Periodic Reporting for period 1 - VIDEC (Visualizing Death Inducing Protein Complexes)
Reporting period: 2020-01-01 to 2023-06-30
The project contributes to the international and inter sectoral integration of European research through a programme that supports researcher mobility between European countries, Third Countries and between industry and academia. This mobility supports knowledge exchange and researcher training through projects focused on human diseases and drug-development, with the aim of finding new treatments for disease. This research is also more widely relevant to areas that include increased food security by improving aquaculture and better protection from chemical hazards by improved methods of toxicity assessment.
The disease research focusses on problems particularly relevant to an aging population (like Europe’s) and include neurodegenerative diseases (e.g. Parkinson’s disease), cancer (e.g. Diffuse Large B Cell Lymphoma), and inflammation. This is being accomplished by studying how proteins bind to each other to cause human disease and then searching form drug-like chemicals to block the binding and cure the disease.
The specific protein-protein interactions being investigated are the binding of Apaf-1 to itself and to caspase-9, the binding of CARD11 to BCL10, the binding of RIPK1 to RIPK1, the binding of NLRP3 to itself and to caspase-1, the binding of ASC to caspase-1 and the binding of caspase-2 to RAIDD. The overall objective is to identify at least one drug-like molecule that blocks a protein-protein interaction and that reduces or prevents disease-associated changes.
The disease research focusses on problems particularly relevant to an aging population (like Europe’s) and include neurodegenerative diseases (e.g. Parkinson’s disease), cancer (e.g. Diffuse Large B Cell Lymphoma), and inflammation. This is being accomplished by studying how proteins bind to each other to cause human disease and then searching form drug-like chemicals to block the binding and cure the disease.
The specific protein-protein interactions being investigated are the binding of Apaf-1 to itself and to caspase-9, the binding of CARD11 to BCL10, the binding of RIPK1 to RIPK1, the binding of NLRP3 to itself and to caspase-1, the binding of ASC to caspase-1 and the binding of caspase-2 to RAIDD. The overall objective is to identify at least one drug-like molecule that blocks a protein-protein interaction and that reduces or prevents disease-associated changes.
Work performed to the end of this period:
1. In silico modelling of protein-protein interactions (Caspase-2 – RAIDD and Caspase-9 – Apaf-1) and the use of the models in AI-guided drug discovery to identify new inhibitors of protein-protein interactions.
The protein-protein interactions (PPIs) important in diverse diseases have been computationally modelled to identify the structural basis of the interaction and to build a tool to search for chemical inhibitors. So far two of a planned four interactions have been successfully modelled and one model has been successfully used to screen a virtual library of chemical to find drug-like molecules. Further testing of these molecules is the first step towards developing new treatments.
2. Development of new assays to quickly and cheaply assess putative inhibitors identified by AI-guided drug discovery
Further testing requires new assays to measure the effect of the drug-like molecules. These include split luciferase assays for Apaf-1 – Caspase-9 interactions, CARD11 – BCL10 interactions, NLRP3 – ASC interactions, RIPK1 interactions and homotypic alpha synuclein interactions, and Bimolecular fluorescence assay for Caspase-2 – RAIDD interactions.
3. Screening chemical libraries using new assays. A unique chemical library built by the consortium was screened to identify PYD – PYD interactions (NLRP3 – ASC binding) and homotypic alpha synuclein interactions.
Main results:
1. Models of Caspase-2 – RAIDD and Caspase-9 – Apaf-1.
2. In silico identification of chemicals that inhibit Caspase-9 – Apaf-1 interaction.
3. Three new assays for high-throughput screening for chemicals that affect protein – protein interactions: Apaf-1 – Caspase-9 assay; CARD11 – BCL10 assay; and an alpha synuclein assay.
4. Discovering that the RIPK assay detects an inactive RIPK complex induced by Type III RIPK1 inhibitors. It does not detect the effect of Type I or Type II RIPK inhibitors.
5. Identification of chemicals that inhibit NLRP3 - ASC binding and alpha synuclein oligomerization.
1. In silico modelling of protein-protein interactions (Caspase-2 – RAIDD and Caspase-9 – Apaf-1) and the use of the models in AI-guided drug discovery to identify new inhibitors of protein-protein interactions.
The protein-protein interactions (PPIs) important in diverse diseases have been computationally modelled to identify the structural basis of the interaction and to build a tool to search for chemical inhibitors. So far two of a planned four interactions have been successfully modelled and one model has been successfully used to screen a virtual library of chemical to find drug-like molecules. Further testing of these molecules is the first step towards developing new treatments.
2. Development of new assays to quickly and cheaply assess putative inhibitors identified by AI-guided drug discovery
Further testing requires new assays to measure the effect of the drug-like molecules. These include split luciferase assays for Apaf-1 – Caspase-9 interactions, CARD11 – BCL10 interactions, NLRP3 – ASC interactions, RIPK1 interactions and homotypic alpha synuclein interactions, and Bimolecular fluorescence assay for Caspase-2 – RAIDD interactions.
3. Screening chemical libraries using new assays. A unique chemical library built by the consortium was screened to identify PYD – PYD interactions (NLRP3 – ASC binding) and homotypic alpha synuclein interactions.
