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...

The tumor suppressor p53 is often inactivated via its interaction with endogenous inhibitors mouse double minute 4 homolog (MDM4 or MDMX) or mouse double minute 2 homolog (MDM2), which are frequently overexpressed in patients with acute myeloid leukemia (AML) and other cancers. Pharmacological disruption of both of these inter-actions has long been sought after as an attractive strategy to fully restore p53-dependent tumor suppressor activity in cancers with wild-type p53. Selective targeting of this pathway has thus far been limited to MDM2-only small-molecule inhibitors, which lack affinity for MDMX. We demonstrate that dual MDMX/MDM2 inhibition with a stapled a-helical peptide (ALRN-6924), which has recently entered phase I clinical testing, produces marked antileukemic effects. ALRN-6924 robustly activates p53-dependent transcription at the single-cell and single-molecule levels and exhibits biochemical and molecular biological on-target activity in leukemia cells in vitro and in vivo. Dual MDMX/MDM2 inhibition by ALRN-6924 inhibits cellular proliferation by inducing cell cycle arrest and apoptosis in cell lines and primary AML patient cells, including leukemic stem cell-enriched populations, and disrupts functional clonogenic and serial replating capacity. Furthermore, ALRN-6924 markedly improves survival in AML xenograft models. Our study provides mechanistic insight to support further testing of ALRN-6924 as a therapeutic approach in AML and other cancers with wild-type p53.

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To realize the full potential of combinatorial chemistry-based drug discovery, generic and efficient tools must be developed that apply the strengths of diversity-oriented chemical synthesis to the identification and optimization of lead compounds for disease-associated protein targets. We report an affinity selection-mass spectrometry (AS-MS) method for protein-ligand affinity ranking and the classification of ligands by binding site. The method incorporates the following steps: (1) an affinity selection stage, where protein-binding compounds are selected from pools of ligands in the presence of varying concentrations of a competitor ligand, (2) a first chromatography stage to separate unbound ligands from protein-ligand complexes, and (3) a second chromatography stage to dissociate the ligands from the complexes for identification and quantification by MS. The ability of the competitor ligand to displace a target-bound library member, as measured by MS, reveals the binding site classification and affinity ranking of the mixture components. The technique requires no radiolabel incorporation or direct biochemical assay, no modification or immobilization of the compounds or target protein, and all reaction components, including any buffers or cofactors required for protein stability, are free in solution. We demonstrate the method for several compounds of wide structural variety against representatives of the most important protein classes in contemporary drug discovery, including novel ATP-competitive and allosteric inhibitors of the Akt-1 (PKB) and Zap-70 kinases, and previously undisclosed antagonists of the M(2) muscarinic acetylcholine receptor, a G-protein coupled receptor (GPCR). The theoretical basis of the technique is analyzed mathematically, allowing quantitative estimation of binding affinities and, in the case of allosteric interaction, absolute determination of binding cooperativity. The method is readily applicable to high-throughput screening hit triage, combinatorial library-based affinity optimization, and developing structure-activity relationships among multiple ligands to a given receptor.

A small molecule nonpeptide inhibitor of beta-secretase has been developed, and its binding has been defined through crystallographic determination of the enzyme-inhibitor complex. The molecule is shown to bind to the catalytic aspartate residues in an unprecedented manner in the field of aspartyl protease inhibition. Additionally, the complex reveals a heretofore unknown S(3) subpocket that is created by the inhibitor. This structure has served an important role in the design of newer beta-secretase inhibitors.