cAMP-dependent signaling regulates multiple physiological responses and its deregulation is involved in many pathological conditions. As such, it represented for many years a major focus for drug discovery; however, the ubiquitous nature of this signaling pathway also presented a burden for drug development due to fear of potential associated side effects. Thus, the development of new approaches for specific targeting is highly needed in the field. The current proposal builds upon the identification of a new complex involving the ERM member Radixin with cAMP effectors, i.e. Epac1 and PKA, and the role of the downstream substrate Rap1 as an integration node. cAMP effectors act synergistically, via Epac1-mediated activation and PKA- dependent phosphorylation of Rap1, to promote cAMP-dependent cell proliferation. Mechanistically, both effectors co-localize in a new sub-membrane compartment, forming a ternary complex with Radixin as the scaffolding unit. Maneuvers that disrupt this compartmentalization abrogate cAMP-mediated proliferation. Interestingly, expression of constitutively active Rap1 but only in its phosphorylated form (G12V-S179D) rescues this inhibition, thus confirming the role of Rap1 as a signal integrator unit of cAMP effector pathways. The overall hypothesis of this proposal is that the synergistic Radixin-Epac1-Rap1 component represents a valid target for pharmacological intervention providing a new rationale towards achieving higher specificity. We have delineated a full pilot screen program to characterize small molecule inhibitors of Epac1-Radixin interaction as part of the assessment of this hypothesis. We will accomplish this task in three integrated aims: 1) To develop and optimize HTS-compatible fluorescence polarization assays to monitor Epac1-Radixin interaction; 2) To validate this polarization assay in qHTS format utilizing a collection o bioactive compounds, and 3) To implement a set of orthogonal, secondary and follow-up assays to assess the significance of primary positive hits. Preliminary studies validate the concept and provide proof-of-principle for its feasibility. We submit that the successful completion of our studies will provide validated assays for the identification of new, selective drugs useful as pharmacological probes for addressing mechanistic aspects and investigating the functional role of Radixin-Epac1 in cAMP signaling that could eventually lead to future new pharmacologic strategies in cAMP-dependent hyperproliferative and other Epac1-mediated disorders. Our research proposal fulfills all of the specifications of PAR-13-364, entitled Development of Assays for High- Throughput Screening for Use in Probe and Pre-therapeutic Discovery namely, developing assays for a new specific biological target (i.e. Epac1-Radixin interaction) and establishing collaboration with established HTS centers (i.e. Dr Inglese, NCATS, NIH). Upon completion of the pilot campaign delineated in the current proposal, the qHTS and validation assays will be submitted via a Fast Track entry mechanism to the NIH Molecular Libraries Probe Production Centers Network (MPLCN) in the Molecular Libraries Program (MLP).
cAMP is a universal second messenger used by several hormones in every cell type. It is involved in multiple aspects of normal physiology and disease and thus a valid target for drug discovery. Our studies demonstrated the need for both effectors, Epac and PKA, for cAMP-mediated responses. Both effectors are found in a complex with the scaffold protein Radixin and we provided evidence that disruption of this Radixin-Epac interaction abolishes cAMP responses. The studies proposed will provide validated assays for screening and identification of new, selective drugs to target Radixin-Epac1 in cAMP signaling that could eventually lead to future new pharmacologic strategies.
