AI-guided drug-discovery of small molecule inhibitors of Apoptotic Protease Activating Factor-1

Apoptotic protease activating factor-1 (Apaf-1) is a central regulator of intrinsic apoptosis and mediates apoptosome assembly following cytochrome c release from mitochondria. Dysregulated activation of this pathway contributes to pathological cell loss in conditions including neurodegeneration, ischemic injury, and ototoxicity, making Apaf-1 a potential therapeutic target. However, pharmacological inhibition of apoptosome signalling remains challenging due to the structural and mechanistic properties of Apaf-1. This thesis investigated two complementary strategies for targeting Apaf-1 signalling: disruption of the Apaf-1:Caspase-9 CARD-CARD interaction and targeting of the Apaf-1 nucleotide-binding pocket. To address these aims, NanoBiT split-luciferase complementation assays were developed to monitor CARD-domain interactions and apoptosome-associated signalling in biochemical and mammalian cell systems. These approaches were combined with recombinant protein purification, peptide inhibition studies, Caspase activity assays, computational docking, alanine-scanning analyses, and AI-assisted virtual screening workflows. NanoBiT assays successfully detected specific interactions between Apaf-1 and Caspase-9 CARD domains and enabled evaluation of peptide and small-molecule inhibitors. Several literature-reported Apaf-1 inhibitors reduced NanoBiT signal intensity; however, additional biochemical analyses demonstrated that some compounds also inhibited Caspase-9 independently of Apaf-1, highlighting the importance of mechanistic specificity controls. Computational analyses further suggested that the Apaf-1:Caspase-9 CARD interface represents a structurally challenging target for conventional small-molecule inhibition. 19 To explore alternative approaches, AI-guided virtual screening was used to identify compounds predicted to interact with the Apaf-1 nucleotide-binding region. Selected compounds were evaluated using mini-apoptosome reconstitution assays and mammalian-cell NanoBiT systems, where several compounds reduced apoptosome-associated signalling outputs. Overall, this work establishes complementary experimental platforms for studying Apaf-1 signalling and provides further insight into the opportunities and limitations associated with pharmacological modulation of the apoptosome. The findings support continued investigation of Apaf-1 as a therapeutic target while emphasising the importance of mechanistic validation and pathway specificity in apoptosome-directed drug discovery.

Funder

University of Galway

Publisher

University of Galway

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CC BY-NC-ND

cbn

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University of Galway Theses (PhD Theses)