FROM VIRTUAL SCREENING TO IN VIVO ANALGESIA: NTS2-SELECTIVE NEUROTENSIN RECEPTOR LIGANDS

Chronic pain is a major public health issue, affecting 20% of the population, underscoring the urgent need for effective painkillers that circumvent opioid-related tolerance, addiction, and adverse effects. The neurotensinergic system induces robust, opioid-independent analgesia in several animal models of chronic pain through activation of its G protein-coupled receptors, NTS1 and NTS2. However, NTS1 activation is associated with adverse effects such as hypothermia and hypotension, making NTS2-selective targeting a potentially safer therapeutic strategy. In this study, we sought to develop new NTS2-selective small-molecule analgesics using a structure-guided drug discovery approach. A chemical library of drug-like molecules was designed based on a receptor–peptide complex, and 14.5 million compounds were computationally docked into the NTS1 orthosteric binding site. The forty top-ranking candidates were synthesized and evaluated in competitive radioligand binding assays using membranes expressing either NTS1 or NTS2. Three compounds, DV1767, DV1776, and DV1967 showed high selectivity for NTS2, with nanomolar affinity (Ki ≈ 10 nM) and minimal binding to NTS1 (Ki > 1000 nM). In vivo, intrathecal administration of these compounds significantly reduced pain behaviors in both the formalin and tail-flick pain tests in rats. Furthermore, pharmacological blockage with nonselective (SR142948A) and NTS2-selective (NTRC-844) antagonists abolished the analgesic effect of DV1767. Moreover, intravenous injection of DV1767 prevented neurotensin-induced hypotension, further supporting its functional selectivity for NTS2. Together, these results identify DV1767 as a promising lead compound and provide a strong foundation for the development of NTS2-selective non-opioid small molecule analgesics to address the substantial unmet need in chronic pain therapy.