New antimycobacterial molecules that kill non-replicating Mycobacterium tuberculosis (Mtb) were identified by screening libraries of synthetic natural products. De novo screening of a 400-membered library of aurachin RE analogs resulted in discovery of UT-317 ((R)-20). UT-317 is a selective vitamin K2 biosynthesis (MenA) inhibitor that killed replicating and non-replicating Mtb at 2.31 μg/mL (MIC) and 0.85 μg/mL, respectively. A 50-membered library of capuramycin analogs was evaluated in their enzymatic inhibitory activities against translocase I (MraY/MurX) and prenyl-phosphate-GlcNAc-1-phosphate transferase (WecA). UT-01320 (45) is identified as a selective WecA inhibitor that kills both replicating and non-replicating Mtb at 1.50 μg/mL (MIC) and 2.58 μg/mL, respectively. UT-01320 killed the intracellular Mtb much faster than the first-line TB drugs such as isoniazid and rifampicin. A strong antimycobacterial agent, UT-800 (64) was identified by screening of a 50-membered library of pleuromutilin derivatives. UT-800 is a protein biosynthesis (50s ribosome) inhibitor which has activity focused against Mtb. UT-800 killed replicating and non-replicating Mtb at 0.83 μg/mL (MIC) and 1.20 μg/mL, respectively. In the course of these works, fluorescent probes, Park’s nucleotide-Nε-C6-FITC (32, for MraY/MurX) and UDP-glucosamine-C6-FITC (46, for WecA) were developed. These fluorescent probes enable us to screen the polyprenyl-phosphate N-acetylhexosamine 1-phosphate transferase enzyme superfamily (e.g. MraY/MurX, WecA, AlgH, and DPAGT1) in high-throughput manner
