptosis in vitro and in vivo 18 .…”

Abrogation of Wip1 expression by RITA-activated p53 potentiates apoptosis induction via activation of ATM and inhibition of HdmX

“…16 Furthermore, activation of p53 after nutlin3a treatment is significantly enhanced upon downregulation of HdmX by siRNA in a number of human tumour cell lines, 16 which is in line with the notion that downregulation of HdmX is necessary for complete p53 activation. 17 There have been reports of short peptides inhibiting HdmX/p53; 18 however, the clinical application of peptides is not optimal due to their proteolytic degradation and insufficient cellular uptake. Recently, the chemical inhibitor of HdmX/p53 interaction, SJ-172550 has been found, which displays additive effects in cancer cells when combined with an Hdm2 inhibitor.…”