Antibacterial Activity of Pharmaceutical-Grade Rose Bengal: An Application of a Synthetic Dye in Antibacterial Therapies.

Rose bengal has been used in the diagnosis of ophthalmic disorders and liver function, and has been studied for the treatment of solid tumor cancers. To date, the antibacterial activity of rose bengal has been sporadically reported; however, these data have been generated with a commercial grade of rose bengal, which contains major uncontrolled impurities generated by the manufacturing process (80-95% dye content). A high-purity form of rose bengal formulation (HP-RBf, >99.5% dye content) kills a battery of Gram-positive bacteria, including drug-resistant strains at low concentrations (0.01-3.13 µg/mL) under fluorescent, LED, and natural light in a few minutes. Significantly, HP-RBf effectively eradicates Gram-positive bacterial biofilms. The frequency that Gram-positive bacteria spontaneously developed resistance to HP-RB is extremely low (less than 1 × 10-13). Toxicity data obtained through our research programs indicate that HP-RB is feasible as an anti-infective drug for the treatment of skin and soft tissue infections (SSTIs) involving multidrug-resistant (MDR) microbial invasion of the skin, and for eradicating biofilms. This article summarizes the antibacterial activity of pharmaceutical-grade rose bengal, HP-RB, against Gram-positive bacteria, its cytotoxicity against skin cells under illumination conditions, and mechanistic insights into rose bengal's bactericidal activity under dark conditions.

Concise Synthesis of Tunicamycin†V and Discovery of a Cytostatic DPAGT1 Inhibitor.

A short total synthesis of tunicamycin V (1), a non-selective phosphotransferase inhibitor, is achieved via a Büchner-Curtius-Schlotterbeck type reaction. Tunicamycin V can be synthesized in 15 chemical steps from D-galactal with 21 % overall yield. The established synthetic scheme is operationally very simple and flexible to introduce building blocks of interest. The inhibitory activity of one of the designed analogues 28 against human dolichyl-phosphate N-acetylglucosaminephosphotransferase 1 (DPAGT1) is 12.5 times greater than 1. While tunicamycins are cytotoxic molecules with a low selectivity, the novel analogue 28 displays selective cytostatic activity against breast cancer cell lines including a triple-negative breast cancer.

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.

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.

Structure determination of lipopeptides from Mycobacterium avium subspecies paratuberculosis and identification of antigenic lipopeptide probes.

Mycobacterium avium subspecies paratuberculosis (MAP) causes chronic illnesses mostly in ruminants. MAP infection of intestinal tissue triggers a fatal inflammatory disorder, Johne's disease (paratuberculosis). Development of fast and reliable diagnostic methods for Johne's disease in clinically suspected ruminants requires the discovery of MAP-specific antigens that induce immune responses. Despite a longtime interest in finding such antigens that can detect serum antibody responses with high sensitivity, the antigens currently used for a diagnosis of the MAP infections are the crude extracts from the whole cell. We performed the serum antibody response assay-guided purification of the ethanol extract from MAP isolated from an infected cow. With the results of extensive fractionations and in vitro assays, we identified that arachidyl-d-Phe-N-Me-l-Val-l-Ile-l-Phe-l-Ala-OH (named lipopeptide IIß, 3) exhibited the highest antibody binding activity in serum of a MAP-infected cattle compared with the other lipopeptides isolated from MAP. The absolute chemistry of 3 was determined unequivocally via our high-performance liquid chromatography (HPLC)-amino acid databases. α-Amino lipopeptide IIß and its fluorescent probes were synthesized and evaluated in serum antibody binding activity assays. Lipopeptide IIß-(2S)-NH2 (9) and its dansyl and fluorescein isothiocyanate (FITC) probes (10 and 11) exhibited antibody-mediated binding activity; thus, such MAP-specific lipopeptide probes can be potential biomarkers for the development of rapid and accurate diagnosis of Johne's disease.

Oxyma-based phosphates for racemization-free peptide segment couplings.

