Cushing’s Syndrome is a disease caused by excess production and excretion of cortisol from the adrenal glands. In the case of ACTH-Independent Cushing’s Syndrome (AICS), the excess cortisol production is caused by adenomas in the adrenal gland. Recent genomics studies of these adrenal adenomas have found that ~40% of patients with AICS share a common genetic mutation in the PRKACA gene (c.617T→G) that results in the expression of the catalytic subunit of cAMP-dependent protein kinase (PKACα) with a point mutation (L205R). The L205R-PKACα mutation leads to loss of binding of the PKA regulatory subunits (PKARIα/β and PKARIIα/β), loss of sensitivity to cAMP signaling, constitutive activation of the mutant PKACα protein, and unregulated phosphorylation of PKACα substrates, including those involved in downstream cortisol biosynthesis. The goals of the presented research are to characterize the enzymology of L205R mutant and to develop mutant selective inhibitors with the use of an in silico model. Using recombinant L205R- and wild type-PKACa, kinetics studies were performed using a fluorophore-labeled substrate in an endpoint-based kinase assay. The kinetic parameters (KM, Vmax, kcat) were determined for both the peptide substrate-binding site and ATP-binding site for both wt- and L205R-PKACa. Inhibition studies were also carried out to determine the IC50 and Ki for the known PKACa inhibitors H89 and PKI(5-24) against both wt- and L205R-PKACa. The results of these kinetics and inhibitor studies will be presented, along with our work to develop new, selective inhibitors of the L205R mutant. Towards this second goal, we have developed an in silico docking model using the reported L205R-PKACa crystal structure, designed and docked potential mutant selective inhibitors into the in silico model, and begun synthesizing and determining the IC50 and Ki for these new inhibitors against both wt- and L205R-PKACa.

Enzymology and drug discovery studies on the L205R mutant of cAMP-dependent Protein Kinase (PKACα)

254th National Meeting (2017)

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