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.

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

Currently, there is a lack of FDA-approved tocolytics for the management of preterm labor (PL). In prior drug discovery efforts, we identified mundulone and its analog mundulone acetate (MA) as inhibitors of in vitro intracellular Ca 2+ -regulated myometrial contractility. In this study, we probed the tocolytic and therapeutic potential of these small molecules using myometrial cells and tissues obtained from patients receiving cesarean deliveries, as well as a mouse model of PL resulting in preterm birth. In a phenotypic assay, mundulone displayed greater efficacy in the inhibition of intracellular-Ca 2+ from myometrial cells; however, MA showed greater potency and uterine-selectivity, based IC 50 and E max values between myometrial cells compared to aorta vascular smooth muscle cells, a major maternal off-target site of current tocolytics. Cell viability assays revealed that MA was significantly less cytotoxic. Organ bath and vessel myography studies showed that only mundulone exerted concentration-dependent inhibition of ex vivo myometrial contractions and that neither mundulone or MA affected vasoreactivity of ductus arteriosus, a major fetal off-target of current tocolytics. A high-throughput combination screen of in vitro intracellular Ca 2+ -mobilization identified that mundulone exhibits synergism with two clinical-tocolytics (atosiban and nifedipine), and MA displayed synergistic efficacy with nifedipine. Of these synergistic combinations, mundulone + atosiban demonstrated a favorable in vitro therapeutic index (TI)=10, a substantial improvement compared to TI=0.8 for mundulone alone. The ex vivo and in vivo synergism of mundulone and atosiban was substantiated, yielding greater tocolytic efficacy and potency on isolated mouse and human myometrial tissue and reduced preterm birth rates in a mouse model of PL compared to each single agent. Treatment with mundulone 5hrs after mifepristone administration (and PL induction) dose-dependently delayed the timing of delivery. Importantly, mundulone in combination with atosiban (FR 3.7:1, 6.5mg/kg + 1.75mg/kg) permitted long-term management of PL after induction with 30 µg mifepristone, allowing 71% dams to deliver viable pups at term (> day 19, 4-5 days post-mifepristone exposure) without any visible maternal and fetal consequences. Collectively, these studies provide a strong foundation for the future development of mundulone as a stand-alone single- and/or combination-tocolytic therapy for management of PL.

Effects of Solvents, Emulsions, Cosolvents, and Complexions on Ex Vivo Mouse Myometrial Contractility.

A great need exists to develop tocolytic and uterotonic drugs that combat poor, labor-related maternal and fetal outcomes. A widely utilized method to assess novel compounds for their tocolytic and uterotonic efficacy is the isometric organ bath contractility assay. Unfortunately, water-insoluble compounds can be difficult to test using the physiological, buffer-based, organ bath assay. Common methods for overcoming solubility issues include solvent variation, cosolvency, surfactant or complexion use, and emulsification. However, these options for drug delivery or formulation can impact tissue function. Therefore, the goal of this study was to evaluate the ability of common solvents, surfactants, cosolvents, and emulsions to adequately solubilize compounds in the organ bath assay without affecting mouse myometrial contractility. We found that acetone, acetonitrile, and ethanol had the least effect, while dimethylacetamide, ethyl acetate, and isopropanol displayed the greatest inhibition of myometrial contractility based on area under the contractile curve analyses. The minimum concentration of surfactants, cosolvents, and human serum albumin required to solubilize nifedipine, a current tocolytic drug, resulted in extensive bubbling in the organ bath assay, precluding their use. Finally, we report that an oil-in-water base emulsion containing no drug has no statistical effect beyond the control (water), while the drug emulsion yielded the same potency and efficacy as the freely solubilized drug.

Drug discovery strategies for the identification of novel regulators of uterine contractility.

Preterm birth and postpartum hemorrhage are the leading causes of neonatal and maternal morbidities worldwide, respectively. Current clinically utilized tocolytics and uterotonics to manage these obstetric conditions are limited due to their off-target effects and/or lack of efficacy. Thus, an ideal tocolytic or uterotonic would be uterine-selective with rapid onset and long-duration efficacy. Here, we discuss strategies for the discovery of new therapeutic targets and compounds that regulate uterine contractility with the aforementioned properties.

Comparative analysis of myometrial and vascular smooth muscle cells to determine optimal cells for use in drug discovery.