Glyceroacetonide-Oxyma [(2,2-dimethyl-1,3-dioxolan-4-yl)methyl 2-cyano-2-(hydroxyimino)acetate (1)] displayed remarkable physico-chemical properties as an additive for peptide-forming reactions. Although racemization-free amide-forming reactions have been established for N-urethane-protected α-amino acids with EDCI, 1, and NaHCO3 in water or DMF-water media, amide-forming reactions of N-acyl-protected α-amino acids and segment couplings of oligopeptides still require further development. Diethylphosphoryl-glyceroacetonide-oxyma (DPGOx 3) exhibits relative stability in aprotic solvents and is an effective coupling reagent for N-acyl-protected α-amino acids and oligo peptide segments. The conditions reported here is also effective in lactam-forming reactions. Unlike most of the reported coupling reagents, simple aqueous work-up procedures can remove the reagents and by-products generated in the reactions.

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.

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.

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.

Mild and convenient N-formylation protocol in water-containing solvents.

We have realized that N-formylations of free amines of some drug leads can improve PK/PD property of parent molecules without decreasing their biological activities. In order to selectively formylate primary amines of polyfunctional molecules, we have sought a mild and convenient formylation reaction. In our screening of N-formylation of an α-amino acid, L-phenylalanine, none of formylation conditions reported to date yielded the desired HCO-L-Phe-OH with satisfactory yield. N-Formylations of amino acids with HCO2H require the reactions in a water-containing media and suppress polymerization reactions due to the competitive reactions among carboxylic acids. We found that N-formylations of α-amino acids could be achieved with a water-soluble peptide coupling additive, an oxyma derivative, (2,2-dimethyl-1,3-dioxolan-4-yl)methyl-2-cyano-2-(hydroxyimino)acetate (2), EDCI, and NaHCO3 in water or a mixture of water and DMF system, yielding N-formylated α-amino acids with excellent yields. Moreover, these conditions could selectively formylate primary amines over secondary amines at a controlled temperature. A usefulness of these conditions was demonstrated by selective formylation of daptomycin antibiotic which contains three different amino groups.

An improved total synthesis of tunicamycin V.

The tunicamycins are important biochemical tools to study N-linked glycosylation and protein misfolding in cancer biochemistry fields. We reported a convergent synthesis of tunicamycin V with 21% overall yield from D-galactal. We have further optimized our original synthetic scheme by increasing the selectivity of azidonitration of the galactal derivative and developing a one-pot Büchner-Curtius-Schlotterbeck reaction. An improved synthetic scheme reported here enables the synthesis of tunicamycin V in 33% overall yield. In this article, we describe detailed procedures for a gram-scale synthesis of the key intermediate 12 and synthesizing 100 mg of tunicamycin V (1) from commercially available D-galctal-4,5-acetonide. All chemical steps have been repeated multiple times.•Highly selective azidonitration of N-(((3aR,4R,7aR)-2,2-dimethyl-3a,7a-dihydro-4H-[1,3]dioxolo[4,5-c]pyran-4-yl)methyl)acetamide (D-galctal-4,5-acetonide) to form 2-azido-2-deoxy-α/ß-D-galactopyranoside derivatives.•Optimized Büchner-Curtius-Schlotterbeck (BCS) reaction procedure for the tunicamycin core structure.•Full detail on the 15-chemical step synthesis of tunicamycin V.

Antibacterial effect of rose bengal against colistin-resistant gram-negative bacteria.

Increasing drug resistance in Gram-negative bacteria presents significant health problems worldwide. Despite notable advances in the development of a new generation of ß-lactams, aminoglycosides, and fluoroquinolones, it remains challenging to treat multi-drug resistant Gram-negative bacterial infections. Colistin (polymyxin E) is one of the most efficacious antibiotics for the treatment of multiple drug-resistant Gram-negative bacteria and has been used clinically as a last-resort option. However, the rapid spread of the transferable gene, mcr-1 which confers colistin resistance by encoding a phosphoethanolamine transferase that modifies lipid A of the bacterial membrane, threatens the efficacy of colistin for the treatment of drug-resistant bacterial infections. Colistin-resistant strains of Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae often reduce their susceptibility to other anti-Gram-negative bacterial agents. Thus, drugs effective against colistin-resistant strains or methods to prevent the acquisition of colistin-resistance during treatment are urgently needed. To perform cell-based screenings of the collected small molecules, we have generated colistin-resistant strains of E. coli, A. baumannii, K. pneumoniae, P. aeruginosa, and S. enterica Typhimurium. In-house MIC assay screenings, we have identified that rose bengal (4,5,6,7-tetrachloro-2',4',5',7'-tetraiodofluorescein) is the only molecule that displays unique bactericidal activity against these strains at low concentrations under illumination conditions. This article reports the antibacterial activity of a pharmaceutical-grade rose bengal against colistin-resistant Gram-negative bacteria.

