Development of a phenotypic screening assay for novel compounds that inhibit peripheral pain-sensing neurons - A promising approach to treat pain is to inhibit peripheral pain-sensing neurons (nociceptors) that are activated in response to noxious (pain-causing) stimuli. For example, local anesthetics are extremely efficacious at blocking pain elicited by a variety of pain conditions that arise in the periphery. Unfortunately, local anesthetics inhibit all sensory, as well as motor, neurons. Our long-term goal is to identify new compounds that specifically inhibit nociceptors but spare other sensory and motor neurons. Such compounds, especially those that are peripherally restricted (do not enter the CNS), would be effective and safer analgesics. The immediate goal of this project, in response to the “Assay Development and Neurotherapeutic Agent Identification” R61/R33 FOA, is to develop an in vitro assay to screen natural product libraries for novel and selective nociceptor inhibitory compounds with much higher throughput than is currently available. We will engineer calcitonin gene-related peptide (CGRP) receptor expressing “sniffer cells” able to detect nociceptor activity. Using co- cultures of nociceptors (derived from adult rat sensory ganglia from an inflammatory pain model) and CGRP sniffer cells, we will screen natural product libraries for compounds that inhibit stimulated nociceptor activity. The use of nociceptors themselves, that are the direct target for newly identified compounds, provides a strong biological rationale with high likelihood that compounds identified will be efficacious analgesic agents in vivo. Further, due to high congruence between mammalian nociceptors, the assay will have high translational validity for efficacy at human nociceptors. The Aims for this application are: Aim 1(R61), engineer sniffer cells that are highly sensitive to the neuropeptide, CGRP; Aim 2 (R61), develop, validate and optimize a co-culture assay (CGRP sniffer cells with sensory neurons derived from rats treated with CFA) to assess known compounds that inhibit nociceptor activity stimulated by a physiological inflammatory soup; and Aim 3 (R33), screen a pre- fractionated natural product library from the Developmental Therapeutics Program (DTP) of the NCI to identify novel compounds that inhibit nociceptor activity. Pre-fractions from the DTP natural products extract library, which currently consists of over 326,000 fractions, will be tested for selective inhibition of activated nociceptors using the assay developed in the R61 phase. Positive hits will be those that reduce nociceptor activity by ≥ 25% without altering fluorescence in sniffer cells (±CGRP) alone. Positive hits will be further refined for nociceptor selectivity by performing a counter screen for fractions that also inhibit the activity of SH-SY5Y neurons. Bioactive compounds will be identified from hit fractions through a process of bioassay-guided fractionation in collaboration with the DTP. These pure compounds will then be assessed for their potency, efficacy and nociceptor specificity in our assay. We expect to screen ~100,000 fractions (limited only by the budgetary restrictions of the grant) to identify 5-10 novel compounds with diverse chemical structures that will enter the NIH Blueprint Neurotherapeutics Network (PAR-20-122) for further development leading to phase 1 clinical testing.
