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Exploiting Protein Complexes that Induce Cell-death

Periodic Reporting for period 2 - EPIC (Exploiting Protein Complexes that Induce Cell-death)

Reporting period: 2018-01-01 to 2019-12-31

Programmed cell death (PCD) occurs in many pathological situations from inflammation to ischaemia. PCD is also the mechanism through which many anti-cancer therapies act. After 20 years of PCD research it is now clear that there are several distinct PCD pathways, including apoptosis, necroptosis and pyroptosis. Many PCD processes appear to be regulated by the formation of large protein complexes that initiate cell the molecular events that eventually kill the cells. For example, the formation of a complex called the apoptosome induces apoptosis, formation of the necrosome induces necroptosis and formation of the inflammasome causes pyroptosis. As these protein complexes determine cell fate, blocking complex formation in diseases where cell death is unwanted or inducing complex formation in diseases where cell death is desirable could be a valuable therapeutic approach.
Realizing this aim is currently limited by the absence of assays for complex formation that are suitable for screening purposes. Solving this problem requires a multi-disciplinary approach and the integration of research activities that are spread across different research groups. Completion of the project will provide new approaches to detect and investigate protein complexes that determine cell fate, providing new strategies for both drug-discovery and for in vitro toxicity testing.
The EPIC project built a multi-disciplinary and intersectoral team of researchers with complementary skills and ideas in the fields of cell death, pharmacology, screening, structural biology and natural product chemistry. The team cooperated to build and test new biosensors to detect the formation, of complexes associated with apoptosis, necroptosis and pyroptosis. The reagents and knowledge were then shared and used to screen small molecule libraries and unique natural product libraries with the aim of discovering novel regulators of cell death.

The key advances made by researchers on the EPIC project are:
• Construction and validation of a screening assay for apoptosome activators and inhibitors
• Identification of a novel apoptosome inhibitor from a new Zooanthid (a deep sea animal) and elucidation of the inhibitor’s chemical structure
• Identification of the mechanism of action of a small molecule inhibitor of apoptosis
• Identification of the binding sites of small molecules through molecular modelling
• Demonstrating the non-apoptotic role of the apoptosome in cell differentiation
• Construction and validation of biosensors for detecting the interaction of necrosome proteins
• Construction and validation of biosensors for detecting the interaction of inflammasome proteins
Progress beyond state of the art

Apaf-1 interactions (NUI Galway, UGOT and Tarbiat Modares University)
1. Construction of Apaf-1 split luciferase reporters as well as truncation and point mutants of Apaf-1 that allow validation of the assay.
2. Validation of the assay in a cell-free format that allows detection of Apaf-1 - Apaf-1 interactions and that allows high throughput screening of small molecule libraries.
3. Screening of a toxicant library to identify toxicants that block Apaf-1 - Apaf-1 interactions.
4. Identification of a toxicant that blocks Apaf-1 - Apaf-1 interactions, a finding that provides the first molecular explanation for the toxicant's reproductive toxicity.
5. Identification of key Apaf-1 residues that lock Apaf-1 in an inactive conformation by a combination of modelling and slit luciferase complementation assay.
6. Screening of a unique library of marine natural products collected in the North Atlantic by an Irish funded marine biodiscovery programme.
7. Identification of a novel natural product Apaf-1 inhibitor produced by an uncharacterised cnidarian collected from the North Atlantic at a depth of 1000 m.
8. Identification of the mechanism of action of the apoptosis inhibitor M50054.
9. Demonstration of apoptosome formation during muscle cell differentiation.

Caspase-9 interactions (NUI Galway and Tarbiat Modares University)
1. Construction of caspase-9 split luciferase biosensors and detection of caspase-9:Apaf-1 interactions using split luciferase complementation assay.
2. Construction of XIAP split luiciferase biosensors and detection of XIAP: caspase-9 interactions using a split luciferase complementation assay.

RIP kinase and necrosome interactions (VIB, NUI Galway and Tarbiat Modares University)
1. Investigation of biosensors for interactions of homotypic and heterotypic interactions between RIPK1, RIPK3, FADD and TRADD and a demonstration that the interactions are sensitive to known RIPk1 and RIPK3 inhibitors.

In silico work to identify small molecule inhibitors and activators (NUI Galway and ProtoQSAR and ProtoQSAR and UGOT)
1. Development of an automated QSAR pipeline using open source tools an initial analysis of Apaf-1 activity assay data. Further exploration of ‘chemical space’ adjacent to active compounds and in silico identification of potential ligand binding sites on Apaf-1 in silico identification of novel Apaf-1 ligands.

Inflammasome interactions (FVIB, NUI Galway and Tarbiat Modares University)
1. Validation of a split luciferase complementation assay for the inflammasome and a demonstration of its utility for highthroughput screening.