ZL006

Neuroprotective effects of ZL006 in Aβ1-42-treated neuronal cells

Amyloid beta (Aβ)-induced neurotoxicity and oxidative stress plays an important role in the pathogenesis of Alzheimer's disease (AD). ZL006 is shown to reduce over-produced nitric oxide and oxidative stress in ischemic stroke by interrupting the interaction of neuronal nitric oxide synthase and postsynaptic density protein 95. However, few studies are reported on the role of ZL006 in AD. To investigate whether ZL006 exerted neuroprotective effects in AD, we used Aβ1-42 to treat primary cortical neurons and N2a neuroblastoma cells as an in vitro model of AD. Cortical neurons were incubated with ZL006 or dimethyl sulfoxide for 2 hours and treated with Aβ1-42 or NH3?H2O for another 24 hours. The results of cell counting Kit-8 (CCK-8) assay and calcein-acetoxymethylester/propidium iodide staining showed that ZL006 pretreatment rescued the neuronal death induced by Aβ1-42. Fluorescence and western blot assay were used to detect oxidative stress and apoptosis-related proteins in each group of cells. Results showed that ZL006 pretreatment decreased neuronal apoptosis and oxidative stress induced by Aβ1-42. The results of CCK8 assay showed that inhibition of Akt or NF-E2-related factor 2 (Nrf2) in cortical neurons abolished the protective effects of ZL006. Moreover, similar results were also observed in N2a neuroblastoma cells. ZL006 inhibited N2a cell death and oxidative stress induced by Aβ1-42, while inhibition of Akt or Nrf2 abolished the protective effect of ZL006. These results demonstrated that ZL006 reduced Aβ1-42-induced neuronal damage and oxidative stress, and the mechanisms might be associated with the activation of Akt/Nrf2/heme oxygenase-1 signaling pathways.

A pain killer without analgesic tolerance designed by co-targeting PSD-95-nNOS interaction and α2-containning GABAARs

Overactivation of N-methyl-D-aspartate receptor (NMDAR) in the spinal cord dorsal horn (SDH) in the setting of injury represents a key mechanism of neuropathic pain. However, directly blocking NMDAR or its downstream signaling, interaction between postsynaptic density-95 (PSD-95) and neuronal nitric oxide synthase (nNOS), causes analgesic tolerance, mainly due to GABAergic disinhibition. The aim of this study is to explore the possibility of preventing analgesic tolerance through co-targeting NMDAR downstream signaling and γ-aminobutyric acid type A receptors (GABA_ARs). Methods: Mechanical/thermal hyperalgesia were quantified to assess analgesic effects. Miniature postsynaptic currents were tested by patch-clamp recording to evaluate synaptic transmission in the SDH. GABA-evoked currents were tested on HEK293 cells expressing different subtypes of recombinant GABA_ARs to assess the selectivity of (+)-borneol and ZL006-05. The expression of α2 and α3 subunits of GABA_ARs and BDNF, and nNOS-PSD-95 complex levels were analyzed by western blotting and coimmunoprecipitation respectively. Open field test, rotarod test and Morris water maze task were conducted to evaluate the side-effect of ZL006-05. Results: (+)-Borneol selectively potentiated α2- and α3-containing GABA_ARs and prevented the disinhibition of laminae I excitatory neurons in the SDH and analgesic tolerance caused by chronic use of ZL006, a nNOS-PSD-95 blocker. A dual-target compound ZL006-05 produced by linking ZL006 and (+)-borneol through an ester bond blocked nNOS-PSD-95 interaction and potentiated α2-containing GABA_AR selectively. Chronic use of ZL006-05 did not produce analgesic tolerance and unwanted side effects. Conclusion: By targeting nNOS-PSD-95 interaction and α2-containing GABA_AR simultaneously, chronic use of ZL006-05 can avoid analgesic tolerance and unwanted side effects. Therefore, we offer a novel candidate drug without analgesic tolerance for treating neuropathic pain.

