Kurosu Lab
Current Projects
Project 1
Validation of new drug targets for discovery of novel antibacterial agents
MraY/MurX/WecA
Bacteria produce multiple glycoconjugates, including lipopolysacchlides (LPS), peptidoglycan (PG), teichoic acids, and capsules. These biopolymers are unique to bacteria, making them ideal antibiotic targets. However, only a few enzymes in PG biosynthesis such as the transpeptidase of penicillin-binding proteins (PBPs) have been studied extensively. Thus, the machinery for PG synthesis is still considered to be a source of unexploited drug targets. We have been studying bacterial phosphotransferases aiming at developing new antibacterial agents for MDR pathogens. The bacterial translocase I (MraY/MurX) and WecA/TagO utilize undecaprenyl or decaprenyl phosphate as the acceptor substrate but use different UDP-2-deoxy-2-N-acetyl-D-hexosamine donor substrates. MraY-type transferases are highly specific for UDP-N-acetylmuramate-pentapeptide, whereas WecA proteins are selective for UDP-N-acetylglucosamine (UDP-GlcNAc). These enzymes are essential in growth of many bacteria listed in NIAID Category A, B, and C Priority Pathogens. Our group identified new inhibitor molecules against translocase I or WecA, and has developed efficient total synthesis schemes for these molecules. The membrane-embedded enzymes (having multiple transmembrane domains) remain significant challenges in performing structure-based drug designs. In this program, we intend to conduct a detailed mechanistic study of translocase I and WecA using structural biology, biochemistry and synthetic chemistry with the goal of novel drug discovery.
Achievements:
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Total chemical syntheses of capuramycin and muraymycin D1 that are amenable to medicinal chemistry
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Discovery of selective WecA inhibitor
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Discovery of the inhibitors with improved activity against MraY/WecA
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Identification of effective MraY/MurX and or WecA inhibitors effective against the dormant form of M. tuberculosis or the spore form of C. difficile
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Convenient assays for MraY, MurG, WecA, and DPAGT1
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Project 2
Menaquinone Biosynthesis
Inhibitors of menaquinone biosynthesis have great potential for the development of novel and selective drugs against MDR Gram-positive pathogens including M. tuberculosis. We have identified a series of MenA (1,4-dihydroxy-2-naphthoate prenyltransferase in menaquinone biosynthesis) enzyme inhibitors and they exhibited strong activity against drug-sensitive and -resistant Mtb. Significantly, it is the first observation that the molecules killed non-replicating Mtb at MICs lower than those for Mtb obtained under aerobic conditions. In order to identify MenA inhibitors possessing antimicrobial spectrum focused against Mycobacterium spp., a series of enzyme inhibitory assays (Mtb MenA, M. smegmatis MenA, and S. aureus MenA), and bacterial growth inhibitory assays (Mtb, M. smegmatis, S. aureus, K. pneumoniae, P. aeruginosa, and E. coli) have been established. Successful completion of this project will result in the discovery of promising preclinical MenA inhibitors that kill non-replicating Mtb at low concentrations. Such molecules are very attractive TB drug leads that can reduce treatment time of current TB chemotherapy. Recently, we discovered that the MenA inhibitors inhibit viability of the spores of C. difficiles.
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Project 3
Discovery of new drug leads towards MDR Gram-negative bacterial infections
Infections caused by antibiotic-resistant bacteria, especially Gram-negative bacteria such as Enterobacter spp., Acinetobacter baumannii, Pseudomonas aeruginosa, and Klebsiella pneumoniae cause significant morbidity and mortality because effective therapeutic options are either very limited or non-existent. As demonstrated by many research groups in academia and drug industries, discovery of drug candidates that are potentially active against MDR Gram-negative bacteria is very difficult. To date, no rational drug discovery for Gram-negative infections has been demonstrated successfully due to molecular mechanisms of action associated with uptake and efflux of drugs. Known protein biosynthesis inhibitors are effective at different stages of prokaryotic mRNA translation into proteins. However, only a few protein biosynthesis inhibitor antibiotics exhibit activity against Gram-negative bacteria, but drug-resistant bacteria are no longer susceptible to FDA-approved protein biosynthesis inhibitor antibiotics. In our program of identifying effective protein biosynthesis inhibitors, the pleuromutilin analogues were identified to exhibit bactericidal activity against K. pneumoniae and A. baumannii with the MIC values of 0.2-6.25 microgram/mL. We established a pharmacological proof-of-concept that the pleuromutilin analogue killed A. baumannii in vivo using a mouse model.
Recently, we discovered a molecule effective against colistin-resistant Gram-negative pathogens. Currently, mechanistic studies of the drug and identification of molecular targets have been performed in our lab.
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Project 4
Identification of novel anticancer agents targeting DPAGT1
AglH/DPAGT1
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WecA enzyme inhibitors have the potential to interfere with a human homologue, dolichyl-phosphate GlcNAc-1-phosphotransferase 1 (DPAGT1), which catalyzes the first step of the protein N-glycosylation process in humans. We have established a counter-selection assay using a thermophilic dolichyl-phosphate GlcNAc-1-phosphotransferase (AglH from Methanocaldococcus jannaschii) to assess toxicity level of antibacterial phosphotransferase inhibitors. Certain cancers overexpress DPAGT1, enhancing intracellular adhesion. DPAGT1 inhibitors potentiate anticancer drugs' efficacy through DPAGT1/Akt/ABCG2 pathway. We have been screening identified WecA inhibitors against L1210, KB, LoVo, SK-OV-3 , AsPC-1, HCT 116, MDA-MB-435S, HepG2, and Caco2 and have identified anticancer DPAGT1 inhibitors without significantly affecting healthy cells. One of our DPAGT1 inhibitors effectively shrunk a metastatic pancreatic cancer, AsPC-1 drafted in nude mice.
