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Measuring Caspase Activity Using a Fluorometric Assay or Flow Cytometry
Published on: March 24, 2023
Pharmacology of smac mimetics; chemotype differentiation based on physical association with caspase regulators and
Randy L Talbott1, Robert M Borzilleri1, Charu Chaudhry1
1Bristol-Myers Squibb Research, PO Box 4000, Princeton, NJ 08543-4000, USA.
Abstract:
Cellular levels of inhibitor of apoptosis (IAP) proteins are elevated in multiple human cancers and their activities often play a part in promoting cancer cell survival by blocking apoptotic pathways, controlling signal transduction pathways and contributing to resistance. These proteins function through interactions of their BIR (baculoviral IAP repeat) protein domains with pathway components and these interactions are endogenously antagonized by Smac/Diablo (second mitochondrial activator of caspases/direct IAP binding protein with low isoelectric point). This report describes development of synthetic smac mimetics (SM) and compares their binding, antiproliferative and anti-tumor activities. All dimeric antagonists inhibit in vitro smac tetrapeptide binding to recombinant IAP proteins, rescue IAP-bound caspase-3 activity and show anti-proliferative activity against human A875 melanoma cells. One heterodimeric SM, SM3, binds tightly to IAP proteins in vitro and slowly dissociates (greater than two hours) from these protein complexes compared to the other antagonists. In addition, in vitro SM anti-proliferation potency is influenced by ABCB1 transporter (ATP-binding cassette, sub-family B; MDR1, P-gp) activities and one antagonist, SM5, does not appear to be an ABCB1 efflux pump substrate. All dimeric smac mimetics inhibit the growth of human melanoma A875 tumors implanted in athymic mice at well-tolerated doses. One antagonist, SM4, shows broad spectrum in vivo anti-tumor activity and modulates known pharmacodynamic markers of IAP antagonism. These data taken together demonstrate the range of diverse dimeric IAP antagonist activities and supports their potential as anticancer agents.
Insights
Synthetic Smac mimetics (SM) were developed to target inhibitor of apoptosis (IAP) proteins, crucial in cancer survival. These SMs demonstrate significant anti-cancer potential by inhibiting IAP activity and reducing tumor growth.
Area of Science:
- Biochemistry
- Molecular Biology
- Oncology
Background:
- Inhibitor of apoptosis (IAP) proteins are upregulated in cancers, promoting cell survival by inhibiting apoptosis.
- IAP proteins interact with cellular components via their baculoviral IAP repeat (BIR) domains.
- Endogenous antagonists, Smac/Diablo, counteract IAP function.
Purpose of the Study:
- To develop synthetic Smac mimetics (SM) as potential anticancer agents.
- To compare the binding, antiproliferative, and anti-tumor activities of novel SMs.
- To evaluate the therapeutic potential of SMs in preclinical cancer models.
Main Methods:
- Development and characterization of dimeric Smac mimetics (SM).
- In vitro assays to assess binding to IAP proteins and rescue of caspase-3 activity.
- In vitro antiproliferative assays using human melanoma cells (A875).
- In vivo studies evaluating anti-tumor efficacy in athymic mouse xenograft models.
Main Results:
- All developed dimeric SMs inhibited Smac/Diablo binding to IAPs and showed antiproliferative effects.
- SM3 exhibited tight binding and slow dissociation from IAP proteins.
- SM5 was identified as not being an ABCB1 efflux pump substrate, potentially overcoming drug resistance.
- SM4 demonstrated broad-spectrum in vivo anti-tumor activity and modulated IAP antagonism markers.
Conclusions:
- Synthetic dimeric Smac mimetics are effective inhibitors of IAP proteins.
- SMs exhibit promising antiproliferative and anti-tumor activities in preclinical models.
- These findings support the potential of Smac mimetics as a novel class of anticancer therapeutics.
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