2019 EHA: Characterization of a novel, potent small molecule MDM2 antagonist which activates wild-type p53 and induces apoptosis in AML

Background: In the presence of various stress signals, p53 acts as a tumor suppressor by regulating the expression of a multitude of genes to elicit cellular responses such as cell cycle arrest and apoptosis. The activity of p53 is tightly regulated by MDM2, an E3 ubiquitin ligase that acts as a primary inhibitor of p53 function by, for example, targeting p53 for proteasomal degradation. Early studies have demonstrated that blocking the MDM2-p53 interaction in tumors carrying wild-type p53 prevents p53 degradation and reactivates it. Small molecule MDM2 antagonists that inhibit the MDM2-p53 interaction, therefore, present a promising strategy for cancer therapy and a number of these compounds are in clinical development.
Aims: Herein, we describe the characterization of a novel, potent small molecule MDM2 antagonist in AML in vitro and in vivo pre-clinical models and in patient-derived AML blast cells.
Methods: A panel of p53 wild-type AML cell lines was tested for reduction in cell proliferation using Alamar Blue assay following treatment with the compound. Induction of apoptosis was measured by flow cytometry using a fluorescent caspase-3 substrate or Annexin V. Target engagement was analyzed by Western Blotting and TaqMan qRT-PCR. The MV-4-11 mouse systemic model was used to test in vivo sensitivity to the compound. Primary AML blasts were isolated from patients using combinations of antibodies against CD34, CD33, CD45, and CD117.
Results: We have applied structure-based design to develop a novel, potent, orally bioavailable MDM2 antagonist. The compound exhibits EC50 <1 nM against the full-length MDM2 protein in a cell-free ELISA and increases p53 levels in a wide range of p53 wild-type cells (e.g. EC50=10 nM for p53 induction in SJSA-1 osteosarcoma cells).
When tested in a panel of p53 wild-type AML cell lines, the compound exerted a strong anti-proliferative effect with GI50 values of <30 nM being observed in 9 out of 11 cell lines. In contrast, the compound had little effect on p53 mutant KG-1 cells (GI50 >10 M). In addition, many of the p53 wild-type AML cell lines showed a strong induction of apoptosis in response to treatment with the compound. Activation of p53 was evident by an increase in the expression of p53 and that of its well-known transcriptional targets such as p21 and MDM2. Consistent with these findings, a detailed study of gene expression changes in MV-4-11 confirmed clear transcriptional activation of several p53 target genes (CDKN1A, MDM2, BBC3, FAS, GADD45, BAX) 2-6 hours after addition of the compound.
In accordance with its potent activity in vitro, the compound displayed significant in vivo efficacy in the MV-4-11 mouse systemic model of AML. Here, QDx14 oral dosing at well tolerated doses demonstrated a clear reduction in tumor burden. Furthermore, p53 activation by the compound triggered apoptosis when tested in primary AML blast cells isolated from patients.
Summary/Conclusion: Taken together, our findings demonstrate that the compound exhibits potent activity against AML cells that retain wild-type p53, thus meriting further clinical investigations.

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Characterization of a novel, potent small molecule MDM2 antagonist which activates wild-type p53 and induces apoptosis in AML

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Johnson et al., “A Fragment-Derived Clinical Candidate for Antagonism of X-Linked and Cellular Inhibitor of Apoptosis Proteins: 1-(6-[(4-Fluorophenyl)methyl]-5-(hydroxymethyl)-3,3-dimethyl-1H,2H,3H-pyrrolo[3,2-b]pyridin-1-yl)-2-[(2R,5R)-5-methyl-2-([(3R)-3-methylmorpholin-4-yl]methyl)piperazin-1-yl]ethan-1-one (ASTX660).” J. Med. Chem., 2018, DOI: 10.1021/acs.jmedchem.8b00900

Abstract
Inhibitor of apoptosis proteins (IAPs) are promising anticancer targets, given their roles in the evasion of apoptosis. Several peptidomimetic IAP antagonists, with inherent selectivity for cellular IAP (cIAP) over X-linked IAP (XIAP), have been tested in the clinic. A fragment screening approach followed by structure-based optimization has previously been reported that resulted in a low-nanomolar cIAP1 and XIAP antagonist lead molecule with a more balanced cIAP–XIAP profile. We now report the further structure-guided optimization of the lead, with a view to improving the metabolic stability and cardiac safety profile, to give the nonpeptidomimetic antagonist clinical candidate 27 (ASTX660), currently being tested in a phase 1/2 clinical trial (NCT02503423).

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Johnson et al., “A Fragment-Derived Clinical Candidate for Antagonism of X-Linked and Cellular Inhibitor of Apoptosis Proteins: 1-(6-[(4-Fluorophenyl)methyl]-5-(hydroxymethyl)-3,3-dimethyl-1H,2H,3H-pyrrolo[3,2-b]pyridin-1-yl)-2-[(2R,5R)-5-methyl-2-([(3R)-3-methylmorpholin-4-yl]methyl)piperazin-1-yl]ethan-1-one (ASTX660).” J. Med. Chem., 2018, DOI: 10.1021/acs.jmedchem.8b00900