Development of Cav1.2-targeting fluorescent probes : from small molecule inhibitor conjugates to de novo cyclic peptide design

The voltage-gated calcium channel Cav1.2 is central to the physiology of neurons and cardiomyocytes. Alternative splicing of Cav1.2 has been linked to Alzheimer’s disease, and its expression and subcellular localization contribute to the pathogenesis of other disorders such as Parkinson’s disease. Existing approaches — quantitative PCR and high-throughput sequencing for transcript level and splicing, immunoblotting for protein expression, and immunofluorescence for spatial distribution — cannot report the real-time distribution of the channel in living cells, and are poorly suited to live-cell and live-tissue samples. This study therefore tests and designs chemical probes targeting Cav1.2 to enable real-time fluorescence imaging of channel expression and distribution in living cells and tissues, under conditions closer to the physiological state. Two objectives were pursued. The first was to validate the existing probe FluoBar2, a small molecule generated in our laboratory by appending a linker and a fluorophore (5-FAM) to the Cav1.2 inhibitor cp-PYT. I constructed an mCherry–Cav1.2 fusion expression vector and expressed it successfully in HEK-293T, U138 and COS-7 cells. Across a range of incubation times and working concentrations, however, FluoBar2 showed insufficient specificity to serve as a Cav1.2-targeted probe. Because the cp-PYT binding pocket lies within the membrane bilayer, we propose that membrane affinity underlies this poor specificity. This validation work is presented in Chapters 2–4. The second objective was to develop a cyclic peptide probe targeting the extracellular domain of Cav1.2. Calciseptine — a small snake-venom protein that binds this domain, and whose complex with Cav1.2 was only recently resolved — was used as a template for de novo ligand design. From this complex structure, a cyclic peptide was designed with RFdiffusion and PyRosetta, and its strong binding potential was confirmed by molecular dynamics simulation. A D-lysine residue in the peptide was then modified to conjugate 5-FAM through an amide bond to the side chain, completing the design of the cyclic peptide fluorescent probe. This work is described in Chapter 5. Owing to time constraints, functional validation of this probe remains for future work.