Synthesis and structure-activity relationship of disubstituted benzamides as a novel class of antimalarial agents.

Malaria is a devastating world health problem. Using a compound library screening approach, we identified a novel series of disubstituted benzamide compounds with significant activity against malaria strains 3D7 and K1. These compounds represent a new antimalarial molecular scaffold exemplified by compound 1, which demonstrated EC(50) values of 60 and 430 nM against strains 3D7 and K1, respectively. Herein we report our findings on the efficient synthesis, structure-activity relationships, and biological activity of this new class of antimalarial agents.

A Convenient Protecting Group for Uridine Ureido Nitrogen: (4,4'-Bisfluorophenyl)methoxymethyl group.

(4,4'-Bisfluorophenyl)methoxymethyl (BFPM) group of uridine ureido nitrogen shows good relative stability in a variety of chemical transformation reactions for uridine. The BFPM group can be cleaved via 2% of TFA in CH2Cl2 without affecting the Boc group.

Application of Mycobacterium smegmatis as a surrogate to evaluate drug leads against Mycobacterium tuberculosis.

Discovery of new anti-tuberculosis (TB) drugs is a time-consuming process due to the slow-growing nature of Mycobacterium tuberculosis (Mtb). A requirement of biosafety level 3 (BSL-3) facility for performing research associated with Mtb is another limitation for the development of TB drug discovery. In our screening of BSL-1 Mycobacterium spp. against a battery of TB drugs, M. smegmatis (ATCC607) exhibits good agreement with its drug susceptibility against the TB drugs under a low-nutrient culture medium (0.5% Tween 80 in Middlebrook 7H9 broth). M. smegmatis (ATCC607) enters its dormant form in 14 days under a nutrient-deficient condition (a PBS buffer), and shows resistance to a majority of TB drugs, but shows susceptibility to amikacin, capreomycin, ethambutol, and rifampicin (with high concentrations) whose activities against non-replicating (or dormant) Mtb were previously validated.

Substrate Tolerance of Bacterial Glycosyltransferase MurG: Novel Fluorescence-Based Assays.

MurG (uridine diphosphate-N-acetylglucosamine/N-acetylmuramyl-(pentapeptide) pyrophosphoryl-undecaprenol N-acetylglucosamine transferase) is an essential bacterial glycosyltransferase that catalyzes the N-acetylglucosamine (GlcNAc) transformation of lipid I to lipid II during peptidoglycan biosynthesis. Park's nucleotide has been a convenient biochemical tool to study the function of MraY (phospho-MurNAc-(pentapeptide) translocase) and MurG; however, no fluorescent probe has been developed to differentiate individual processes in the biotransformation of Park's nucleotide to lipid II via lipid I. Herein, we report a robust assay of MurG using either the membrane fraction of a M. smegmatis strain or a thermostable MraY and MurG of Hydrogenivirga sp. as enzyme sources, along with Park's nucleotide or Park's nucleotide-Nε-C6-dansylthiourea and uridine diphosphate (UDP)-GlcN-C6-FITC as acceptor and donor substrates. Identification of both the MraY and MurG products can be performed simultaneously by HPLC in dual UV mode. Conveniently, the generated lipid II fluorescent analogue can also be quantitated via UV-Vis spectrometry without the separation of the unreacted lipid I derivative. The microplate-based assay reported here is amenable to high-throughput MurG screening. A preliminary screening of a collection of small molecules has demonstrated the robustness of the assays and resulted in rediscovery of ristocetin A as a strong antimycobacterial MurG and MraY inhibitor.

DPAGT1 Inhibitors of Capuramycin Analogues and Their Antimigratory Activities of Solid Tumors.

