A click chemistry‐mediated approach to understanding survivin:caspase‐9 protein‐protein interactions

Survivin, a 16.5 kDa mammalian cytosolic protein, is upregulated in multiple cancers and is a promising therapeutic target. As its name implies, survivin facilitates cell survival by inhibiting apoptosis. Survivin is thought to inhibit the apoptotic initiator caspase‐9, however, the specific mechanisms of and amino acids involved in caspase‐9 inhibition have yet to be fully elucidated. Identifying the amino acid residues involved in the interaction between survivin and caspase‐9 will facilitate the rational design of small molecule protein‐protein interaction inhibitors. Click chemistry provides a facile approach to covalently link disparate azide‐ and terminal alkyne‐ containing molecules, thereby allowing for site‐specific incorporation of a fluorescent tag into a polypeptide sequence. Using site‐directed mutagenesis with the unnatural amino acid para ‐azidophenylalanine (paF), we synthesized an azide‐containing mutant survivin protein. We then used copper‐free click chemistry to covalently modify the mutant survivin azide with an alkyne‐containing fluorophore dibenzylcyclooctyne‐488 (DBCO488) to generate a triazole‐containing cycloadducted product. Using western blotting, UV illumination, and Coomassie blue staining, we demonstrated the ability to site‐specifically cycloadduct the para ‐azidophenylalanine in the survivin protein to the fluorescent cyclooctyne, DBCO488. This technology will now enable us to perform fluorescence analyses of the survivin:caspase‐9 complex in order to identify the specific amino acid residues involved in this interaction. This novel approach to monitor protein‐protein interactions holds untapped potential to improve rational drug design for survivin:caspase‐9‐targeted anticancer therapeutic agents. Funding: Environmental Toxicology Training Program 5T32ES007079–32 (SCB), COBRE P20 RR021940 ‐06 (JNL), R01 GM099959 ‐01 (PI, Collaborator John Karanicolas)