Development of DART (Drugs Acutely Restricted by Tethering) Probes for Targeting Opioid Receptors and Voltage-Gated Sodium Channels

Abstract

The brain, comprising billions of neurons and glial cells, forms intricate networks that transmit information via chemical neurotransmission and electrical coupling. These sophisticated systems coordinate a wide spectrum of functions, such as movement, emotion, and memory. Despite decades of neuroanatomical work establishing core principles of circuit organization, definitively assigning functions to specific neuronal cell types remains difficult, owing to network complexity and the heterogeneity of cell types. To meet this challenge, investigators have developed various approaches such as photocontrollable or receptor–ligand pairs to interrogate cell-type-specific functions; however, achieving temporal precision, endogenous-protein targeting, and cell-type specificity simultaneously is still challenging with current approaches. The DART (Drugs Acutely Restricted by Tethering) strategy, developed by our collaborators in the Tadross lab, addresses this gap by leveraging HaloTag protein (HTP), a self-labeling enzyme expressed selectively in defined cell types. DART attains cell-type specificity through restricted HTP expression, enabling rapid, spatially precise modulation of endogenous proteins, uniquely offering all aspects in contrast to earlier approaches. The DART system was applied to diverse neuronal populations and demonstrated its versatility through successfully identifying and isolating underlying mechanisms of behavior, such as AMPAR-mediated pathways of motor control in specific cell types in Parkinsonian mice. This work involves the extension of the DART tool to valuable targets, Opioid Receptors (ORs) and voltage-gated sodium channels (Navs), which are widely expressed across the central and peripheral nervous systems and contribute to essential functions, such as pain processing and reward regulation of ORs and excitability and disorders such as epilepsy of Navs. Our objective is to develop DART probes targeting ORs and Navs and delineate their specific roles in neuronal circuits and behavior. For the application of DART to ORs, we successfully developed potent and subtype-selective DART probes, naltrexone2DART.2 (2.15a) for μ-OR, naltrindole3DART.2 (2.31c) for δ-OR, and benzamideDART.2 (2.41) for κ-OR. Based on the starting scaffold of naltrexone·HCl, we achieved naltrexone2DART.2 (2.15a) by modifying the C3 phenolic hydroxyl group with monofluoromethoxy or difluoromethoxy groups to obtain desired activity and selectivity in DART system and resulted in a potent and μ-selective probe over δ- and κ-OR. Naltrindole3DART.2 retained the naltrindole scaffold and incorporated an N-benzamide linker on the indole ring, yielding a selective δ-OR antagonist with improved subtype selectivity. BenzamideDART.2 achieved a highly selective κ-OR antagonistic profile by grafting a benzamide scaffold from a known κ-OR antagonist, guided by docking studies. Collectively, the strong efficacy and robust subtype selectivity of these OR-DART probes establish their utility as precise tools for dissecting the distinct behavioral functions of each OR subtype. For the application of DART to Navs, we next developed potent Nav-DART probes that silence neuronal activity by cell-specific modulation of Navs. We achieved JHNav2DART.2 (3.2) and JHNav3DART.2 (3.42), which effectively antagonize Navs and reduce neuronal firing. JHNav2DART.2 (3.2) employs a potent Nav1.7-derived core with a polyamine-linker strategy targeting acidic residues identified from the Nav1.6 homology model. JHNav3DART.2 (3.42) adapted the potent Nav1.6 core of the reported potent Nav1.6 blocker, given that the predominant distribution of Nav1.6 is in the hippocampal neurons. Silencing neuronal activity of Nav-DART probes was evaluated by whole-cell patch clamp recording. These probes, which enable the modulation of Navs with cell-type specificity, offer promising avenues for future research on essential functions contributed by Navs, such as epilepsy. We expect these newly developed probes, efficient and subtype-selective OR-DART probes, and potent Nav-DART probes, to offer a powerful tool for identifying each of the targets' neuronal functions that shape behaviors by cell-specific modulation of targets. Eventually, we hope these probes employing the DART platform to expand and advance the knowledge of cell-specific functions of ORs and Navs in physiology and neuronal disease.