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.

Inhibition of the interaction between the tumor suppressor protein p53 and its negative regulators MDM2 and MDMX is of great interest in cancer biology and drug design. We previously reported a potent duodecimal peptide inhibitor, termed PMI (TSFAEYWNLLSP), of the p53-MDM2 and -MDMX interactions. PMI competes with p53 for MDM2 and MDMX binding at an affinity roughly two orders of magnitude higher than that of 17–28p53 (ETFSDLWKLLPE) of the same length; both peptides adopt nearly identical α-helical conformations in the complexes, where the three highlighted hydrophobic residues Phe, Trp and Leu dominate PMI or 17–28p53 binding to MDM2 and MDMX. To elucidate the molecular determinants for PMI activity and specificity, we performed a systematic Ala scanning mutational analysis of PMI and 17–28p53. The binding affinities for MDM2 and MDMX of a total of 35 peptides including 10 truncation analogs were quantified, affording a complete dissection of energetic contributions of individual residues of PMI and 17–28p53 to MDM2 and MDMX association. Importantly, the N8A mutation turned PMI into the most potent dual specific antagonist of MDM2 and MDMX reported to date, registering respective Kd values of 490 pM and 2.4 nM. The co-crystal structure of N8A-PMI-25–109MDM2 was determined at 1.95 Å, affirming that high-affinity peptide binding to MDM2/MDMX necessitates, in addition to optimized inter-molecular interactions, enhanced helix stability or propensity contributed by non-contact residues. The powerful empirical binding data and crystal structures present a unique opportunity for computational studies of peptide inhibition of the p53-MDM2/MDMX interactions.

Despite the small size and conserved tertiary structure of defensins, little is known at a molecular level about the basis of their functional versatility. For insight into the mechanism(s) of defensin function, we prepared enantiomeric pairs of four human defensins, HNP1, HNP4, HD5, and HBD2, and studied their killing of bacteria, inhibition of anthrax lethal factor, and binding to HIV-1 gp120. Unstructured HNP1, HD5, and HBD3 and several other human ␣-and ␤-defensins were also examined. Crystallographic analysis showed a plane of symmetry that related L HNP1 and D HNP1 to each other. Either D-enantiomerization or linearization significantly impaired the ability of HNP1 and HD5 to kill Staphylococcus aureus but not Escherichia coli. In contrast, L HNP4 and D HNP4 were equally bactericidal against both bacteria. D-Enantiomers were generally weaker inhibitors or binders of lethal factor and gp120 than their respective native, all-L forms, although activity differences were modest, particularly for HNP4. A strong correlation existed among these different functions. Our data indicate: (a) that HNP1 and HD5 kill E. coli by a process that is mechanistically distinct from their actions that kill S. aureus and (b) that chiral molecular recognition is not a stringent prerequisite for other functions of these defensins, including their ability to inhibit lethal factor and bind gp120 of HIV-1.

The oncoproteins MDM2 and MDMX negatively regulate the activity and stability of the tumor suppressor protein p53, conferring tumor development and survival. Antagonists targeting the p53-binding domains of MDM2 and MDMX kill tumor cells both in vitro and in vivo by reactivating the p53 pathway, promising a class of antitumor agents for cancer therapy. Aided by native chemical ligation and mirror image phage display, we recently identified a D-peptide inhibitor of the p53-MDM2 interaction termed The tumor suppressor protein p53 is a transcription factor that transactivates, in response to cellular stresses, the expression of various target genes that mediate cell cycle arrest, senescence, or apoptosis (1). Dubbed the "guardian of the genome" (2), p53 is critical for maintaining genetic stability and preventing tumor development (3). Not surprisingly, loss of p53 activity resulting from point mutations in the TP53 gene is responsible for approximately 50% of human tumors. Although p53 retains WT status in many other tumors, its tumor suppressor activity and in vivo stability are abrogated by regulatory molecules such as the E3 ubiquitin ligase MDM2 and its homologue MDMX (4, 5). Amplified or over-expressed in a significant fraction of cancers without concomitant TP53 mutation, MDM2 and MDMX directly contribute to p53 inactivation and tumor survival.MDM2 itself is transcriptionally inducible by p53 in a negative feedback loop (6). MDM2 binds the N-terminal transactivation domain of p53 with high affinity to block p53 regulating responsive gene expression (7). More importantly, MDM2 controls p53 stability by targeting the tumor suppressor protein for ubiquitinmediated constitutive degradation (8-10). Although MDMX lacks E3 ubiquitin ligase activity, the MDM2 homologue acts as an effective transcriptional antagonist of p53, and nonredundantly impedes p53-induced growth inhibitory and apoptotic responses (4, 5). In addition, MDMX forms heterodimers with MDM2 through their C-terminal RING finger domains, stimulating MDM2-mediated ubiquitination and degradation of p53 and MDMX itself (11-13). The interplay between MDM2 and MDMX confers a robust p53 inactivation in tumorigenesis (14).Recent studies show that restoring endogenous p53 activity can halt the growth of cancerous tumors in mice through cell typedependent multiple mechanisms, including apoptosis, senescence, and senescence-triggered innate inflammatory responses (15-17). Thus, antagonists of MDM2 and MDMX that activate the p53 pathway can potentially be developed into a class of therapeutic agents for cancer treatment (14). Much of the current efforts have been focused on combinatorial library search for and structurebased rational design of low molecular weight antagonists of MDM2 (18). Successful examples include a cis-imidazoline analogue, termed nutlin-3, and, a spiro-oxindole-derived compound, termed 20). For optimal efficacy, however, dual specific inhibitors may be needed to target both MDM2 and MDMX (14).We previously reported the synthesis of the p53...

The increasing prevalence of antibacterial resistance globally underscores the urgent need to the update of antibiotics. Here, we describe a strategy for inducing the self-assembly of a host-defense antimicrobial peptide (AMP) into nanoparticle antibiotics (termed nanobiotics) with significantly improved pharmacological properties. Our strategy involves the myristoylation of human alpha-defensin 5 (HD5) as a therapeutic target and subsequent self-assembly in aqueous media in the absence of exogenous excipients. Compared with its parent HD5, the C-terminally myristoylated HD5 (HD5-myr)-assembled nanobiotic exhibited significantly enhanced broad-spectrum bactericidal activity in vitro. Mechanistically, it selectively killed Escherichia coli (E. coli) and methicillin-resistant Staphylococcus aureus (MRSA) through disruption of the cell wall and/or membrane structure. The in vivo results further demonstrated that the HD5-myr nanobiotic protected against skin infection by MRSA and rescued mice from E. coli-induced sepsis by lowering the systemic bacterial burden and alleviating organ damage. The self-assembled HD5-myr nanobiotic also showed negligible hemolytic activity and substantially low toxicity in animals. Our findings validate this design rationale as a simple yet versatile strategy for generating AMP-derived nanobiotics with excellent in vivo tolerability. This advancement will likely have a broad impact on antibiotic discovery and development efforts aimed at combating antibacterial resistance.