Thursday, April 25, 2024
4/25/2024
PROJECT SUMMARY The opioid crisis has driven home the need for new, non-addictive analgesics to treat both severe acute and chronic pain. Over the past 25 years, the search for such novel analgesics has had only a few successes and many failures. There are two drawbacks of modern analgesic screening that we aim to correct with our screening assay. First, screening for novel analgesics typically relies on a biochemical readout that is readily amenable to high throughput screening (HTS). While such an approach has advantages (e.g., speed), it does not account for the complexity and heterogeneity of nociceptor sensory neuron biology. Second, many new analgesic targets and efficacy studies from HTS rely on rodent behavior and cellular physiology. For clear ethical reasons, rodent efficacy studies have made complete sense, and yet, the differences between homo sapiens and murine organisms have led to a failure to translate exciting preclinical targets to human pain. The mechanisms for these failures are now being illuminated as multiple research groups, including our own, have begun to study sensory neuron physiology and pharmacology in primary human dorsal root ganglia (DRG). Differences in expression of “standard” nociceptor markers between mice, rats, and humans illustrate the importance of moving into human cells as early as possible in the development of novel analgesics. In this proposal, we address both issues above by performing screening of natural product fractions for their ability to reduce hyperexcitability in human induced pluripotent stem cell-derived nociceptors. In the past, the promise of derived nociceptors has fallen short of true human nociceptor replication. Based on our preliminary and published data, we believe that our cells, RealDRGs, truly are “nociceptors.” In this proposal, we will combine this abundant, replicable, and reliable source of human “nociceptors” with cutting-edge multi-electrode array (MEA) technology to screen natural product fractions for analgesic-like effects. Over the last several years, we have used mouse DRGs with MEAs to show proof-of- concept (PoC) analgesic screening including documenting and validating a Z’-factor assay quality assessment process. We will use the R61 portion to determine (1) the optimal stable baseline after plating of RealDRGs to estimate the active electrode yield, (2) the stability in firing rate after exogenous sensitization, (3) the Z’ factor, and (4) PoC screening of known nociceptor inhibitors. Upon reaching our quantitative milestones, we will then progress in the R33 phase to (1) screen natural product fractions from the National Cancer Institute with (2) final testing of subfractions in RealDRGs and in human primary DRGs.
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