Molecular Targets Of Immunosuppressive Pyrazole Derivatives: From Trpc Channels To Mtor

Pyrazole‐containing immunosuppressive compounds exemplified in the literature by BTP2 have been identified in several labs. Published studies demonstrated that BTP2 can inhibit both receptor and store‐operated Ca2+‐influx in both native cells and cells transfected with members of the TRPC ion channel family. Here, we show the results of pull‐down studies performed with a biotinylated BTP2 analog attached to avidin beads. Lysates were prepared from Jurkat cells and HEK293 stably transfected with TRPC6, TRPC3, or TRPC5. Binding proteins were identified by mass spec, Western blots, or both. Identified binding proteins included TRPC3, TRPC5, TRPC6, STIM1, eIF3, PKCα, FKBP52, FKBP12, mTOR, calcineurin, and p70 S6 kinase. Many of the proteins in this group are known to complex with or regulate other members so it is not clear which proteins have actual binding interactions with the inhibitor probe. A potential link is the presence of FKBP proteins which are involved in regulation of both the TRPC and mTOR pathways. We observed direct probe binding to recombinant FKBP12 and an ability of PKC inhibitors to reverse Ca2+‐influx inhibition by BTP2 in HEK293‐TRPC6 cells. PKC phosphorylation of TPRC molecules inhibits their activity and activates TRPM4 activity. We propose that BTP2‐like compounds inhibit dephosphophorylation of TRPC to help impart their immunosuppressive effects.