Structure-Based Design of PDZ Ligands as Inhibitors of 5-HT_2AReceptor/PSD-95 PDZ1 Domain Interaction Possessing Anti-hyperalgesic Activity

Abstract

Disrupting the interaction between the PDZ protein PSD-95 and the C-terminal domain of the 5-HT2A serotonin receptor has been shown to reduce hyperalgesia in a rodent model of neuropathic pain. Here, we designed and synthesized PDZ ligands capable of binding to the first PDZ domain (PDZ1) of the PSD-95 protein and evaluated their biological activity in vitro and in vivo. A series of substituted indoles was identified by docking simulations, and six novel analogues were synthesized. Three analogues displayed strong interactions with the first PDZ domain (PDZ1) of PDZ-95 in (1)H-(15)N heteronuclear single-quantum coherence (HSQC) experiments and two of them were able to inhibit the interaction between PSD-95 and the 5-HT2A receptor in vitro. We identified compound 8b as the analogue able to significantly suppress mechanical hyperalgesia in an experimental model of traumatic neuropathic pain in the rat. This effect was suppressed by the coadministration of the 5-HT2A receptor antagonist M100907, consistent with an inhibitory effect upon 5-HT2A receptor/PSD-95 interaction. Finally, we determined an NMR-restraint driven model structure for the PSD95 PDZ1/8b complex, which confirms that indole 8b binds to the putative PDZ-ligand binding site.

Biochemical investigations of the mechanism of action of small molecules ZL006 and IC87201 as potential inhibitors of the nNOS-PDZ/PSD-95-PDZ interactions

“…6b-c ), none of which appear to be from residues Asp62, Leu107, Phe111 and Arg121, which are residues proposed to bind or to be affected by ligand-binding 24 25 . Typically, chemical shift perturbations of >0.1 ppm and up to 0.3 ppm are observed on binding even very weak ligands with K d ~ 300–500 μM 37 . Here, the maximum shift changes observed were less than 0.05 and are, therefore, considered non-specific.…”

Emerging Themes in PDZ Domain Signaling

“…One particular analogue (8b) was effective at suppressing the mechanical hyperalgesia in a rat model of neuropathic pain (Fig. 7A.3) (Boucherle et al, 2011;Vogrig et al, 2013).…”

Design and synthesis of triazole-based peptidomimetics of a PSD-95 PDZ domain inhibitor

“…15 To compare the triazole compounds with current leading small-molecule PSD-95 PDZ inhibitors we synthesized Indole 1 and 2 ( Fig. 1C) according to literature 24 and tested them in the FP assay. Among 9-18, compound 10 showed robust inhibition of the PDZ2/probe interaction with a K i of 289 μM (Fig.…”

“…On the other hand, 10 is several fold more potent than Indole 1 and about 2-fold more potent than Indole 2. Vogrig et al 24 demonstrated that Indole 1 inhibits the PSD-95-PDZ1/ 5-HT 2A interaction with an IC 50 of 190 μM in an in vitro pulldown assay. Indole 2 was not tested in this assay, as it was estimated to be a weak binder based on 1 H-15 N HSQC NMR.…”

“…13 Until recently, no small-molecule N heteronuclear single-quantum coherence, HSQC, NMR), inhibit the PSD-95-PDZ/5-HT 2A receptor interaction (pull-down) and relieve pain in hyperalgesic rats. 24 Herein, we pursue a peptidomimetic strategy introducing a rigid scaffold into a short PSD-95 peptide ligand with the potential of improving affinity, selectivity, and stability and provide novel chemotypes targeting PSD-95. We used the tripeptide Thr-Ala-Val (TAV) as starting point (Fig.…”