Allosteric Modulation of Cannabinoid Type 1 Receptor by ZCZ011: Integration of In Vitro Pharmacology and Molecular Dynamics Simulation

Over the years, the cannabinoid type 1 receptor (CB₁R) has emerged as a promising therapeutic target for addressing various neurodegenerative diseases including HIV-associated neurocognitive disorders (HAND). However, the therapeutic application of direct CB₁R activation is often hindered by undesirable psychoactive side effects. To mitigate this issue, research has focused on utilizing positive allosteric modulators (PAMs) to enhance the CB₁R activity indirectly. Preclinical studies have highlighted the efficacy of CB₁R PAMs, such as ZCZ011 and GAT211, in mouse models of Huntington's disease, neuropathic pain, and, more recently, HAND. Building on this evidence, we employed primary frontal cortex neuronal cultures and whole brain microglial cultures to investigate the direct and indirect effects of racemic ZCZ011 against the HIV-1 trans-activator of transcription (Tat)-induced excitotoxicity. In parallel, molecular modeling and molecular dynamics simulations were conducted using the ZCZ011 enantiomers (<i>R</i>)-ZCZ011 and (<i>S</i>)-ZCZ011, to elucidate their binding profiles at CB₁R. Our in vitro studies revealed that ZCZ011 demonstrated neuroprotective effects against Tat-induced excitotoxicity in the presence of <i>N</i>-arachidonoylethanolamine (AEA) in a dose-dependent manner. Interestingly, racemic ZCZ011 exhibited neuroprotective properties even in the absence of AEA, deviating from the classical behavior of a true PAM. This prompted further investigation into the binding profiles of the enantiomers. Molecular modeling revealed that (<i>R</i>)-ZCZ011 and (<i>S</i>)-ZCZ011 bind to distinct sites on CB₁R, aligning with the binding profiles of other CB₁R allosteric modulators, GAT228 and GAT229. Notably, (<i>R</i>)-ZCZ011 exhibited a higher number of hydrogen bonds and both polar and nonpolar interactions with CB₁R, enhancing the stabilization of AEA binding to CB₁R. In summary, these findings suggest that (<i>R</i>)-ZCZ011 may function as both a PAM and an allosteric agonist, like GAT228, while (<i>S</i>)-ZCZ011 may act as a pure PAM, resembling GAT229. This dual functionality underscores the therapeutic potential of ZCZ011 in modulating CB₁R activity for a number of neuroprotective applications.