Aurachin RE (1) is a strong antibiotic that was recently found to possess 1,4-dihydroxy-2-naphthoate prenyltransferase (MenA) and bacterial electron transport inhibitory activities. Aurachin RE is the only molecule in a series of aurachin natural products that has the chiral center in the alkyl side chain at C9′-position. To identify selective MenA inhibitors against Mycobacterium tuberculosis, a series of chiral molecules were designed based on the structures of previously identified MenA inhibitors and 1. The synthesized molecules were evaluated in in vitro assays, including MenA enzyme and bacterial growth inhibitory assays. We could identify novel MenA inhibitors that showed significant increase in potency of killing nonreplicating M. tuberculosis in the low oxygen recovery assay (LORA) without inhibiting other Gram-positive bacterial growth even at high concentrations. The MenA inhibitors reported here are useful new pharmacophores for the development of selective antimycobacterial agents with strong activity against nonreplicating M. tuberculosis.

Capuramycin (1) and its analogs are strong translocase I (MurX/MraY) inhibitors. In our SAR studies of capuramycin analogs against M. tuberculosis (Mtb), we observed for the first time that a capuramycin analog, UT-01320 (3) killed non-replicating (dormant) Mtb at low concentrations under low-oxygen conditions, whereas selective MurX inhibitors killed only replicating Mtb under aerobic conditions. Interestingly, 3 did not exhibit MurX enzyme inhibitory activity even at high concentrations, however, 3 inhibited bacterial RNA polymerases with the IC50 values of 100-150 nM range. A new RNA polymerase inhibitor 3 displayed strong synergistic effects with a MurX inhibitor SQ 641 (2), a promising preclinical TB drug.

Translocase I (MraY/MurX) is an essential enzyme in growth of the vast majority of bacteria that catalyzes the transformation from UDP-MurNAc-pentapeptide (Park’s nucleotide) to prenyl-MurNAc-pentapeptide (lipid I), the first membrane-anchored peptidoglycan precursor. MurX has been received considerable attentions to the development of new TB drugs due to the fact that the MurX inhibitors kill exponentially growing Mycobacterium tuberculosis (Mtb) much faster than clinically used TB drugs. Lipid I isolated from Mtb contains the C50-prenyl unit that shows very poor water-solubility, and thus, this chemical characteristic of lipid I renders MurX enzyme assays impractical for screening and lacks reproducibility of the enzyme assays. We have established a scalable chemical synthesis of Park’s nucleotide-Nε-dansylthiourea 2 that can be used as a MurX enzymatic substrate to form lipid I analogues. In our investigation of minimum structure requirement of the prenyl phosphate in the MraY/MurX-catalyzed lipid I analogue synthesis with 2, we found that neryl phosphate (C10-phosphate) can be recognized by MraY/MurX to generate the water-soluble lipid I analogue in quantitative yield under the optimized conditions. Herein, we report a rapid and robust analytical method for quantifying MraY/MurX inhibitory activity of library molecules.

A stereocontrolled first total synthesis of muraymycin D1 (1) has been achieved. The synthetic route is highly stereoselective, featuring 1) selective β-ribosylation of the C2-methylated amino ribose, 2) selective Strecker reaction, 3) ring-opening reaction of a diastereomeric mixture of a diaminolactone to synthesize muraymycidine (epi-capreomycidine). The acid-cleavable protecting groups for secondary alcohol and uridine ureido nitrogen are applied for simultaneous deprotections with the Boc and tBu groups. Muraymycin D1 (1) and its amide derivatives (2 and 3) exhibited growth inhibitory activity against Mycobacterium tuberculosis (MIC50 1.56–6.25 μg/mL) and strong enzyme inhibitory activities against the bacterial phosphotransferases (MurX and WecA) (IC50 0.096−0.69 μM).

