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EPIC Report Summary

Project ID: 690939
Funded under: H2020-EU.1.3.3.

Periodic Reporting for period 1 - EPIC (Exploiting Protein Complexes that Induce Cell-death)

Reporting period: 2016-01-01 to 2017-12-31

Summary of the context and overall objectives of the project

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 and necroptosis. All PCD processes appear to be regulated by the formation of large protein complexes that are required to initiate cell death processes. Thus, the formation of a complex called the apoptosome induces apoptosis and formation of the necrosome induces necroptosis. 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.

The EPIC project is bringing together researchers with complementary skills and ideas in the fields of cell death, pharmacology, screening, structural biology and natural product chemistry in a network that will exchange reagents, technologies and knowledge. This cross-sectoral network is building a synergistic environment and making new tools for investigating cell-death processes involved in human disease. It has supported the generation of innovative assays to enable the identification of new classes of therapeutics and is providing international, cross-sectoral and multi-disciplinary training opportunities.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

In the first twenty four months of the project teams working mainly in the National University of Ireland Galway (NUIG) and the Flanders Institute for Biotechnology (VIB) but also in a company (ProtoQSAR, Spain) and at the University of Gothenberg (Sweden) have hosted visiting researchers from Iran, Spain and Ireland. The exchange of staff and expertise has allowed the development of new tools to detect the interaction between proteins involved in apoptosis (Apaf-1 and caspase-9) and necroptosis (RIP Kinases but also other proteins involved in necroptosis) using split luciferase complementary assays. These new experimental tools will form the basis of new assays that can be used to discover new drug-like chemicals that are activators or inhibitors of cell death processes.

The main results so far are:

Apaf-1 interactions (NUI Galway 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. Screening of a unique library of marine natural products collected in the North Atlantic by an Irish funded marine biodiscovery programme.
6. Identification of an Apaf-1 inhibitor produced by an uncharacterised cnidarian collected at 1000 m.

Caspase-9 interactions (NUI Galway and Tarbiat Modares University)

1. Construction of caspase-9 split luciferase reporters that allow validation of the assay.
2. Testing the reporters shows that the reporters do not detect either caspase-9 - caspase -9 nor caspase-9 - Apaf-1 interactions. Further work is underway to modify and then re-test the reporters.

RIP kinase and necrosome interactions (VIB and Tarbiat Modares University)

1. Construction of a panel of reporters (RIPK1, RIPK3, FADD, TRADD).
2. Testing the reporters shows that the reporters do not detect either interactions associated with necroptosis. Further work is underway to modify and then re-test the reporters.

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.
2. Initial analysis of Apaf-1 activity assay data. Further exploration of ‘chemical space’ adjacent to active compounds is needed to increase discriminative power.
3. In silico identification of potential ligand binding sites on Apaf-1 and in silico screening of a 10,000 compound diversity set.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

Apaf-1 has been shown to play roles in different cell events such as cell death, neuronal development, cell cycle arrest and spermatogenesis. In the current study, we established an assay based on split luciferase reporters to investigate the homotypic Apaf-1 interactions in vitro. The initial aim of this study was to develop a new tool to directly study the apoptosome complex formation which could be further used for identification of new inhibitors. The new tools are a significant advance over the current state of the art as they permit high throughput screening of molecular libraries; an approach that was previously impossible.

The new tools have allowed us to identify a persistent organic pollutant and known reproductive toxicant as an Apaf-1 inhibitor, a new discovery that provides a molecular explanation for this compound's toxicity. Thus the new tools allow detection of a whole new class of reproductive toxicants that could not be previously detected by high throughput screening and the new assay can be added to the in vitro tests used in hazard identification and risk assessment.

The new tools have also allowed us to identify a novel chemical produced by a deep-sea marine animal that is also an Apaf-1 inhibitor. This molecule will be further investigated to determine whether it can be a lead molecule for the development of a new therapeutic agent to treat deafness caused by cancer chemotherapy.

All this work has been complemented by computational approaches to identify new ligands for Apaf-1. These in silico discoveries are now being validated in the real world using the new experimental tools we have developed.

The progress made with Apaf-1 reporters has demonstrated the utility of the approach taken by EPIC researchers, who will now build on this success to make and test new reporters for other cell death pathways.
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