Friday, December 6, 2024
12/6/2024
PROJECT SUMMARY Relevance to public health. Microglia, the inflammatory cells in the brain and spinal cord, participate in the development of neuropathic pain (NeuP) after nerve injury by releasing reactive oxygen species (ROS) and proinflammatory cytokines. NeuP affects >16 million Americans, is associated with a severe reduction in quality of life. NeuP is often refractory to conventional analgesics, and opioids have only limited efficacy increasing the risk of their misuse. Medications not typically used as analgesics are often the first-line treatment of NeuP. How- ever, <50% of patients report pain relief with current treatment, and side effects are common. We offer evidence that suppressing the voltage-gated proton channel (Hv1) in microglia can reduce inflammatory mediators release and attenuate NeuP. We propose to identify small-molecule Hv1 blockers by high-throughput screening (HTS) of plant extracts and botanical compounds as a step toward new drugs for NeuP. Brief background. This application builds on: (i) our publication that Hv1 controls ROS and proinflammatory cytokines release in microglia and contributes to the development of NeuP, (ii) our preliminary finding that C6, a designer peptide blocker of Hv1, suppresses ROS and proinflammatory cytokine release by microglia and atten- uates NeuP after peripheral nerve injury in mice, and (iii) our successful pilot screening of a small compound library and eight exemplar plant extracts using a novel live-cell, fluorescence-based HTS assay. The HTS assay employs a pH-sensitive fluorescent protein genetically linked to the human Hv1 channel (hHv1-VFP-H148G), allowing real-time monitoring of Hv1 operation and the identification of Hv1 blockers. Unique features and innovation. Our target choice is innovative because no clinically approved drugs se- lectively target microglia and Hv1 lacks potent and specific small-molecule blockers. Our HTS assay is innovative because there is no reported HTS method for proton channels. We identified unique reagents: C6 (the first specific blocker of Hv1), C6 variants, F6 (a small molecule that inhibits Hv1 in the HTS), and Lavender (a plant extract that inhibits Hv1 in the HTS) to facilitate the development, refinement, and validation of the HTS assay. We will employ the HTS assay to screen a unique plant extract library with >1,500 species (with >15,000 esti- mated compounds), many of which have been used historically for anti-inflammation and pain-relief. Three specific aims. (1) Optimize the HTS assay (R61 phase) seeks to improve and validate the HTS assay for plant extracts and botanical compounds. (2) Select leads from a unique library of plant extracts (R33 phase) seeks to screen the plant extracts library and identify potent and specific botanical Hv1 blockers. (3) Study sup- pression of the microglial inflammatory response and NeuP with identified botanical Hv1 blockers (R33 phase) seeks to validate the efficacy of identified botanical Hv1 blockers in microglia cells and a mouse model of NeuP. Significance. This work addresses an unmet medical need for NeuP therapeutics and has a broader influ- ence because Hv1 in microglia is complicit in additional inflammatory disorders, such as ischemic stroke.
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