Polyprenyl phosphate-GlcNAc-1-phosphate transferase (WecA) is an essential enzyme for the growth of Mycobacterium tuberculosis (Mtb) and some other bacteria. Mtb WecA catalyzes the transformation from UDP-GlcNAc to decaprenyl-P-P-GlcNAc, the first membrane-anchored glycophospholipid that is responsible for the biosynthesis of mycolylarabinogalactan in Mtb. Inhibition of WecA will block the entire biosynthesis of essential cell wall components of Mtb in both replicating and non-replicating states, making this enzyme a target for development of novel drugs. Here, we report a fluorescence-based method for the assay of WecA using a modified UDP-GlcNAc, UDP-Glucosamine-C6-FITC (1), a membrane fraction prepared from an M. smegmatis strain, and the E. coli B21WecA. Under the optimized conditions, UDP-Glucosamine-C6-FITC (1) can be converted to the corresponding decaprenyl-P-P-Glucosamine-C6-FITC (3) in 61.5% yield. Decaprenyl-P-P-Glucosamine-C6-FITC is readily extracted with n-butanol and can be quantified by ultraviolet-visible (UV-Vis) spectrometry. Screening of the compound libraries designed for bacterial phosphotransferases resulted in the discovery of a selective WecA inhibitor, UT-01320 (12) that kills both replicating and non-replicating Mtb at low concentration. UT-01320 (12) also kills the intracellular Mtb in macrophages. We conclude that the WecA assay reported here is amenable to medium- and high-throughput screening, thus facilitating the discovery of novel WecA inhibitors.

Introduction Methicillin-resistant Staphylococcus aureus (MRSA) have been on the increase during the past decade, due to the steady growth of the elderly and immunocompromised patients, and the emergence of multi-drug-resistant (MDR) bacterial strains. Although, only a limited number of anti-MRSA drugs are available, a number of different combination antimicrobial drug regimens have been used to treat serious MRSA infections. Thus, addition of several new antistaphylococcal drugs into clinical practice should broaden therapeutic options. Because MRSA is one of the most common and problematic bacteria associated with increasing antimicrobial resistance, continuous efforts on discovery of lead compounds as well as development of alternative therapies and faster diagnostics to ensure effective antistaphylococcal therapy are required. Areas covered This article summarizes the FDA approved drugs to treat MRSA infections, the drugs in clinical trials, and the drug leads for MRSA and related Gram-positive bacterial infections. In addition, the mode of action of antistaphylococcal molecules and resistant mechanisms of some molecules are briefly discussed. Expert opinion The number of pipeline drugs presently undergoing clinical trials is not particularly encouraging. There are limited and rather expensive therapeutic options for the infections by MRSA in the critically ill. This review article provides an update on antistaphylococcal drugs in clinical trials and antibacterial molecules effective against Gram-positive bacteria including MRSA. The structural and biological information of antibacterials summarized here are very useful for designing drug leads to develop into new anti-MRSA drugs.

Capuramycin and its congeners have been considered important lead molecules for the development of a new drug for multidrug-resistant (MDR) Mycobacterium tuberculosis infections. Extensive structure-activity relationship studies of capuramycin to improve the efficacy have been limited due to difficulty in selective chemical modifications of the desired position(s) of the natural product with biologically interesting functional groups. We have developed efficient syntheses of capuramycin and its analogs using new protecting groups, which are derived from the chiral (chloro-4-methoxyphenyl) (chlorophenyl) methanols, for the uridine ureido nitrogen and primary alcohol. The chiral non-racemic (2,6-dichloro-4-methoxyphenyl) (2,4-dichlorophenyl) methanol derivative is a useful reagent to resolve rac-3-amino-1,3-dihydro-5-phenyl-2H-1,4-benzodiazepin-2-one, whose (S)-configuration isomer plays a significant role in improving the mycobactericidal activity of capuramycin.

Peptidoglycan (PG) is unique to bacteria, and thus, the enzymes responsible for its biosynthesis are promising antibacterial drug targets. The membrane-embedded enzymes in PG remain significant challenges in studying their mechanisms due to the fact that preparations of suitable enzymatic substrates require time-consuming biological transformations or chemical synthesis. Lipid I (prenyl diphosphoryl-MurNAc-pentapeptide) is an important PG biosynthesis intermediate to study the central enzymes, translocase I (MraY/MurX) and MurG. Lipid I isolated from nature contains the C50-or C55-prenyl unit that shows extremely poor water-solubility that renders studies of translocase I and MurG enzymes difficult. We have studied biological transformation of water soluble lipid I fluorescent probes using bacterial membrane fractions and purified MraY enzymes. In our investigation of the minimum structural requirements of the prenyl phosphates in MraY-catalyzed lipid I synthesis, we found that (2Z,6E)-farnesyl phosphate (C15-phosphate) can be recognized by E. coli MraY to generate the water-soluble lipid I fluorescent probes in high-yield. Under the optimized conditions, the same reaction was performed by using the purified MraY from Hydrogenivirga spp. to afford the lipid I analog with high-yield in a short reaction time.