MDM2 binds the p53 tumor suppressor protein with high affinity and negatively modulates its transcriptional activity and stability. Overexpression of MDM2, found in many human tumors, effectively impairs p53 function. Inhibition of MDM2-p53 interaction can stabilize p53 and may offer a novel strategy for cancer therapy. Here, we identify potent and selective small-molecule antagonists of MDM2 and confirm their mode of action through the crystal structures of complexes. These compounds bind MDM2 in the p53-binding pocket and activate the p53 pathway in cancer cells, leading to cell cycle arrest, apoptosis, and growth inhibition of human tumor xenografts in nude mice.

Stapled α−helical peptides have emerged as a promising new modality for a wide range of therapeutic targets. 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. 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. 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. 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. Overall, ATSP-7041 demonstrates in vitro and in vivo proofof-concept that stapled peptides can be developed as therapeutically relevant inhibitors of protein-protein interaction and may offer a viable modality for cancer therapy.T he 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). 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). 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).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). Consequently, aberrant MDM2 overexpression and gene amplification contribute to accelerated cancer development and growth (1, 8). The other negative regulator, MDMX, possesses a similar p53-binding activity and also effectively inhibits p53 transcriptional activity. 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). 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. Given these functional differences, MDM2 and MDMX are each unable to compensate for the loss of the other, and they regulate nonoverlapping fu...

No abstract

Restoration of p53 activity by inhibition of the p53-MDM2 interaction has been considered an attractive approach for cancer treatment. However, the hydrophobic protein-protein interaction surface represents a significant challenge for the development of small-molecule inhibitors with desirable pharmacological profiles. RG7112 was the first small-molecule p53-MDM2 inhibitor in clinical development. Here, we report the discovery and characterization of a second generation clinical MDM2 inhibitor, RG7388, with superior potency and selectivity.