Structural Determinants of the Neuronal Glycine Transporter 2 for the Selective Inhibitors ALX1393 and ORG25543.

Cristina Benito-Muñoz,Raquel Felipe,Almudena Perona,Gonzalo Pérez-Siles,Carmen Aragón,Beatriz López-Corcuera,Enrique Núñez

doi:10.1021/acschemneuro.0c00602

Abstract

The neuronal glycine transporter GlyT2 modulates inhibitory glycinergic neurotransmission by controlling the extracellular concentration of synaptic glycine and the supply of neurotransmitter to the presynaptic terminal. Spinal cord glycinergic neurons present in the dorsal horn diminish their activity in pathological pain conditions and behave as gate keepers of the touch-pain circuitry. The pharmacological blockade of GlyT2 reduces the progression of the painful signal to rostral areas of the central nervous system by increasing glycine extracellular levels, so it has analgesic action. O-[(2-benzyloxyphenyl-3-fluorophenyl)methyl]-l-serine (ALX1393) and N-[[1-(dimethylamino)cyclopentyl]methyl]-3,5-dimethoxy-4-(phenylmethoxy)benzamide (ORG25543) are two selective GlyT2 inhibitors with nanomolar affinity for the transporter and analgesic effects in pain animal models, although with deficiencies which preclude further clinical development. In this report, we performed a comparative ligand docking of ALX1393 and ORG25543 on a validated GlyT2 structural model including all ligand sites constructed by homology with the crystallized dopamine transporter from Drosophila melanogaster. Molecular dynamics simulations and energy analysis of the complex and functional analysis of a series of point mutants permitted to determine the structural determinants of ALX1393 and ORG25543 discrimination by GlyT2. The ligands establish simultaneous contacts with residues present in transmembrane domains 1, 3, 6, and 8 and block the transporter in outward-facing conformation and hence inhibit glycine transport. In addition, differential interactions of ALX1393 with the cation bound at Na1 site and ORG25543 with TM10 define the differential sites of the inhibitors and explain some of their individual features. Structural information about the interactions with GlyT2 may provide useful tools for new drug discovery.

Highlights

Pain sensation is transmitted by afferent fibers connecting the peripheral tissues to the central nervous system
Inhibitory glycinergic interneurons in the dorsal spinal cord regulate the transmission of pain signals to the brain by operating through glycine receptors (GlyR) containing the α3 subunit at spinal cord synapses.[1,2]
ALX1393 inhibits the glycine transport by recombinant GlyT2 expressed in COS7 cells with an IC50 = 31 ± 2.7 nM and displays about 2 orders of magnitude selectivity for GlyT2 over GlyT1 (IC50 in the low μM range)

Summary

■ INTRODUCTION

Pain sensation is transmitted by afferent fibers connecting the peripheral tissues to the central nervous system. It is worth noting that the two cysteines introduced in the double mutant substituted two amino acids which were predicted to have a role in the binding of the two inhibitors, but more prominent in that of ORG25543 (Tables 1 and 2) For this reason, the labeling of the transporter mutant was reduced in basal conditions (vehicle), but especially in the presence of ORG25543 (Figure 6B,C). A second piece of evidence came from experiments using the A223C mutant of GlyT2 that contains an exogenous cysteine in EL1, a region sensitive to the different conformations of the transporter induced by the substrates during the transport cycle.[45] As reported previously, the MTSEA-biotin labeling of the A223C mutant is protected by glycine in a buffer containing NaCl but not in a medium containing choline chloride, indicating the accessibility of the cysteine is reduced when sodium is bound (Figure 7B,C).

■ METHODS

■ CONCLUSIONS

■ ACKNOWLEDGMENTS

■ REFERENCES