Deciphering the Inhibition of the Neuronal Calcium Sensor 1 and the Guanine Exchange Factor Ric8a with a Small Phenothiazine Molecule for the Rational Generation of Therapeutic Synapse Function Regulators

Abstract

Protein-protein interactions (PPIs) are known to play an essential role between the neuronal calcium sensor 1 (NCS-1) and the guanine exchange factor Ric8a to regulate synapse function, emerging as a druggable interface for synaptopathies such as the fragile X syndrome (FXS). Recently, the phenothiazine FD44 has been identified as an inhibitor of this PPI, decreasing the abnormally high synapse number and enhancing associative learning in a FXS animal model. Here, we have integrated advanced experimental and computational studies to obtain important structural insights into Drosophila NCS-1/FD44 recognition to understand the basis of its affinity and specificity and generate improved PPI regulators. This has allowed the identification of a new small drug-like molecule, IGS-1.76, which efficiently inhibits the human NCS-1/Ric8a complex with improved binding potency. The crystal structure of the Drosophila NCS-1/IGS-1.76 complex demonstrates that the new inhibitor, although chemically different from FD44, shares the same mechanism of action and constitutes a new hit candidate for FXS.

Insights into real-time chemical processes in a calcium sensor protein-directed dynamic library

“…Interestingly, 3b targets the same region as other inhibitors (Fig. 8) 18,19 , displaying a contact area of 303.4 Å 2 32 .…”

“…The comparison of h NCS-1/ 3b structure with the reported structures of d NCS-1 bound to strong (FD44, IGS-1.76) 18,19 and mild (CPZ) inhibitors (Fig. 8) shows that 3b indole group is placed upper in the cavity enabling D37 participation.…”

“…a , b Structural comparison of 3b with strong inhibitors. Superimposition of the structure of h NCS-1/ 3b complex (yellow ribbons/sticks) with that of d NCS-1/IGS-1.76 (pink ribbons/sticks) (PDB code 6epa) 19 . The structure of d NCS-1/FD44 has also been superimposed 18 , but only the small compound is represented (white sticks).…”

“…In fact, inhibitors of this protein-protein interaction (PPI) target the NCS-1 crevice and stabilize the orientation that presents the helix H10 inside the crevice. This topology in turn, decreases synapse number and enhances associative learning in a Fragile X syndrome animal model 18,19 . Following the same reasoning, it would be tempting to hypothesize that an stabilizer of this PPI would permit to enhance synapse number and therefore constitute a pharmacological target of neurodegenerative diseases, where synapse number is abnormally low (Fig.…”

“…1) 20,21 . The low number of ligands reported for d NCS-1 18,19 makes the DCC approach attractive as a genuine discovery tool for modulators able to unveil the mechanism to control neurotransmission 22 and synaptogenesis.

Fig. 1The complex between NCS-1 and Ric8a as a target for synaptopathies.

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