Disruption of nNOS‐PSD95 protein–protein interaction inhibits acute thermal hyperalgesia and chronic mechanical allodynia in rodents

Abstract

Background and purpose: Post-synaptic density protein 95 (PSD95) contains three PSD95/Dosophilia disc large/ZO-1 homology domains and links neuronal nitric oxide synthase (nNOS) with the N-methyl-D-aspartic acid (NMDA) receptor. This report assesses the effects of disruption of the protein-protein interaction between nNOS and PSD95 on pain sensitivity in rodent models of hyperalgesia and neuropathic pain. Experimental approach: We generated two molecules that interfered with the nNOS-PSD95 interaction: IC87201, a small molecule inhibitor; and tat-nNOS (residues 1-299), a cell permeable fusion protein containing the PSD95 binding domain of nNOS. We then characterized these inhibitors using in vitro and in vivo models of acute hyperalgesia and chronic allodynia, both of which are thought to require nNOS activation. Key results: IC87201 and tat-nNOS (1-299) inhibited the in vitro binding of nNOS with PSD95, without inhibiting nNOS catalytic activity. Both inhibitors also blocked NMDA-induced 3′,5′-cyclic guanosine monophosphate (cGMP) production in primary hippocampal cultures. Intrathecal administration of either inhibitor potently reversed NMDA-induced thermal hyperalgesia in mice. At anti-hyperalgesic doses, there was no effect on acute pain thresholds or motor coordination. Intrathecal administration of IC87201 and tat-nNOS also reversed mechanical allodynia induced by chronic constriction of the sciatic nerve. Conclusions and implications: nNOS-PSD95 interaction is important in maintaining hypersensitivity in acute and chronic pain. Disruption of the nNOS-PSD95 interaction provides a novel approach to obtain selective anti-hyperalgesic compounds.

Small-Molecule Inhibitors at the PSD-95/nNOS Interface have Antidepressant-Like Properties in Mice

“…Specifically, IC87201 peak plasma levels were previously reported to occur 15 min following i.p. administration of 1 or 2 mg/kg in mice and rats, respectively, and behavioural effects were delayed by 45 min, an interval thought to reflect the time needed for distribution of IC87201 to its site of action (Florio et al, 2009). Alternatively, IC87201 may produce its effects by a process of neurobiological adaptation.…”

“…Reports to date have provided evidence that IC87201 had no effects on binding to a panel of 34 targets that include receptors, transporters and ion channels (Florio et al, 2009) and that the structurally related compound ZL006 did not disrupt the interaction between CAPON/nNOS, synGAP/ nNOS and NR2B/PSD-95, which are all PDZ-related interactions (Zhou et al, 2010). The serotonin transporter SERT interacts with nNOS via a PDZ motif/PDZ domain interaction, an interaction that may well be of relevance to depression and antidepressant action.…”

“…The selection of doses was determined from a published report indicating that IC87201 was effective in treating NMDA-induced thermal hyperalgesia in mice with an EC 50 of 0.1 mg/kg, i.p. (Florio et al, 2009). …”

“…Retention latencies were measured 24 h and 72 h after the training session. The dose of IC87201 was selected from a previous report indicating an attenuation of mechanical allodynia in rats subjected to chronic constriction injury (Florio et al, 2009).…”

“…In rats, IC87201 abolished mechanical allodynia when administered intrathecally (at 50 and 100 nmol doses) or i.p. (2 mg/kg) (Florio et al, 2009). 4-(3,5-Dichloro-2-hydroxy-benzylamino)-2-hydroxybenzoic acid (ZL006) is a molecule structurally related to IC87201, which inhibits NMDA-R-dependent NO synthesis in cortical neurons with an IC 50 of 82 nM and crosses the blood brain barrier after systemic administration (Zhou et al, 2010).…”