(1104-A) The ultra-HTS cell lysate-based platform to discover small molecule modulators of p38/MK2 protein-protein interaction for therapeutic discovery
Monday, February 5, 2024
12:00 PM – 1:00 PM EST
Location: Exhibit Halls AB
Abstract: The mitogen-activated protein kinase 14 (MAPK14), known as p38, is a key regulator of inflammation and is a well-established therapeutic target in cancer, neurodegenerative disorders, and metabolic diseases. However, targeting the kinase activity of p38 or its primary substrate, MK2, for therapeutic development is challenging. While many p38 and MK2 inhibitors have advanced to clinical trials, none have been approved as a drug. This lack of success is largely attributed to the limited selectivity of the inhibitors against other kinases and a wide range of p38 substrates affected by the inhibition of p38 kinase activity. Such limitations lead to a wide range of adverse side effects and opposite therapeutic responses. New approaches to regulate p38/MK2-dependent inflammation for therapeutic discovery are urgently needed. Small molecule inhibitors of protein-protein interaction between p38 and MK2 may provide a new strategy to target p38-signaling in human diseases. To discover the first inhibitors of the p38/MK2 complex, we have developed a cell lysate-based time-resolved fluorescence resonance energy transfer (TR-FRET) assay. We have optimized and miniaturized the assay into a 1,536-well format for ultra-high-throughput screening (uHTS) with Z’> 0.5 and signal-to-background ratio (S/B) > 20. The pilot uHTS of more than 10,000 structurally diverse pharmacologically active compounds followed by the dose-response confirmatory screen has revealed multiple low-micromolar inhibitors of p38/MK2 interaction. Furthermore, several compounds appeared as potent small molecule stabilizers of the p38/MK2 complex. We further validated the most potent compounds in orthogonal biochemical and biophysical assays and demonstrated a high selectivity against other p38 binding partners, including its main activator MKK3. Functionally, the p38/MK2 complex disruption correlates with decreased MK2 phosphorylation and suppression of the p38-dependent inflammatory cytokines, such as TNF and IL-6. Using the in vitro and cellular thermal shift assays, we have determined the target engagement of the inhibitors with p38 and MK2. Based on these data, we used computational modeling to determine the compound binding modes and the SAR to guide further structure optimization. Together, our studies have defined the p38/MK2 interaction as a druggable target for therapeutic discovery, provided a robust uHTS assay for screening small molecule p38/MK2 inhibitors, and revealed the first small molecules to control this pathogenic axis.