title: Small molecule inhibitors of PSD95-nNOS protein-protein interactions as novel analgesics.
abstract: Aberrant increases in NMDA receptor (NMDAR) signaling contributes to central nervous system sensitization and chronic pain by activating neuronal nitric oxide synthase (nNOS) and generating nitric oxide (NO). Because the scaffolding protein postsynaptic density 95kDA (PSD95) tethers nNOS to NMDARs, the PSD95-nNOS complex represents a therapeutic target. Small molecule inhibitors IC87201 (EC5O: 23.94 μM) and ZL006 (EC50: 12.88 μM) directly inhibited binding of purified PSD95 and nNOS proteins in AlphaScreen without altering binding of PSD95 to ErbB4. Both PSD95-nNOS inhibitors suppressed glutamate-induced cell death with efficacy comparable to MK-801. IC87201 and ZL006 preferentially suppressed phase 2A pain behavior in the formalin test and suppressed allodynia induced by intraplantar complete Freund's adjuvant administration. IC87201 and ZL006 suppressed mechanical and cold allodynia induced by the chemotherapeutic agent paclitaxel (ED50s: 2.47 and 0.93 mg/kg i.p. for IC87201 and ZL006, respectively). Efficacy of PSD95-nNOS disruptors was similar to MK-801. Motor ataxic effects were induced by MK-801 but not by ZL006 or IC87201. Finally, MK-801 produced hyperalgesia in the tail-flick test whereas IC87201 and ZL006 did not alter basal nociceptive thresholds. Our studies establish the utility of using AlphaScreen and purified protein pairs to establish and quantify disruption of protein-protein interactions. Our results demonstrate previously unrecognized antinociceptive efficacy of ZL006 and establish, using two small molecules, a broad application for PSD95-nNOS inhibitors in treating neuropathic and inflammatory pain. Collectively, our results demonstrate that disrupting PSD95-nNOS protein-protein interactions is effective in attenuating pathological pain without producing unwanted side effects (i.e. motor ataxia) associated with NMDAR antagonists.
authors.affiliation: Biochemistry Interdisciplinary Graduate Program, Molecular and Cellular Biochemistry Department, Indiana University, Bloomington, IN, USA.
authors.forename: Wan-Hung
authors.initials: WH
authors.lastname: Lee
authors.affiliation: Department of Psychological and Brain Sciences, Indiana University, Bloomington, IN, USA.
authors.forename: Zhili
authors.initials: Z
authors.lastname: Xu
authors.affiliation: Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN, USA.
authors.forename: Nicole M
authors.initials: NM
authors.lastname: Ashpole
authors.affiliation: Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN, USA.
authors.forename: Andy
authors.initials: A
authors.lastname: Hudmon
authors.affiliation: Center for Drug Discovery, and Department of Pharmaceutical Sciences, Northeastern University, Boston, MA, USA.
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, USA.
authors.forename: Ganesh A
authors.initials: GA
authors.lastname: Thakur
authors.affiliation: Department of Psychological and Brain Sciences, Indiana University, Bloomington, IN, USA.
authors.forename: Yvonne Y
authors.initials: YY
authors.lastname: Lai
authors.affiliation: Biochemistry Interdisciplinary Graduate Program, Molecular and Cellular Biochemistry Department, Indiana University, Bloomington, IN, USA; Department of Psychological and Brain Sciences, Indiana University, Bloomington, IN, USA; Gill Center for Biomolecular Science, Bloomington, IN, USA. Electronic address: hohmanna@indiana.edu.
authors.forename: Andrea G
authors.initials: AG
authors.lastname: Hohmann
mesh_terms: D000636:Aminosalicylic Acids / Q000494:pharmacology*; D000700:Analgesics / Q000494:pharmacology*; D000818:Animals; D001259:Ataxia / Q000139:chemically induced; D001596:Benzylamines / Q000494:pharmacology*; D001921:Brain / Q000187:drug effects / Q000502:physiology; D016923:Cell Death / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D002733:Chlorophenols / Q000494:pharmacology*; D004195:Disease Models, Animal; D016291:Dizocilpine Maleate / Q000009:adverse effects / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D006930:Hyperalgesia / Q000188:drug therapy / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D008297:Male; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D008810:Mice, Inbred C57BL; D009474:Neurons / Q000187:drug effects / Q000502:physiology; D018696:Neuroprotective Agents / Q000009:adverse effects / Q000494:pharmacology; D052248:Nitric Oxide Synthase Type I / Q000037:antagonists & inhibitors / Q000378:metabolism; D059226:Nociceptive Pain / Q000188:drug therapy / Q000503:physiopathology; D017207:Rats, Sprague-Dawley; D014230:Triazoles / Q000494:pharmacology*
chemical_list: C546030:2-((1H-benzo(d)(1,2,3)triazol-5-ylamino)methyl)-4,6-dichlorophenol; D000636:Aminosalicylic Acids; D000700:Analgesics; D001596:Benzylamines; D002733:Chlorophenols; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D018696:Neuroprotective Agents; D014230:Triazoles; C000607694:ZL006 compound; D016291:Dizocilpine Maleate; D052248:Nitric Oxide Synthase Type I
keywords: Allodynia; Central sensitization; Complete Freund's adjuvant; NMDAR; Neuronal nitric oxide synthase; Neuropathic pain; Postsynaptic density 95; motor ataxia; paclitaxel