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

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.