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

Development of DART (Drugs Acutely Restricted by Tethering) Probes for Targeting Opioid Receptors and Voltage-Gated Sodium Channels The brain, comprising billions of neurons and glial cells, forms intricate networks that coordinate diverse functions such as movement, emotion, and memory through sophisticated communication systems. Assigning functions to specific neuronal cell types is essential for understanding underlying mechanisms but remains challenging with currently available tools. The DART (Drugs Acutely Restricted by Tethering) system is a newly developed approach that predominate over existing methods by uniquely enabling spatiotemporal modulation of endogenous proteins with cell-type specificity. DART employs HaloTag protein (HTP), a self-labeling enzyme, to achieve specificity through restricted expression in defined cell types. Its versatility has been demonstrated, for example, in delineating AMPAR-mediated motor control pathways in Parkinsonian mice. Building on this foundation, the present work extends DART to opioid receptors (ORs) and voltage-gated sodium channels (Navs), both of which mediate essential functions but remain incompletely understood. This dissertation focuses on developing DART probes targeting OR subtypes and Navs to define their specific roles, such as pain processing and reward regulation for ORs, and excitability and pathological states such as epilepsy for Navs. Guided by X-ray crystal structures and docking studies, we successfully developed subtype-selective OR-DART probes: naltrexone2DART.2 for μ-OR, naltrindole3DART.2 for δ-OR, and benzamideDART.2 for κ-OR. In parallel, we generated potent Nav-DART probes, JHNav2DART.2 and JHNav3DART.2, which effectively suppressed neuronal firing through cell-specific Navs modulation in cultured hippocampal neurons. We anticipate that these newly developed probes, efficient and subtype-selective OR-DART probes and potent Nav-DART probes, will serve as powerful tools for uncovering target-specific neuronal functions that shape behavior via cell-specific modulation. Ultimately, we expect that applying these probes within the DART platform will expand and advance knowledge of the cell-specific roles of ORs and Navs in both normal physiology and neurological disease.