P104

We conducted an extensive screening of 1 million compounds targeting Mycobacterium tuberculosis under low pH conditions. Initially, we optimized a high-throughput screening assay for low pH by utilizing 1536-well plates and incorporating a resazurin read-out, which enhanced our throughput capabilities. Furthermore, we refined the protocol to shorten the incubation time and enhance assay reproducibility. The screening process involved evaluating the inhibitory effects of 1 million compounds at low pH using a single-dose approach. Compounds exhibiting inhibition above 30% underwent confirmation screening, where their activity was assessed in dose-response experiments under both low pH and normal conditions. Promising compounds underwent further in vitro evaluation to determine their bactericidal efficacy across additional relevant culture conditions, such as cholesterol and hypoxia. Simultaneously, in silico methodologies aided in hit clustering based on chemical structure and predicted compound liabilities using machine learning algorithms.