Capuramycin displays a narrow spectrum of antibacterial activity by targeting bacterial translocase I (MraY). In our program of development of new N-acetylglucosaminephosphotransferase1 (DPAGT1) inhibitors, we have identified that a capuramycin phenoxypiperidinylbenzylamide analogue (CPPB) inhibits DPAGT1 enzyme with an IC50 value of 200 nM. Despite a strong DPAGT1 inhibitory activity, CPPB does not show cytotoxicity against normal cells and a series of cancer cell lines. However, CPPB inhibits migrations of several solid cancers including pancreatic cancers that require high DPAGT1 expression in order for tumor progression. DPAGT1 inhibition by CPPB leads to a reduced expression level of Snail but does not reduce E-cadherin expression level at the IC50 (DPAGT1) concentration. CPPB displays a strong synergistic effect with paclitaxel against growth-inhibitory action of a patient-derived pancreatic adenocarcinoma, PD002: paclitaxel (IC50: 1.25 µM) inhibits growth of PD002 at 0.0024-0.16 µM in combination with 0.10-2.0 µM CPPB (IC50: 35 µM).

A practical synthesis of a novel DPAGT1 inhibitor, aminouridyl phenoxypiperidinbenzyl butanamide (APPB) for in vivo studies.

Immunotherapy that targets N-linked glycans has not yet been developed due in large part to the lack of specificity of N-linked glycans between normal and malignant cells. N-Glycan chains are synthesized by the sequential action of glycosyl transferases in the Golgi apparatus. It is an overwhelming task to discover drug-like inhibitors of glycosyl transferases that block the synthesis of specific branching processes in cancer cells, killing tumor cells selectively. It has long been known that N-glycan biosynthesis can be inhibited by disruption of the first committed enzyme, dolichyl-phosphate N-acetylglucosaminephosphotransferase 1 (DPAGT1). Selective DPAGT1 inhibitors have the promising therapeutic potential for certain solid cancers that require increased branching of N-linked glycans in their growth progressions. Recently, we discovered that an anti-Clostridium difficile molecule, aminouridyl phenoxypiperidinbenzyl butanamide (APPB) showed DPAGT1 inhibitory activity with the IC50 value of 0.25 µM. It was confirmed that APPB inhibits N-glycosylation of ß-catenin at 2.5 nM concentration. A sharp difference between APPB and tunicamycin was that the hemolytic activity of APPB is significantly attenuated (IC50 > 200 µM RBC). Water solubility of APPB is >350-times greater than that of tunicamycin (78.8 mg/mL for APPB, <0.2 mg/mL for tunicamycin). A novel DPAGT1 inhibitor, APPB selectively inhibits growth of the solid tumors (e.g. KB, LoVo, SK-OV-3, MDA-MB-432S, HCT116, Panc-1, and AsPC-1) at low µM concentrations, but does not inhibit growth of a leukemia cell (L1210) and the healthy cells (Vero and HPNE) at these concentrations. In vitro metabolic stability using rat liver microsomes indicated that a half-life (t 1/2) of APPB is sufficiently long (>60 min) for in vivo studies (PK/PD, safety profiles, and in vivo efficacy) using animal models. We have refined all steps in the previously reported synthesis for APPB for larger-scale. This article summarizes protocols of gram-scale synthesis of APPB and its physicochemical data, and a convenient DPAGT1 assay. •Remember that the abstract is what readers see first in electronic abstracting & indexing services.•This is the advertisement of your article. Make it interesting, and easy to be understood.•Be accurate and specific, keep it as brief as possible.

Semisynthesis of an Anticancer DPAGT1 Inhibitor from a Muraymycin Biosynthetic Intermediate.

We have explored a method to convert a muraymycin biosynthetic intermediate 3 to an anticancer drug lead 2 for in vivo and thorough preclinical studies. Cu(OAc)2 forms a stable complex with the amide 4 and prevents electrophilic reactions at the 2-((3-aminopropyl)amino)acetamide moiety. Under the present conditions, the desired 5″-primary amine was selectively protected with (Boc)2O to yield 6. The intermediate 6 was converted to 2 in two steps with 90% yield.