Novel therapeutic regulators of uterine contractility are needed to manage preterm labor, induce labor and control postpartum hemorrhage. Therefore, we previously developed a high-throughput assay for large-scale screening of small molecular compounds to regulate calcium-mobilization in primary mouse uterine myometrial cells. The goal of this study was to select the optimal myometrial cells for our high-throughput drug discovery assay, as well as determine the similarity or differences of myometrial cells to vascular smooth muscle cells (VSMCs)-the most common off-target of current myometrial therapeutics. Molecular and pharmacological assays were used to compare myometrial cells from four sources: primary cells isolated from term pregnant human and murine myometrium, immortalized pregnant human myometrial (PHM-1) cells and immortalized non-pregnant human myometrial (hTERT-HM) cells. In addition, myometrial cells were compared to vascular SMCs. We found that the transcriptome profiles of hTERT-HM and PHM1 cells were most similar (r = 0.93 and 0.90, respectively) to human primary myometrial cells. Comparative transcriptome profiling of primary human myometrial transcriptome and VSMCs revealed 498 upregulated (p ≤ 0.01, log2FC≥1) genes, of which 142 can serve as uterine-selective druggable targets. In the high-throughput Ca2+-assay, PHM1 cells had the most similar response to primary human myometrial cells in OT-induced Ca2+-release (Emax = 195% and 143%, EC50 = 30 nM and 120 nM, respectively), while all sources of myometrial cells showed excellent and similar robustness and reproducibility (Z' = 0.52 to 0.77). After testing a panel of 61 compounds, we found that the stimulatory and inhibitory responses of hTERT-HM cells were highly-correlated (r = 0.94 and 0.95, respectively) to human primary cells. Moreover, ten compounds were identified that displayed uterine-selectivity (≥5-fold Emax or EC50 compared to VSMCs). Collectively, this study found that hTERT-HM cells exhibited the most similarity to primary human myometrial cells and, therefore, is an optimal substitute for large-scale screening to identify novel therapeutic regulators of myometrial contractility. Moreover, VSMCs can serve as an important counter-screening tool to assess uterine-selectivity of targets and drugs given the similarity observed in the transcriptome and response to compounds.

An antimycobacterial pleuromutilin analogue effective against dormant bacilli.

Pleuromutilin is a promising pharmacophore to design new antibacterial agents for Gram-positive bacteria. However, there are limited studies on the development of pleuromutilin analogues that inhibit growth of Mycobacterium tuberculosis (Mtb). In screening of our library of pleuromutilin derivatives, UT-800 (1) was identified to kill replicating- and non-replicating Mtb with the MIC values of 0.83 and 1.20 µg/mL, respectively. UT-800 also kills intracellular Mtb faster than rifampicin at 2× MIC concentrations. Pharmacokinetic studies indicate that 1 has an oral bioavailability with an average F-value of 27.6%. Pleuromutilin may have the potential to be developed into an orally administered anti-TB drug.

A New Combination of a Pleuromutilin Derivative and Doxycycline for Treatment of Multidrug-Resistant Acinetobacter baumannii.

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

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

A stereocontrolled first total synthesis of muraymycin D1 (1) has been achieved. The synthetic route is highly stereoselective, featuring (1) selective ß-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 (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).

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 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.

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 (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.

Discovery of a capuramycin analog that kills nonreplicating Mycobacterium tuberculosis and its synergistic effects with translocase I inhibitors.

Capuramycin (1) and its analogs are strong translocase I (MurX/MraY) inhibitors. In our structure-activity relationship studies of capuramycin analogs against Mycobacterium tuberculosis (Mtb), we observed for the first time that a capuramycin analog, UT-01320 (3) killed nonreplicating (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 tuberculosis drug.

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. We have established a scalable chemical synthesis of Park's nucleotide-N(ε)-dansylthiourea 2 that can be used as a MurX enzymatic substrate to form lipid I analogues. In our investigation of the 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. Here, we report a rapid and robust analytical method for quantifying MraY/MurX inhibitory activity of library molecules.

One-pot protection-glycosylation reactions for synthesis of lipid†II analogues.

(2,6-Dichloro-4-methoxyphenyl)(2,4-dichlorophenyl)methyl trichloroacetimidate (3) and its polymer-supported reagent 4 can be successfully applied to a one-pot protection-glycosylation reaction to form the disaccharide derivative 7 d for the synthesis of lipid II analogues. The temporary protecting group or linker at the C-6 position and N-Troc protecting group of 7 d can be cleaved simultaneously through a reductive condition. Overall yields of syntheses of lipid II (1) and neryl-lipid II N(ε)-dansylthiourea are significantly improved by using the described methods.

Improved synthesis of capuramycin and its analogues.

Capuramycin and its congeners are considered to be 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 because of difficulties in selectively chemically modifying the desired position(s) of the natural product with biologically interesting functional groups. We have developed efficient syntheses of capuramycin and its analogues by using new protecting groups, derived from the chiral (chloro-4-methoxyphenyl)(chlorophenyl)methanols, for the uridine ureido nitrogen and primary alcohol. The chiral nonracemic (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, the (S)-configuration isomer of which plays a significant role in improving the mycobactericidal activity of capuramycin.

Advances in MRSA drug discovery: where are we and where do we need to be?

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 multidrug-resistant (MDR) bacterial strains. Although there are a limited number of anti-MRSA drugs available, a number of different combination antimicrobial drug regimens have been used to treat serious MRSA infections. Thus, the addition of several new antistaphylococcal drugs into clinical practice should broaden clinician's therapeutic options. As MRSA is one of the most common and problematic bacteria associated with increasing antimicrobial resistance, continuous efforts for the discovery of lead compounds as well as development of alternative therapies and faster diagnostics 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 article discusses the mode of action of antistaphylococcal molecules and the resistant mechanisms of some molecules. EXPERT OPINION: The number of pipeline drugs presently undergoing clinical trials is not particularly encouraging. There are limited and rather expensive therapeutic options for MRSA infections in the critically ill. Further research efforts are required for effective phage therapy on MRSA infections in clinical use, which seem to be attractive therapeutic options for the future.

Discovery of selective menaquinone biosynthesis inhibitors against Mycobacterium tuberculosis.

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.