Chessari at al. Structure-Based Design of Potent and Orally Active Isoindolinone Inhibitors of MDM2-p53 Protein–Protein Interaction; Med Chem 2021
Chessari at al., “Structure-Based Design of Potent and Orally Active Isoindolinone Inhibitors of MDM2-p53 Protein–Protein Interaction”; Med Chem 2021
2020 EHA: ASTX295, a novel small molecule MDM2 antagonist, demonstrates potent activity in AML in combination with decitabine
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ASTX295, a novel small molecule MDM2 antagonist, demonstrates potent activity in AML in combination with decitabine
Abstract:
Background: The tumour suppressor p53 is activated in response to various stress signals to induce transcriptional changes leading to cellular responses such as cell cycle arrest and apoptosis. Activity of p53 is tightly regulated by the E3 ubiquitin ligase MDM2, which inhibits p53 function by, for example, targeting it for proteasomal degradation. Targeting the MDM2-p53 interaction to restore p53 function, is therefore, a promising strategy for cancer therapy and a number of these compounds are in clinical development including ASTX295 (NCT03975387). ASTX295 is a novel, orally bioavailable MDM2 antagonist developed through structure-based drug design that has demonstrated potent activity in a range of p53 wild-type pre-clinical models.
Aims: We investigated the therapeutic potential of ASTX295 alone and in combination with decitabine, a DNA- hypomethylating agent, in AML.
Methods: Primary blasts were isolated from AML patient samples using a combination of antibodies against CD34, CD33, CD45 and CD117. A panel of AML cell lines and primary AML blasts were treated with decitabine and ASTX295 at a range of concentrations, alone and in combination. After treatment, viability was assessed by Alamar blue assay or induction of apoptosis by flow cytometry using a fluorescent caspase substrate or Annexin V.
Effects of drug combinations were analysed using the Combenefit software based on different mathematical models (Loewe, bliss & HSA). Target engagement was confirmed by western blotting.
Results: When tested in a panel of p53 wild-type AML cell lines, ASTX295 exerted a strong anti-proliferative effect in which GI50 <30 nM was observed in 9 out of 11 cell lines. Additionally, p53 activation by ASTX295 triggered apoptosis in both AML cell lines, and primary AML blasts isolated from patients.
Activity of ASTX295 was further enhanced by combining with decitabine. Treatment of AML cell lines with ASTX295 and decitabine showed an increase in growth inhibitory effect and apoptosis compared to respective single agent treatments. This combinatory effect, as assessed by Combenefit, was also observed in primary AML blasts in which 7 of 12 samples tested demonstrated increased apoptosis at or above 300 nM ASTX295 and 100 nM decitabine. Target engagement of ASTX295 and decitabine was confirmed by upregulation of p53 transcriptional targets and decreased DNMT-1 expression.
Summary/Conclusion: Our findings demonstrate that the combination of ASTX295 with decitabine exhibits potent activity against p53 wild-type AML cells, and thus merits further investigation.
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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