Donna McEachern

Temporal activation of p53 by a specific MDM2 inhibitor is selectively toxic to tumors and leads to complete tumor growth inhibition

We have designed MI-219 as a potent, highly selective and orally active small-molecule inhibitor of the MDM2–p53 interaction. MI-219 binds to human MDM2 with a Ki value of 5 nM and is 10,000-fold selective for MDM2 over MDMX. It disrupts the MDM2–p53 interaction and activates the p53 pathway in cells with wild-type p53, which leads to induction of cell cycle arrest in all cells and selective apoptosis in tumor cells. MI-219 stimulates rapid but transient p53 activation in established tumor xenograft tissues, resulting in inhibition of cell proliferation, induction of apoptosis, and complete tumor growth inhibition. MI-219 activates p53 in normal tissues with minimal p53 accumulation and is not toxic to animals. MI-219 warrants clinical investigation as a new agent for cancer treatment.

Endocrine-Therapy-Resistant ESR1 Variants Revealed by Genomic Characterization of Breast-Cancer-Derived Xenografts

To characterize patient-derived xenografts (PDXs) for functional studies, we made whole-genome comparisons with originating breast cancers representative of the major intrinsic subtypes. Structural and copy number aberrations were found to be retained with high fidelity. However, at the single-nucleotide level, variable numbers of PDX-specific somatic events were documented, although they were only rarely functionally significant. Variant allele frequencies were often preserved in the PDXs, demonstrating that clonal representation can be transplantable. Estrogen-receptor-positive PDXs were associated with ESR1 ligand-binding-domain mutations, gene amplification, or an ESR1/YAP1 translocation. These events produced different endocrine-therapy-response phenotypes in human, cell line, and PDX endocrine-response studies. Hence, deeply sequenced PDX models are an important resource for the search for genome-forward treatment options and capture endocrine-drug-resistance etiologies that are not observed in standard cell lines. The originating tumor genome provides a benchmark for assessing genetic drift and clonal representation after transplantation.

A potent and orally active antagonist (SM-406/AT-406) of multiple inhibitor of apoptosis proteins (IAPs) in clinical development for cancer treatment.

We report the discovery and characterization of SM-406 (compound 2), a potent and orally bioavailable Smac mimetic and an antagonist of the inhibitor of apoptosis proteins (IAPs). This compound binds to XIAP, cIAP1, and cIAP2 proteins with K(i) of 66.4, 1.9, and 5.1 nM, respectively. Compound 2 effectively antagonizes XIAP BIR3 protein in a cell-free functional assay, induces rapid degradation of cellular cIAP1 protein, and inhibits cancer cell growth in various human cancer cell lines. It has good oral bioavailability in mice, rats, non-human primates, and dogs, is highly effective in induction of apoptosis in xenograft tumors, and is capable of complete inhibition of tumor growth. Compound 2 is currently in phase I clinical trials for the treatment of human cancer.

SM-164: a novel, bivalent Smac mimetic that induces apoptosis and tumor regression by concurrent removal of the blockade of cIAP-1/2 and XIAP.

Small-molecule Smac mimetics are being developed as a novel class of anticancer drugs. Recent studies have shown that Smac mimetics target cellular inhibitor of apoptosis protein (cIAP)-1/2 for degradation and induce tumor necrosis factor-alpha (TNFalpha)-dependent apoptosis in tumor cells. In this study, we have investigated the mechanism of action and therapeutic potential of two different types of novel Smac mimetics, monovalent SM-122 and bivalent SM-164. Our data showed that removal of cIAP-1/2 by Smac mimetics or small interfering RNA is not sufficient for robust TNFalpha-dependent apoptosis induction, and X-linked inhibitor of apoptosis protein (XIAP) plays a critical role in inhibiting apoptosis induction. Although SM-164 is modestly more effective than SM-122 in induction of cIAP-1/2 degradation, SM-164 is 1,000 times more potent than SM-122 as an inducer of apoptosis in tumor cells, which is attributed to its much higher potency in binding to and antagonizing XIAP. SM-164 induces rapid cIAP-1 degradation and strong apoptosis in the MDA-MB-231 xenograft tumor tissues and achieves tumor regression, but has no toxicity in normal mouse tissues. Our study provides further insights into the mechanism of action for Smac mimetics and regulation of apoptosis by inhibitor of apoptosis proteins. Furthermore, our data provide evidence that SM-164 is a promising new anticancer drug for further evaluation and development.

