Split luciferase reporters of apoptosome formation: a bio-tool to identify new drug-like molecules

Formation of apoptosome, a key multiprotein complex in mitochondrial-mediated apoptosis, is an essential step during normal development and deregulation of this event is associated with pathological conditions. The apoptosome contains seven Apoptotic Protease‐Activating Factor‐1 (Apaf‐1) molecules and induces cell death by activating caspase‐9. Apoptosome is a protein target for therapeutic approaches. However, the lack of suitable screening assay to directly identify new modulators of apoptosome has limited the field. Here, I define a new split luciferase reporters of apoptosome for in vitro studying the apoptosome. In this assay, upon apoptosome formation Apaf‐1 fused to an N‐terminal fragment of luciferase binds to Apaf‐1 fused to a C‐terminal fragment of luciferase and reconstitutes luciferase activity. The assay was validated by showing the cytochrome c/dATP-dependent luciferase activity, inhibition of the luciferase activity by apoptosome inhibitor (NS3694) and contribution of the fusion proteins in the complex with the expected molecular weight. This assay was then used to screen a panel of compounds to identify the inhibitors of apoptosome. Screening a persistent organic pollutant library indicated that pentachlorophenol (PCP) inhibits apoptosome formation, and further investigation revealed that PCP binds to cytochrome c. Screening a library of natural products also identified a new chemical with strong inhibitory effect, although at high concentrations. This reporter was also used to study the mechanism of inhibition by NS3694 and M50054. NS3694 is an inhibitor of apoptosome. I showed that the main target of NS3694 is cytochrome c rather than apoptosome. M50054 was also known to inhibit caspase activation. However, the mechanism and the direct target was unknown. I showed that, unlike NS3694, M50054 mainly targets apoptosome complex. The data demonstrate the utility of the new assay in identifying apoptosome inhibitors, and I suggest that the assay may be useful in screening a large chemical collection. Luciferase-based reporters provide a quick, easy, and cost-effective in vitro assay.

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NUI Galway

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Attribution-NonCommercial-NoDerivs 3.0 Ireland

cbn

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University of Galway Theses (PhD Theses)