Small molecule inhibitors of PSD95–nNOS protein–protein interactions suppress formalin-evoked Fos protein expression and nociceptive behavior in rats

Abstract

Excessive activation of NMDA receptor (NMDAR) signaling within the spinal dorsal horn contributes to central sensitization and the induction and maintenance of pathological pain states. However, direct antagonism of NMDARs produces undesirable side effects which limit their clinical use. NMDAR activation produces central sensitization, in part, by initiating a signaling cascade that activates the enzyme neuronal nitric oxide synthase (nNOS) and generates the signaling molecule nitric oxide (NO). NMDAR-mediated activation of nNOS requires a scaffolding protein, postsynaptic density protein 95 kDa (PSD95), which tethers nNOS to NMDARs. Thus, disrupting the protein-protein interaction between PSD95 and nNOS may inhibit pro-nociceptive signaling mechanisms downstream of NMDARs and suppress central sensitization while sparing unwanted side effects associated with NMDAR antagonists. We examined the impact of small molecule PSD95-nNOS protein-protein interaction inhibitors (ZL006, IC87201) on both nociceptive behavior and formalin-evoked Fos protein expression within lumbar spinal dorsal horn of rats. Comparisons were made with ZL007, an inactive analog of ZL006, and the NMDAR antagonist MK-801. IC87201 and ZL006, but not ZL007, suppressed phase 2 of formalin-evoked pain behavior and decreased the number of formalin-induced Fos-like immunoreactive cells in spinal dorsal horn regions associated with nociceptive processing. MK-801 suppressed Fos protein expression in both dorsal and ventral horns. MK-801 produced motor ataxia in the rotarod test whereas IC87201 and ZL006 failed to do so. ZL006 but not ZL007 suppressed paclitaxel-induced mechanical and cold allodynia in a model of chemotherapy-induced neuropathic pain. Co-immunoprecipitation experiments revealed the presence of the PSD95-nNOS complex in lumbar spinal cord of paclitaxel-treated rats, although ZL006 did not reliably disrupt the complex in all subjects. The present findings validate use of putative small molecule PSD95-nNOS protein-protein interaction inhibitors as novel analgesics and demonstrate, for the first time, that these inhibitors suppress inflammation-evoked neuronal activation at the level of the spinal dorsal horn.

ZLc002, a putative small-molecule inhibitor of nNOS interaction with NOS1AP, suppresses inflammatory nociception and chemotherapy-induced neuropathic pain and synergizes with paclitaxel to reduce tumor cell viability

“…Adult male Sprague Dawley rats, weighing 285–446 g (Envigo, Indianapolis, IN, USA), were used in the formalin study. The positive control (MK-801) and vehicle-treated groups that appear in the rat formalin study described here (vehicle and MK801 data points in Figure 5 only 6 ) were published previously by our group as comparators in a separate evaluation of structurally distinct PSD95-nNOS protein–protein interaction disruptors (i.e. IC87201 and ZL006) and are included here with permission from the publisher.…”

“… 6 The vehicle- and MK-801-treated groups were tested, perfused, and processed concurrently, under blinded conditions, with the ZLc002-treated groups described for the first time in the present report, in accordance with our obligations to comply with the guidelines from the Association for Assessment and Accreditation of Laboratory Animal Care to reduce unnecessary animal use. 6 Tissue from all subjects was processed concurrently for Fos immunohistochemistry. Animals were housed in a temperature-controlled facility (73 ± 2°F), 45% humidity under a 12-h light/dark cycle with standard rodent chow and water ad libitum.…”

“…Composite pain scores (CPS) were calculated for every 5 min time bin as described in our previous work. 6 , 26 No pain behavior was scored as 0, lifting of the paw was scored as 1, and shaking/biting/flinching was scored as 2. The area under the curve (AUC) for formalin-evoked pain was calculated for the early phase of pain behavior (phase 1, 0–10 min) and the late phase (phase 2, 10–60 min) for each subject.…”

“…Immunohistochemical experiments were conducted as described previously. 6 , 27 – 30 Briefly, transverse sections (30 μm) of the L4-L5 lumbar spinal cord were cut on a cryostat and kept in an antifreeze solution (50% sucrose in ethylene glycol and 0.1 M PBS) prior to staining. Every fourth section was processed for immunostaining to avoid counting the same cell twice in adjacent sections.…”

“…NR2B-PSD95, PSD95-nNOS, and nNOS–NOS1AP) produces antinociceptive efficacy without unwanted side effects associated with NMDAR antagonists. 6 – 10 We recently proposed that the nNOS–NOS1AP protein–protein interface was a previously unrecognized target for analgesic drug development. 11 We showed that TAT-GESV, a peptide inhibitor of the nNOS–NOS1AP interface, disrupted binding between nNOS and NOS1AP in vitro, suppressed glutamate-induced cell death in cultured cortical neurons, and produced antinociceptive efficacy in mechanistically distinct models of neuropathic pain.…”