A potent small-molecule inhibitor of the MDM2-p53 interaction (MI-888) achieved complete and durable tumor regression in mice.

We previously reported the discovery of a class of spirooxindoles as potent and selective small-molecule inhibitors of the MDM2-p53 interaction (MDM2 inhibitors). We report herein our efforts to improve their pharmacokinetic properties and in vivo antitumor activity. Our efforts led to the identification of 9 (MI-888) as a potent MDM2 inhibitor (Ki = 0.44 nM) with a superior pharmacokinetic profile and enhanced in vivo efficacy. Compound 9 is capable of achieving rapid, complete, and durable tumor regression in two types of xenograft models of human cancer with oral administration and represents the most potent and efficacious MDM2 inhibitor reported to date.

Potent and Orally Active Small-Molecule Inhibitors of the MDM2−p53 Interaction

We report herein the design of potent and orally active small-molecule inhibitors of the MDM2-p53 interaction. Compound 5 binds to MDM2 with a K(i) of 0.6 nM, activates p53 at concentrations as low as 40 nM, and potently and selectively inhibits cell growth in tumor cells with wild-type p53 over tumor cells with mutated/deleted p53. Compound 5 has a good oral bioavailability and effectively inhibits tumor growth in the SJSA-1 xenograft model.

Diastereomeric spirooxindoles as highly potent and efficacious MDM2 inhibitors.

Small-molecule inhibitors that block the MDM2-p53 protein-protein interaction (MDM2 inhibitors) are being intensely pursued as a new therapeutic strategy for cancer treatment. We previously published a series of spirooxindole-containing compounds as a new class of MDM2 small-molecule inhibitors. We report herein a reversible ring-opening-cyclization reaction for some of these spirooxindoles, which affords four diastereomers from a single compound. Our biochemical binding data showed that the stereochemistry in this class of compounds has a major effect on their binding affinities to MDM2, with >100-fold difference between the most potent and the least potent stereoisomers. Our study has led to the identification of a set of highly potent MDM2 inhibitors with a stereochemistry that is different from that of our previously reported compounds. The most potent compound (MI-888) binds to MDM2 with a Ki value of 0.44 nM and achieves complete and long-lasting tumor regression in an animal model of human cancer.

SAR405838: An Optimized Inhibitor of MDM2–p53 Interaction That Induces Complete and Durable Tumor Regression

Blocking the oncoprotein murine double minute 2 (MDM2)-p53 protein-protein interaction has long been considered to offer a broad cancer therapeutic strategy, despite the potential risks of selecting tumors harboring p53 mutations that escape MDM2 control. In this study, we report a novel small-molecule inhibitor of the MDM2-p53 interaction, SAR405838 (MI-77301), that has been advanced into phase I clinical trials. SAR405838 binds to MDM2 with K(i) = 0.88 nmol/L and has high specificity over other proteins. A cocrystal structure of the SAR405838:MDM2 complex shows that, in addition to mimicking three key p53 amino acid residues, the inhibitor captures additional interactions not observed in the p53-MDM2 complex and induces refolding of the short, unstructured MDM2 N-terminal region to achieve its high affinity. SAR405838 effectively activates wild-type p53 in vitro and in xenograft tumor tissue of leukemia and solid tumors, leading to p53-dependent cell-cycle arrest and/or apoptosis. At well-tolerated dose schedules, SAR405838 achieves either durable tumor regression or complete tumor growth inhibition in mouse xenograft models of SJSA-1 osteosarcoma, RS4;11 acute leukemia, LNCaP prostate cancer, and HCT-116 colon cancer. Remarkably, a single oral dose of SAR405838 is sufficient to achieve complete tumor regression in the SJSA-1 model. Mechanistically, robust transcriptional upregulation of PUMA induced by SAR405838 results in strong apoptosis in tumor tissue, leading to complete tumor regression. Our findings provide a preclinical basis upon which to evaluate SAR405838 as a therapeutic agent in patients whose tumors retain wild-type p53.

