HINT1 Inhibitors as Selective Modulators of MOR–NMDARCross-Regulation and Non-Opioid Analgesia

Human histidine triad nucleotide-binding protein 1 (HINT1) has recently become a protein of interest due to its involvement in several CNS processes, including neuroplasticity and the development of several neuropsychiatric disorders. Crucially, HINT1 behaves as a mediator for cross-regulation of the mu-opioid receptor (MOR) and <i>N</i>-methyl-d-aspartate receptor (NMDAR). Active site inhibition of HINT1 using small-molecule inhibitors has been demonstrated to have a significant impact on this cross-regulatory relationship in vivo. Herein, we describe the development of a series of ethenoadenosine HINT1 inhibitors to further evaluate the effect of HINT1 inhibition on morphine’s blockade of NMDA-evoked behaviors, the development of acute endomorphin-2 tolerance, and analgesia. X-ray crystallographic analysis and HINT1 binding experiments demonstrate that modifications to the inhibitor nucleobase greatly impact the inhibitor binding interactions with HINT1. Our results reveal a complex structure–activity relationship for HINT1 inhibitors, in which minor modifications to the ethenoadenosine scaffold resulted in dramatic changes to their activity in these assays modeling MOR–NMDAR interaction. Specifically, we observed the ability of HINT1 inhibitors to selectively affect individual pathways of MOR–NMDAR crosstalk. Furthermore, we observed that a carbamate ethenoadenosine inhibitor of HINT1 can induce analgesia while not affecting opioid tolerance. Additionally, although past studies have indicated that the loss of HINT1 expression can result in the downregulation of p53, we have shown that the inhibition of HINT1 has no effect on either the expression of HINT1 or p53. These studies highlight the critical role of HINT1 in MOR–NMDAR crosstalk and demonstrate the intriguing potential of using HINT1 active-site inhibitors as tools to probe its role in these biochemical pathways and its potential as a novel pain target.