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

Abstract

Elevated N-methyl-D-aspartate receptor activity contributes to central sensitization. Our laboratories and others recently reported that disrupting protein–protein interactions downstream of N-methyl-D-aspartate receptors suppresses pain. Specifically, disrupting binding between the enzyme neuronal nitric oxide synthase and either its upstream (postsynaptic density 95 kDa, PSD95) or downstream (e.g. nitric oxide synthase 1 adaptor protein, NOS1AP) protein partners suppressed inflammatory and/or neuropathic pain. However, the lack of a small-molecule neuronal nitric oxide synthase-NOS1AP inhibitor has hindered efforts to validate the therapeutic utility of disrupting the neuronal nitric oxide synthase-NOS1AP interface as an analgesic strategy. We, therefore, evaluated the ability of a putative small-molecule neuronal nitric oxide synthase-NOS1AP inhibitor ZLc002 to disrupt binding between neuronal nitric oxide synthase and NOS1AP using ex vivo, in vitro, and purified recombinant systems and asked whether ZLc002 would suppress inflammatory and neuropathic pain in vivo. In vitro, ZLc002 reduced co-immunoprecipitation of full-length NOS1AP and neuronal nitric oxide synthase in cultured neurons and in HEK293T cells co-expressing full-length neuronal nitric oxide synthase and NOS1AP. However, using a cell-free biochemical binding assay, ZLc002 failed to disrupt the in vitro binding between His-neuronal nitric oxide synthase1-299 and glutathione S-transferase-NOS1AP400-506, protein sequences containing the required binding domains for this protein–protein interaction, suggesting an indirect mode of action in intact cells. ZLc002 (4–10 mg/kg i.p.) suppressed formalin-evoked inflammatory pain in rats and reduced Fos protein-like immunoreactivity in the lumbar spinal dorsal horn. ZLc002 also suppressed mechanical and cold allodynia in a mouse model of paclitaxel-induced neuropathic pain. Anti-allodynic efficacy was sustained for at least four days of once daily repeated dosing. ZLc002 also synergized with paclitaxel when administered in combination to reduce breast (4T1) or ovarian (HeyA8) tumor cell line viability but did not alter tumor cell viability without paclitaxel. Our results verify that ZLc002 disrupts neuronal nitric oxide synthase-NOS1AP interaction in intact cells and demonstrate, for the first time, that systemic administration of a putative small-molecule inhibitor of neuronal nitric oxide synthase-NOS1AP suppresses inflammatory and neuropathic pain.

A peripheral CB2 cannabinoid receptor mechanism suppresses chemotherapy-induced peripheral neuropathy: evidence from a CB2 reporter mouse

“…on day 0, 2, 4, and 6 (approximately 48 hours between injections); cumulative dose 16 mg/kg) to produce a CIPN condition, as described previously. 14,19,21,42,[48][49][50]52,54,[83][84][85][86][87][88] Control mice received an equal volume of cremophor-based vehicle on the same treatment schedule. Development of paclitaxel-induced allodynia was assessed on day 0, 4, 7, 11, and 14.…”

Targeting PSD95/nNOS by ZL006 alleviates social isolation-induced heightened attack behavior in mice

“…The actions observed in mice treated with ZL006 in our study have paved the way for future dose optimization in preclinical trials and subsequent clinical trials. Besides, we also inspired other studies that employ similar agents targeting the PSD95/nNOS or its downstream node in the pathway, including IC87201 or ZLc002 (Florio et al 2009 ; Lee et al 2018 ; Lee et al 2015 ; Zhu et al 2014 ).…”

nNOS‐CAPON blockers produce anxiolytic effects by promoting synaptogenesis in chronic stress‐induced animal models of anxiety

“…Also, stroke, neuropathic pain and early‐stage Alzheimer's disease are associated with the co‐morbid anxiety (Bierman, Comijs, Jonker, Scheltens, & Beekman, 2009; Campbell Burton et al, 2013; Knapp et al, 2017; Kwak, Yang, & Koo, 2017; Nicholson & Verma, 2004). Reportedly, nNOS–CAPON association mediates amyloid‐β‐induced neurotoxicity (Zhang et al, 2018), ischaemia‐induced impairment of structural plasticity (Ni et al, 2018) and inflammatory nociception and chemotherapy‐induced neuropathic pain (Lee, Carey, et al, 2018; Lee, Li, et al, 2018). We previously reported that dissociation of nNOS with CAPON by ZLc‐002 produces rapid onset of anxiolytic‐like effect (Zhu et al, 2014).…”