Initial client request
“To support the positive therapeutic evidence obtained through the existing cell-based screening assay at Cellomatics we are looking for a supplementary method to validate the effects seen for certain drug candidates. This must confirm target engagement is specific for the protein target and the effects observed are directly related to this engagement.”
Hypothesis of mode of action
Proposed experimental approach
Expression assessment
Endogenous expression levels of proteins A, B, and C were assessed by western blot using two commercially available antibodies at the recommended dilutions. An inactive non-specific drug control was utilised.
Functional assessment: drug candidate- target validation
Validation of ‘chaperone mechanism’ for target protein interactions
- Protein B is essential: Knockout of Protein B abrogates Co-IP of Protein A by Protein C.
- Drug Candidate 2 modulates the interaction between Protein A and Protein C in a Wild-type system.
Summary and next steps
- Optimised western blot conditions for three proteins of interest.
- Demonstrated that suspected protein interaction were present between protein A and protein C.
- Confirmed that the interaction was dependent on the presence of protein B.
- Utilised Co-IP system to demonstrate that drug candidates designed to perturb protein A-B interactions, reduce protein A-C interactions.
