Efficient Characterization of GPCRs Allosteric Modulation: Application to the Rational Design of De Novo S1PR1 Allosteric Modulators.

G-protein-coupled receptors (GPCRs) constitute the largest superfamily of integral membrane proteins in the human genome that mediate most transmembrane signaling processes. Malfunction of these signaling processes is related to many human pathologies (Parkinson's, heart diseases, etc.), causing GPCRs to be the largest family of druggable proteins. Traditionally, GPCRs were targeted by orthosteric ligands. However, this regulation usually causes side effects, provoking many GPCRs-associated pathologies to remain without an effective treatment. Allosteric regulation offers a promising alternative to circumvent this problem, and consequently, comprehending its details is of utmost importance. For this reason, we developed in the present work a methodology to study the allosteric modulation in a comprehensive way. Specifically, this methodology allows calculating the free energy profiles (Δ) for activation processes of GPCRs and their derived complexes combining the usage of targeted molecular dynamics (TMD) simulations to generate the intermediate structures of a given activation process, with the protein-dipole Langevin-dipole (PDLD) method within the linear response approximation (LRA) framework (PDLD/S-LRA-2000) and our refined coarse-grained (CG) model for GPCRs to calculate the binding and conformational free energy contributions (Δ, Δ), respectively, which take into account the cellular membrane effects by an implicit membrane. Sphingosine 1-phosphate receptor 1 (S1PR1) has been chosen as a case study due to its available data for benchmark purposes. Apart from validating our developed methodology, the conducted S1PR1 study has partially filled its knowledge gap regarding allosteric modulation and has allowed rational design of a de novo pure positive allosteric modulator for one of its prospective allosteric cavities according to our calculations. The methodology presented in this paper provides a very useful tool to study the GPCRs allosteric modulation, and the GPCRs activation processes in general, which will hopefully encourage a more thorough exploration of the topic.