Main results:
1. Models of Caspase-2 – RAIDD and Caspase-9 – Apaf-1.
2. In silico identification of chemicals that inhibit Caspase-9 – Apaf-1 interaction.
3. Three new assays for high-throughput screening for chemicals that affect protein – protein interactions: Apaf-1 – Caspase-9 assay; CARD11 – BCL10 assay; and an alpha synuclein assay.
4. Discovering that the RIPK assay detects an inactive RIPK complex induced by Type III RIPK1 inhibitors. It does not detect the effect of Type I or Type II RIPK inhibitors.
5. Identification of chemicals that inhibit NLRP3 - ASC binding and alpha synuclein oligomerization.
Progress beyond the state of the art:
1. The first AI-guided identification of an inhibitor of the CARD – CARD interaction.
2. The first assays for CARD – CARD interactions have been built and validated for high-throughput screening.
3. The first assay that specifically detects the effect of Type III kinase inhibitors.
Expected results to the end of the project:
1. Investigate Caspase-2 activation using a BiFC reporter in a zebrafish model of epilepsy.
2. Investigate apoptosome activation using a BiFC reporter in a zebrafish model of epilepsy.
3. Complete modelling of CARD11 – BCL10 interaction and Caspase-1 – ASC interaction.
4. Complete in silico screening for inhibitors of CARD11 – BCL10 interaction and Caspase-1 – ASC interaction.
5. Test putative inhibitors in cell culture and zebrafish models of neurodegeneration, cancer and inflammation.
Potential impacts (including the socio-economic impact and the wider societal implications of the project so far):
1. New career experiences and fostering entrepreneurship
The exchange of information and experiences between the academic and non-academic sectors in VIDEC will educate researchers in the processes involved in commercializing new assays for basic research, toxicology and drug-discovery. This experience will equip and encourage VIDEC members to engage in future entrepreneurial activity. The expectation of is that this activity will help researchers to realize their personal and economic potential while at the same time contributing to the improvement of human health and EU economic development. The CARD11 – BCL10 and Apaf-1 – Caspase-9 work has already generated data that is the basis for applications for additional drug-discovery funding from the Irish government and will be the basis of future applications to the European Commission.
2. Industrial outreach and economic growth
Outreach to industry is aimed at raising Industry’s awareness of VIDEC value. Forging relationships between VIDEC and the Biotechnology and Pharmaceutical sectors is designed to make new therapy paradigms available to industry, enhance cross-sectoral researcher training and drive the appearance of new therapies for currently intractable diseases. Pre-existing relationships with the pharmaceutical industry in the EU and globally will be developed with a view to licensing VIDEC IP to drive further development of lead compounds. The expectation is that all VIDEC member organizations will benefit from such licensing. The project has fostered additional industry relationships beyond those envisioned in the original project plan. These include a well developed collaboration with an Irish technology SMA (Hooke Bio), which has secured more than €5M in funding from the Irish government and the European Commission to develop a new cell imaging and drug discovery platform, and developing collaborations with a cell-imaging company in Germany (Phio AG), and a drug-discovery company in Norway (Theracule AS).
1. The first AI-guided identification of an inhibitor of the CARD – CARD interaction.
2. The first assays for CARD – CARD interactions have been built and validated for high-throughput screening.
3. The first assay that specifically detects the effect of Type III kinase inhibitors.
Expected results to the end of the project:
1. Investigate Caspase-2 activation using a BiFC reporter in a zebrafish model of epilepsy.
2. Investigate apoptosome activation using a BiFC reporter in a zebrafish model of epilepsy.
3. Complete modelling of CARD11 – BCL10 interaction and Caspase-1 – ASC interaction.
4. Complete in silico screening for inhibitors of CARD11 – BCL10 interaction and Caspase-1 – ASC interaction.
5. Test putative inhibitors in cell culture and zebrafish models of neurodegeneration, cancer and inflammation.
Potential impacts (including the socio-economic impact and the wider societal implications of the project so far):
1. New career experiences and fostering entrepreneurship
The exchange of information and experiences between the academic and non-academic sectors in VIDEC will educate researchers in the processes involved in commercializing new assays for basic research, toxicology and drug-discovery. This experience will equip and encourage VIDEC members to engage in future entrepreneurial activity. The expectation of is that this activity will help researchers to realize their personal and economic potential while at the same time contributing to the improvement of human health and EU economic development. The CARD11 – BCL10 and Apaf-1 – Caspase-9 work has already generated data that is the basis for applications for additional drug-discovery funding from the Irish government and will be the basis of future applications to the European Commission.
2. Industrial outreach and economic growth
Outreach to industry is aimed at raising Industry’s awareness of VIDEC value. Forging relationships between VIDEC and the Biotechnology and Pharmaceutical sectors is designed to make new therapy paradigms available to industry, enhance cross-sectoral researcher training and drive the appearance of new therapies for currently intractable diseases. Pre-existing relationships with the pharmaceutical industry in the EU and globally will be developed with a view to licensing VIDEC IP to drive further development of lead compounds. The expectation is that all VIDEC member organizations will benefit from such licensing. The project has fostered additional industry relationships beyond those envisioned in the original project plan. These include a well developed collaboration with an Irish technology SMA (Hooke Bio), which has secured more than €5M in funding from the Irish government and the European Commission to develop a new cell imaging and drug discovery platform, and developing collaborations with a cell-imaging company in Germany (Phio AG), and a drug-discovery company in Norway (Theracule AS).