title: 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. NMDAR-mediated activation of nNOS requires a scaffolding protein, postsynaptic density protein 95kDa (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 the lumbar spinal cord 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.
authors.affiliation: Department of Psychological and Brain Sciences, Indiana University, Bloomington, IN, United States; Program in Neuroscience, Indiana University, Bloomington, IN, United States.
authors.forename: Lawrence M
authors.initials: LM
authors.lastname: Carey
authors.affiliation: Interdisciplinary Biochemistry Program, Molecular and Cellular Biochemistry Department, Indiana University, Bloomington, IN, United States.
authors.forename: Wan-Hung
authors.initials: WH
authors.lastname: Lee
authors.affiliation: Department of Psychological and Brain Sciences, Indiana University, Bloomington, IN, United States.
authors.forename: Tannia
authors.initials: T
authors.lastname: Gutierrez
authors.affiliation: Center for Drug Discovery, and Department of Pharmaceutical Sciences, Northeastern University, Boston, MA, United States.
authors.forename: Pushkar M
authors.initials: PM
authors.lastname: Kulkarni
authors.affiliation: Center for Drug Discovery, and Department of Pharmaceutical Sciences, Northeastern University, Boston, MA, United States.
authors.forename: Ganesh A
authors.initials: GA
authors.lastname: Thakur
authors.affiliation: Department of Psychological and Brain Sciences, Indiana University, Bloomington, IN, United States; Anagin, Inc., Indianapolis, IN, United States.
authors.forename: Yvonne Y
authors.initials: YY
authors.lastname: Lai
authors.affiliation: Department of Psychological and Brain Sciences, Indiana University, Bloomington, IN, United States; Program in Neuroscience, Indiana University, Bloomington, IN, United States; Interdisciplinary Biochemistry Program, Molecular and Cellular Biochemistry Department, Indiana University, Bloomington, IN, United States; Gill Center for Biomolecular Science, Indiana University, Bloomington, IN, United States. Electronic address: hohmanna@indiana.edu.
authors.forename: Andrea G
authors.initials: AG
authors.lastname: Hohmann
mesh_terms: D000818:Animals; D001522:Behavior, Animal / Q000187:drug effects*; D000076122:Disks Large Homolog 4 Protein / Q000037:antagonists & inhibitors*; D016291:Dizocilpine Maleate / Q000494:pharmacology*; D005557:Formaldehyde / Q000494:pharmacology; D008297:Male; D052248:Nitric Oxide Synthase Type I / Q000187:drug effects* / Q000378:metabolism; D009619:Nociceptors / Q000187:drug effects* / Q000378:metabolism; D017207:Rats, Sprague-Dawley; D013116:Spinal Cord / Q000187:drug effects* / Q000378:metabolism
chemical_list: D000076122:Disks Large Homolog 4 Protein; C501449:Dlg4 protein, rat; D005557:Formaldehyde; D016291:Dizocilpine Maleate; D052248:Nitric Oxide Synthase Type I
keywords: N-methyl-d-aspartate receptor; central sensitization; dorsal horn; neuronal nitric oxide synthase; postsynaptic density protein 95 kDa; protein–protein interaction inhibitor