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.

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.

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.

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.

Role of TGF-ß in pancreatic ductal adenocarcinoma progression and PD-L1 expression.

PURPOSE: The transforming growth factor-beta (TGF-ß) pathway plays a paradoxical, context-dependent role in pancreatic ductal adenocarcinoma (PDAC): a tumor-suppressive role in non-metastatic PDAC and a tumor-promotive role in metastatic PDAC. We hypothesize that non-SMAD-TGF-ß signaling induces PDAC progression. METHODS: We investigated the expression of non-SMAD-TGF-ß signaling proteins (pMAPK14, PD-L1, pAkt and c-Myc) in patient-derived tissues, cell lines and an immunocompetent mouse model. Experimental models were complemented by comparing the signaling proteins in PDAC specimens from patients with various survival intervals. We manipulated models with TGF-ß, gemcitabine (DNA synthesis inhibitor), galunisertib (TGF-ß receptor inhibitor) and MK-2206 (Akt inhibitor) to investigate their effects on NF-κB, ß-catenin, c-Myc and PD-L1 expression. PD-L1 expression was also investigated in cancer cells and tumor associated macrophages (TAMs) in a mouse model. RESULTS: We found that tumors from patients with aggressive PDAC had higher levels of the non-SMAD-TGF-ß signaling proteins pMAPK14, PD-L1, pAkt and c-Myc. In PDAC cells with high baseline ß-catenin expression, TGF-ß increased ß-catenin expression while gemcitabine increased PD-L1 expression. Gemcitabine plus galunisertib decreased c-Myc and NF-κB expression, but induced PD-L1 expression in some cancer models. In mice, gemcitabine plus galunisertib treatment decreased metastases (p = 0.018), whereas galunisertib increased PD-L1 expression (p < 0.0001). In the mice, liver metastases contained more TAMs compared to the primary pancreatic tumors (p = 0.001), and TGF-ß increased TAM PD-L1 expression (p < 0.05). CONCLUSIONS: In PDAC, the non-SMAD-TGF-ß signaling pathway leads to more aggressive phenotypes, TAM-induced immunosuppression and PD-L1 expression. The divergent effects of TGF-ß ligand versus receptor inhibition in tumor cells versus TAMs may explain the TGF-ß paradox. Further evaluation of each mechanism is expected to lead to the development of targeted therapies.

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.

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).

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.

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.

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.

Novel FR-900493 Analogues That Inhibit the Outgrowth of Clostridium difficile Spores.

The spectrum of antibacterial activity for the nucleoside antibiotic FR-900493 (1) can be extended by chemical modifications. We have generated a small focused library based on the structure of 1 and identified UT-17415 (9), UT-17455 (10), UT-17460 (11), and UT-17465 (12), which exhibit anti-Clostridium difficile growth inhibitory activity. These analogues also inhibit the outgrowth of C. difficile spores at 2× minimum inhibitory concentration. One of these analogues, 11, relative to 1 exhibits over 180-fold and 15-fold greater activity against the enzymes, phospho-MurNAc-pentapeptide translocase (MraY) and polyprenyl phosphate-GlcNAc-1-phosphate transferase (WecA), respectively. The phosphotransferase inhibitor 11 displays antimicrobial activity against several tested bacteria including Bacillus subtilis, Clostridium spp., and Mycobacterium smegmatis, but no growth inhibitory activity is observed against the other Gram-positive and Gram-negative bacteria. The selectivity index (Vero cell cytotoxicity/C. difficileantimicrobial activity) of 11 is approximately 17, and 11 does not induce hemolysis even at a 100 µM concentration.

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.

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.