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

Rationale: Deregulated attack behaviors have devastating social consequences; however, satisfactory clinical management for the behavior is still an unmet need so far. Social isolation (SI) has been common during the COVID-19 pandemic and may have detrimental effects on mental health, including eliciting heightened attack behavior.
Objectives: This study aims to explore whether injection of ZL006 can alleviate SI-induced escalation of attack behavior in mice.
Methods: Pharmacological tools, biochemical methods, and behavioral tests were used to explore the potential therapeutic effects of ZL006 targeting postsynaptic density 95 (PSD95)/neuronal nitric oxide synthase (nNOS) pathway on escalation of attack behavior induced by SI in mice.
Results: ZL006 mitigated SI-induced escalated attack behaviors and elevated nitric oxide (NO) level in the cortex of the SI mice. The beneficial effects of ZL006 lasted for at least 72 h after a single injection of ZL006. Potentiation of NO levels by L-arginine blocked the effects of ZL006. Moreover, a sub-effective dose of 7-NI in combination with a sub-effective dose of ZL006 decreased both SI-induced escalated attack behaviors and NO levels in mice subjected to SI.
Conclusions: Our study highlights the importance of the PSD95/nNOS pathway in mediating SI-induced escalation of attack behavior. ZL006 may be a promising therapeutic strategy for treating aggressive behaviors.

ZL006 promotes migration and differentiation of transplanted neural stem cells in male rats after stroke

New strategies must be developed to resolve the problems of stroke treatment. In recent years, stem cell-based therapy after stroke has come into the public and academic lens. Previously we have shown that uncoupling neuronal nitric oxide synthase (nNOS) from the postsynaptic density protein-95 (PSD-95) by ZL006, a small molecular compound, can ameliorate ischemic damage and promote neuronal differentiation of endogenous neural stem cells (NSCs) in focal cerebral ischemic male rats. In this study, we transplanted exogenous NSCs into the ipsilateral hemisphere of male rats in combination with ZL006 treatment after ischemic stroke. We show that ZL006 treatment facilitates the migration of transplanted NSCs into the ischemia-injured area and promotes neuronal differentiation of these cells, which is not due to a direct effect of ZL006 on exogenous NSCs but is associated with increased phosphorylation of cAMP response element-binding protein (CREB) in neurons and favorable microenvironment. Moreover, improved functional outcome in the ZL006-treated group was also found. Taken together, our data indicate that ZL006, uncoupling nNOS-PSD-95 in neurons, positively regulates the fate of transplanted NSCs and benefits the functional outcome after stroke in male rats.

ZL006 protects spinal cord neurons against ischemia-induced oxidative stress through AMPK-PGC-1α-Sirt3 pathway

Spinal cord ischemia (SCI) induces a range of cellular and molecular cascades, including activation of glutamate receptors and downstream signaling. Post-synaptic density protein 95 (PSD-95) links neuronal nitric oxide synthase (nNOS) with the N-methyl-d-aspartic acid (NMDA) receptors to form a ternary complex in the CNS. This molecular complex-mediated cytotoxicity has been implicated in brain ischemia, but its role in SCI has not been determined. The goal of the study was to investigate the potential protective effects of ZL006, a small-molecule inhibitor of the PSD-95/nNOS interaction, in an in vitro SCI model induced by oxygen and glucose deprivation (OGD) in cultured spinal cord neurons. We found that ZL006 reduced OGD-induced lactate dehydrogenase (LDH) release, neuronal apoptosis and loss of cell viability. This protection was accompanied by the preservation of mitochondrial function, as evidenced by reduced mitochondrial oxidative stress, attenuated mitochondrial membrane potential (MMP) loss, and enhanced ATP generation. In addition, ZL006 stimulated mitochondrial enzyme activities and SOD2 deacetylation in a Sirt3-dependent manner. The results of western blot analysis showed that ZL006 increased the activation of AMPK-PGC-1α-Sirt3 pathway, and the beneficial effects of ZL006 was partially abolished by AMPK inhibitor and PGC-1α knockdown. Therefore, our present data showed that, by the AMPK-PGC-1α-Sirt3 pathway, ZL006 protects spinal cord neurons against ischemia through reducing mitochondrial oxidative stress to prevent apoptosis.