Reactivation of p53 by a specific MDM2 antagonist (MI-43) leads to p21-mediated cell cycle arrest and selective cell death in colon cancer

MDM2 oncoprotein binds directly to the p53 tumor suppressor and inhibits its function in cancers retaining wild-type p53. Blocking this interaction using small molecules is a promising approach to reactivate p53 function and is being pursued as a new anticancer strategy. The spiro-oxindole MI-43, a small-molecule inhibitor of the MDM2-p53 interaction, was designed and examined for its cellular mechanism of action and therapeutic potential in colon cancer. MI-43 binds to MDM2 protein with a Ki value of 18 nmol/L and is 300 times more potent than a native p53 peptide. MI-43 blocks the intracellular MDM2-p53 interaction and induces p53 accumulation in both normal and cancer cells, with wild-type p53 without causing p53 phosphorylation. Induction of p53 leads to modulation of the expression of p53 target genes, including up-regulation of p21 and MDM2 in normal primary human cells and in colon cancer cells with wild-type p53. Using HCT-116 isogenic colon cancer cell lines differing only in p53 status or RNA interference to knockdown expression of p53 in the RKO colon cancer cell line, we show that the cell growth inhibition and cell death induction by MI-43 is p53 dependent. Furthermore, induction of cell cycle arrest by MI-43 is dependent on p53 and p21. In normal cells, MI-43 induces cell cycle arrest but not apoptosis. This study suggests that p53 activation by a potent and specific spiro-oxindole MDM2 antagonist may represent a promising therapeutic strategy for the treatment of colon cancer and should be further evaluated in vivo and in the clinic. [Mol Cancer Ther 2008;7(6):1533–42]

Therapeutic potential and molecular mechanism of a novel, potent, nonpeptide, Smac mimetic SM-164 in combination with TRAIL for cancer treatment

Smac mimetics are being developed as a new class of anticancer therapies. Because the single-agent activity of Smac mimetics is very limited, rational combinations represent a viable strategy for their clinical development. The combination of Smac mimetics with TNF-related apoptosis inducing ligand (TRAIL) may be particularly attractive because of the low toxicity of TRAIL to normal cells and the synergistic antitumor activity observed for the combination. In this study, we have investigated the combination synergy between TRAIL and a potent Smac mimetic, SM-164, in vitro and in vivo and the underlying molecular mechanism of action for the synergy. Our study shows that SM-164 is highly synergistic with TRAIL in vitro in both TRAIL-sensitive and TRAIL-resistant cancer cell lines of breast, prostate, and colon cancer. Furthermore, the combination of SM-164 with TRAIL induces rapid tumor regression in vivo in a breast cancer xenograft model in which either agent is ineffective. Our data show that X-linked IAP (XIAP) and cellular IAP 1 (cIAP1), but not cIAP2, work in concert to attenuate the activity of TRAIL; SM-164 strongly enhances TRAIL activity by concurrently targeting XIAP and cIAP1. Moreover, although RIP1 plays a minimal role in the activity of TRAIL as a single agent, it is required for the synergistic interaction between TRAIL and SM-164. This study provides a strong rationale to develop the combination of SM-164 and TRAIL as a new therapeutic strategy for the treatment of human cancer. Mol Cancer Ther; 10(5); 902–14. ©2011 AACR.