ABSTRACT
ZL006 and IC87201 have been presented as efficient inhibitors of the nNOS/PSD-95 protein-protein interaction and shown great promise in cellular experiments and animal models of ischemic stroke and pain. Here, we investigate the proposed mechanism of action of ZL006 and IC87201 using biochemical and biophysical methods, such as fluorescence polarization (FP), isothermal titration calorimetry (ITC), and (1)H-(15)N HSQC NMR. Our data show that under the applied in vitro conditions, ZL006 and IC87201 do not interact with the PDZ domains of nNOS or PSD-95, nor inhibit the nNOS-PDZ/PSD-95-PDZ interface by interacting with the ß-finger of nNOS-PDZ. Our findings have implications for further medicinal chemistry efforts of ZL006, IC87201 and analogues, and challenge the general and widespread view on their mechanism of action.
Subject(s)
Aminosalicylic Acids/pharmacology , Benzylamines/pharmacology , Chlorophenols/pharmacology , Intracellular Signaling Peptides and Proteins/antagonists & inhibitors , Membrane Proteins/antagonists & inhibitors , Nitric Oxide Synthase Type I/antagonists & inhibitors , PDZ Domains/drug effects , Triazoles/pharmacology , Calorimetry , Disks Large Homolog 4 Protein , Fluorescence Polarization , Humans , Magnetic Resonance SpectroscopyABSTRACT
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.
Subject(s)
Analgesics/chemistry , Hyperalgesia/drug therapy , PDZ Domains , Receptor, Serotonin, 5-HT2A/metabolism , Serotonin 5-HT2 Receptor Antagonists/chemistry , Amino Acid Sequence , Analgesics/chemical synthesis , Analgesics/pharmacology , Animals , Computer Simulation , Disease Models, Animal , Ligands , Magnetic Resonance Spectroscopy , Molecular Structure , Rats , Serotonin 5-HT2 Receptor Antagonists/chemical synthesis , Serotonin 5-HT2 Receptor Antagonists/pharmacology , Structure-Activity RelationshipABSTRACT
Disrupting the interaction between the PDZ protein, PSD-95, and its target ligands (such as the glutamate NMDA receptor or the serotonin 5-HT2A receptor) was found to reduce hyperalgesia in various models of neuropathic pain. Here, we set out to identify lead molecules which would interact with PSD-95, and hence, would potentially display analgesic activity. We describe the virtual screening of the Asinex and Cambridge databases which together contain almost one million molecules. Using three successive docking filters and visual inspection, we identified three structural classes of molecules and synthesized a potential lead compound from each class. The binding of the molecules with the PDZ domains of PSD-95 was assessed by (1)H-(15)N HSQC NMR experiments. The analgesic activity of the best ligand, quinoline 2, was evaluated in vivo in a model of neuropathic pain and showed promising results.
