(535) Identification of microglial immunomodulatory compounds by high-throughput proteomics: insights from quantification of 1000 proteins in >6,500 samples

Introduction/Rationale: Microglia are critical for maintaining neural homeostasis. However, under pathological conditions, they can adopt a chronically pro-inflammatory phenotype contributing to neurodegenerative diseases including Alzheimer’s, Parkinson’s, and multiple sclerosis (MS). Identifying potential therapies requires capturing the breath of inflammatory proteins produced by microglia, and signals of potential toxicities to minimize adverse events (AEs).

Methods: The high cost of capturing these diverse signals at scale limits typical drug discovery efforts to simple low-plex readouts. To address this gap, we previously described the Nomic platform, a proteomics tool capable of quantifying >1000 proteins simultaneously. Here, we leverage the Nomic’s Omni 1000 to screen 510 bioactive small molecules in microglia stimulated with LPS, generating >6,500 samples and >6.5 million data points.

Results: LPS led to expected increased expression of TNF-alpha, IL-1β, and IL-6, as well as ISG15 and IL-12 p40, indicators of chronic inflammation associated with neurodegenerative diseases. Approximately ⅓ of the compounds screened blocked inflammation, of which ¼ presented clear signs of toxicity, characterized by leaking of intracellular content into the supernatant. To further assess potential toxicities, we treated cardiomyocytes and hepatocytes with these compounds, identifying expected and novel drugs with promising properties. For example, Berberine (BBR) is being developed to revert M1 microglia to M2 by blocking NF-κB signaling. We observed significantly reduced IL-12 p40, IL-10, and CXCL6 upon addition of BBR, with minimal signs of toxicities. Methylprednisolone was similarly anti-inflammatory, but paradoxically led to increased SAA and IL-6, consistent with clinical reports of hepatic AEs with high MP exposure.

Conclusion: Our results demonstrate the value of high-throughput proteomics to simultaneously identify immunomodulatory compounds and characterize their efficacy and safety profile.