During programmed cell death (PCD), the formation of specific protein complexes triggers distinct pathways within cells. Inhibiting the activity of some of these protein complexes with small molecules can reduce disease-associated damage and show therapeutic efficacy. However, drug discovery efforts have been hampered by the lack of tools and assays that measure complex formation.
Innovative assays for screening PCD protein complexes
The EPIC project, with the support of the Marie Skłodowska-Curie programme, addressed these technological problems and developed new approaches for screening PCD protein complexes. “Delineation of PCD mechanisms will facilitate the development of small molecules that can activate or deactivate protein complexes depending on the condition,” explains the MSC research fellow Howard Fearnhead. The project team was multi-disciplinary and included experts in the fields of cell death, pharmacology and screening, structural biology and natural product chemistry. Researchers focused on apoptosis, a PCD approach that is triggered by mitochondrial stress or cellular damage and involves the release of the mitochondrial protein cytochrome c. This protein induces the self-association of the apoptotic protease activating factor-1 (Apaf-1) which, together with the protease caspase-9, activates the cell killing process. Moreover, scientists studied another PCD mechanism, necroptosis, that is activated in response to tumour necrosis factor, anti-cancer drugs and pathogens. Necroptosis involves the oligomerisation of the mixed lineage kinase domain like pseudokinase (MLKL) initiated by receptor-interacting protein (RIP) kinases, which affects membrane permeability and causes necrotic cell death.
Small molecules targeting PCD
According to Fearnhead, “activators of Apaf-1 have the potential to work as anti-cancer agents, but such molecules have received little attention due to the lack of assays that measure Apaf-1 activity.” EPIC filled this gap by developing an assay that can detect the function and protein interaction in the apoptotic complex or apoptosome. The reaction produces luminescence upon protein interaction which is sensed by a specially designed biosensor. Similarly, EPIC constructed and validated biosensors for detecting the interaction of necrosome proteins. This will be extremely helpful for the identification and testing of small molecule inhibitors of necroptosis which can protect against ischaemia-associated injury, neurodegeneration and chemotherapy-induced nephrotoxicity. The new experimental tools from the EPIC team have helped scientists elucidate the mechanism of action and determine the binding sites of two previously identified small molecules. They also discovered a new protein complex inhibitor from a previously undescribed deep-sea organism collected from the floor of the north Atlantic and elucidated its chemical structure.
EPIC high-throughput tools will be exploited for drug-discovery purposes to characterise the pharmacological properties of cell death-inducing protein complex inhibitors. Using these tools to screen large chemical libraries will culminate in the identification of new lead molecules. “The next steps are to extend assay applications to visualise the protein complexes in cells and tissues in vivo,” continues Fearnhead. EPIC researchers are working to achieve these goals through collaboration with drug-discovery experts in the Netherlands, developmental biologists in Spain and Norway, and experts in visualising luminescence in Switzerland and Japan.
EPIC, PCD, apoptosis, necroptosis, protein complex, inhibitor, biosensor