We recently reported a potent and selective small molecule, RO-5963, that effectively inhibits p53 binding to both MDM2 and MDMX via a protein dimerization mechanism of action ( 14). Despite the structural similarity between MDM2 and MDMX, there is sufficient diversity in the p53-binding regions of these proteins to make the development of small-molecule dual antagonists challenging. Although the relative contributions of MDM2 and MDMX to regulation of p53 are not completely understood, several lines of evidence suggest that selective MDM2 antagonists will not be optimally effective in tumors that express high levels of MDMX ( 1, 6, 10, 13). Although three different classes of small-molecule MDM2 antagonists are currently under clinical investigation, one potential limitation of these molecules is that they are all practically inactive against MDMX. The first potent and selective small-molecule inhibitors of the p53–MDM2 interaction, the Nutlins, provided proof of concept that restoration of p53 activity is feasible and may have application in cancer therapy ( 11, 12). Given these functional differences, MDM2 and MDMX are each unable to compensate for the loss of the other, and they regulate nonoverlapping functions of p53 ( 4, 6). MDMX does not have the intrinsic E3 ubiquitin ligase activity of MDM2 and cannot affect p53 stability, but MDM2/MDMX heterodimers can increase ubiquitin ligase activity relative to the MDM2 monomer. Amplification of MDMX is seen in many tumors, including melanoma, breast, head and neck, hepatocellular, and retinoblastoma, and, interestingly, amplification of MDMX appears to correlate with both p53WT status and an absence of MDM2 amplification ( 6, 9, 10). The other negative regulator, MDMX, possesses a similar p53-binding activity and also effectively inhibits p53 transcriptional activity. Consequently, aberrant MDM2 overexpression and gene amplification contribute to accelerated cancer development and growth ( 1, 8). MDM2 negatively regulates p53 function through multiple mechanisms, including direct binding that masks the p53 transactivation domain, impairing nuclear import of the p53 protein, and ubiquitination and proteasomal degradation of the p53 protein ( 6, 7). Cancers that overexpress the inhibitory proteins MDM2 and MDMX also possess wild-type p53 (p53WT), and thus pharmacological disruption of the interactions between p53 and MDM2 and MDMX offers the opportunity to restore p53-dependent cell-cycle arrest and apoptosis in this important class of tumors ( 3 – 6). Inactivation of this guardian of the genome either by deletion or mutation or through overexpression of inhibitory proteins is the most common defect in human cancers ( 1, 2). The human transcription factor protein p53 induces cell-cycle arrest and apoptosis in response to DNA damage and cellular stress and thereby plays a critical role in protecting cells from malignant transformation ( 1, 2). Overall, ATSP-7041 demonstrates in vitro and in vivo proof-of-concept that stapled peptides can be developed as therapeutically relevant inhibitors of protein–protein interaction and may offer a viable modality for cancer therapy. Most importantly, ATSP-7041 demonstrates robust p53-dependent tumor growth suppression in MDM2/MDMX-overexpressing xenograft cancer models, with a high correlation to on-target pharmacodynamic activity, and possesses favorable pharmacokinetic and tissue distribution properties. A high resolution (1.7-Å) X-ray crystal structure reveals its molecular interactions with the target protein MDMX, including multiple contacts with key amino acids as well as a role for the hydrocarbon staple itself in target engagement. Specifically, ATSP-7041 binds both MDM2 and MDMX with nanomolar affinities, shows submicromolar cellular activities in cancer cell lines in the presence of serum, and demonstrates highly specific, on-target mechanism of action. Here, we report a potent and selective dual inhibitor of MDM2 and MDMX, ATSP-7041, which effectively activates the p53 pathway in tumors in vitro and in vivo. Stapled α−helical peptides have emerged as a promising new modality for a wide range of therapeutic targets.
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