Discovery of Natural Product-based Antimycobacterial Agents Effective against Non-replicating Bacilli

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

Chemoenzymatic syntheses of water-soluble lipid I fluorescent probes

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 (MurNAc(pentapeptide)-pyrophosphoryl prenol) is an important PG biosynthesis intermediate to study the central enzymes, translocase I (MraY/MurX) and MurG. Lipid I isolated from nature contains the C_(50)- or C_(55)-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 (C_(15)-phosphate) can be recognized by Escherichia coli MraY to generate the water-soluble lipid I fluorescent probe in high-yields. 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-yields in a short reaction time

Biosynthesis of a water-soluble lipid I analogue and a convenient assay for translocase I

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 received considerable attention in the development of new tuberculosis (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; thus, this chemical characteristic of lipid I renders MurX enzyme assays impractical for screening and lacks reproducibility of the enzyme assays.Wehave established a scalable chemical synthesis of Park’s nucleotide-Ne-dansylthiourea 2 that can be used as a MurX enzymatic substrate to form lipid I analogues. In our investigation of the minimumstructure 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. Here, we report a rapid and robust analytical method for quantifying MraY/MurX inhibitory activity of library molecule

Stereocontrolled Total Synthesis of Muraymycin D1 Having a Dual Mode of Action against Mycobacterium tuberculosis

A stereocontrolled first total synthesis of muraymycin D1 (<b>1</b>) has been achieved. The synthetic route is highly stereoselective, featuring (1) selective β-ribosylation of the C2-methylated amino ribose, (2) selective Strecker reaction, and (3) ring-opening reaction of a diastereomeric mixture of a diaminolactone to synthesize muraymycidine (<i>epi</i>-capreomycidine). The acid-cleavable protecting groups for <i>secondary</i> alcohol and uridine ureido nitrogen are applied for simultaneous deprotections with the Boc and ^<i>t</i>Bu groups. Muraymycin D1 (<b>1</b>) and its amide derivatives (<b>2</b> and <b>3</b>) 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)

Fluorescence-based assay for polyprenyl phosphate-GlcNAc-1-phosphate transferase (WecA) and identification of novel antimycobacterial WecA inhibitors.

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 fluorescencebased 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 nbutanol and can be quantified by ultravioletevisible (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

A New Combination of a Pleuromutilin Derivative and Doxycycline for Treatment of Multidrug-Resistant <i>Acinetobacter baumannii</i>

Multidrug-resistant (MDR) <i>Acinetobacter baumannii</i> is one of the most difficult Gram-negative bacteria to treat and eradicate. In a cell-based screening of pleuromutilin derivatives against a drug sensitive <i>A. baumannii</i> strain, new molecules (<b>2</b>–<b>4</b>) exhibit bacteriostatic activity with 3.13 μg/mL concentration and <b>1</b> shows bactericidal activity with an MBC of 6.25 μg/mL. The pleuromutilin derivative <b>1</b> displays strong synergistic effects with doxycycline in a wide range of concentrations. A 35/1 ratio of <b>1</b> and doxycycline (<b>1-</b>Dox 35/1) kills drug susceptible <i>A. baumannii</i> with the MBC of 2.0 μg/mL and an MDR <i>A. baumannii</i> with the MBC of 3.13 μg/mL. In vitro anti-<i>Acinetobacter</i> activity of <b>1-</b>Dox 35/1 is superior to that of clinical drugs such as tobramycin, tigecycline, and colistin. The efficacy of <b>1-</b>Dox 35/1 is evaluated in a mouse septicemia model; treatment of the infected C57BL/6 mice with <b>1-</b>Dox 35/1 protects from lethal infection of <i>A. baumannii</i> with an ED₅₀ value of <2.0 mg/kg

Arrest of mouse preterm labor until term delivery by combination therapy with atosiban and mundulone, a natural product with tocolytic efficacy

There is a lack of FDA-approved tocolytics for the management of preterm labor (PL). In prior drug discovery efforts, we identified mundulone and mundulone acetate (MA) as inhibitors of in vitro intracellular Ca2+-regulated myometrial contractility. In this study, we probed the tocolytic potential of these compounds using human myometrial samples and a mouse model of preterm birth. In a phenotypic assay, mundulone displayed greater efficacy, while MA showed greater potency and uterine-selectivity in the inhibition of intracellular-Ca2+ mobilization. Cell viability assays revealed that MA was significantly less cytotoxic. Organ bath and vessel myography studies showed that only mundulone exerted inhibition of myometrial contractions and that neither compounds affected vasoreactivity of ductus arteriosus. A high-throughput combination screen identified that mundulone exhibits synergism with two clinical-tocolytics (atosiban and nifedipine), and MA displayed synergistic efficacy with nifedipine. Of these combinations, mundulone+atosiban demonstrated a significant improvement in the in vitro therapeutic index compared to mundulone alone. The ex vivo and in vivo synergism of mundulone+atosiban was substantiated, yielding greater tocolytic efficacy and potency on myometrial tissue and reduced preterm birth rates in a mouse model of PL compared to each single agent. Treatment with mundulone after mifepristone administration dose-dependently delayed the timing of delivery. Importantly, mundulone+atosiban permitted long-term management of PL, allowing 71% dams to deliver viable pups at term (>day 19, 4–5 days post-mifepristone exposure) without visible maternal and fetal consequences. Collectively, these studies provide a strong foundation for the development of mundulone as a single or combination tocolytic for management of PL