Virtual Screening Assisted Search for Inhibitors of the Translocated Intimin Receptor of Enteropathogenic Escherichia Coli.

This study aimed to identify inhibitors of the translocated intimin receptor (Tir) of enteropathogenic Escherichia coli (EPEC). EPEC is an intestinal pathogen that causes diarrhea and is a major health concern worldwide. Because Tir is a key virulence factor involved in EPEC pathogenesis, inhibiting its function is a potential strategy for controlling EPEC infections. Virtual screening was applied to chemical libraries to search for compounds that inhibit Tir-mediated bacterial adherence to host cells. Three sites were targeted using the cocrystal structure published earlier. A selection of compounds was then assessed in a cell-based infection model and fluorescence microscopy assay. The results of this study provide a basis for further optimization and testing of Tir inhibitors as potential therapeutic agents for EPEC infections.

Selected strategies to fight pathogenic bacteria.

Natural products and analogues are a source of antibacterial drug discovery. Considering drug resistance levels emerging for antibiotics, identification of bacterial metalloenzymes and the synthesis of selective inhibitors are interesting for antibacterial agent development. Peptide nucleic acids are attractive antisense and antigene agents representing a novel strategy to target pathogens due to their unique mechanism of action. Antisense inhibition and development of antisense peptide nucleic acids is a new approach to antibacterial agents. Due to the increased resistance of biofilms to antibiotics, alternative therapeutic options are necessary. To develop antimicrobial strategies, optimised in vitro and in vivo models are needed. In vivo models to study biofilm-related respiratory infections, device-related infections: ventilator-associated pneumonia, tissue-related infections: chronic infection models based on alginate or agar beads, methods to battle biofilm-related infections are discussed. Drug delivery in case of antibacterials often is a serious issue therefore this review includes overview of drug delivery nanosystems.

Synthesis and Cytotoxicity Evaluation of Spirocyclic Bromotyrosine Clavatadine C Analogs.

Marine-originated spirocyclic bromotyrosines are considered as promising scaffolds for new anticancer drugs. In a continuation of our research to develop potent and more selective anticancer compounds, we synthesized a library of 32 spirocyclic clavatadine analogs by replacing the agmatine, i.e., 4-(aminobutyl)guanidine, side chain with different substituents. These compounds were tested for cytotoxicity against skin cancer using the human melanoma cell line (A-375) and normal human skin fibroblast cell line (Hs27). The highest cytotoxicity against the A-375 cell line was observed for dichloro compound 18 (CC50 0.4 ± 0.3 µM, selectivity index (SI) 2). The variation of selectivity ranged from SI 0.4 to reach 2.4 for the pyridin-2-yl derivative 29 and hydrazide analog of 2-picoline 37. The structure-activity relationships of the compounds in respect to cytotoxicity and selectivity toward cancer cell lines are discussed.

A New Cell-Based AI-2-Mediated Quorum Sensing Interference Assay in Screening of LsrK-Targeted Inhibitors.

Quorum sensing (QS), a bacterial communication strategy, has been recognized as one of the control mechanisms of virulence in bacteria. Thus, targeting QS offers an interesting opportunity to impair bacterial pathogenicity and develop antivirulence agents. Aiming to enhance the discovery of QS inhibitors, we developed a bioreporter Escherichia coli JW5505 pET-Plsrlux and set up a cell-based assay for identifying inhibitors of autoinducer-2 (AI-2)-mediated QS. A comparative study on the performance of target- versus cell-based assays was performed, and 91 compounds selected with the potential to target the ATP binding pocket of LsrK, a key enzyme in AI-2 processing, were tested in an LsrK inhibition assay, providing 36 hits. The same set of compounds was tested by the AI-2-mediated QS interference assay, resulting in 24 active compounds. Among those, six were also found to be active against LsrK, whereas 18 might target other components of the pathway. Thus, this AI-2-mediated QS interference cell-based assay is an effective tool for complementing target-based assays, yet also stands as an independent assay for primary screening.

Anti-influenza virus activity of benzo[d]thiazoles that target heat shock protein 90.

Seasonal or pandemic influenza virus infections are a worldwide health problem requiring antiviral therapy. Since virus resistance to the established neuraminidase inhibitors and novel polymerase inhibitors is growing, new drug targets are needed. Heat shock protein 90 (Hsp90) is associated with several aspects of the influenza virus life cycle, and is considered a relevant host cell target. We report here on a series of benzo[d]thiazole and 4,5,6,7-tetrahydrobenzo[d]thiazole derivatives with robust and selective activities against influenza A (H1N1, H3N2) and influenza B viruses. Two compounds, 1 and 4, have low micromolar EC50 values and show high binding affinities for Hsp90, which suggests that inhibition of Hsp90 is the mechanism underlying their antiviral effects. These compounds represent suitable scaffolds for designing novel Hsp90 inhibitors with favourable activities against influenza virus.

Design, synthesis and biological evaluation of novel DNA gyrase inhibitors and their siderophore mimic conjugates.

Bacterial DNA gyrase is an important target for the development of novel antibacterial drugs, which are urgently needed because of high level of antibiotic resistance worldwide. We designed and synthesized new 4,5,6,7-tetrahydrobenzo[d]thiazole-based DNA gyrase B inhibitors and their conjugates with siderophore mimics, which were introduced to increase the uptake of inhibitors into the bacterial cytoplasm. The most potent conjugate 34 had an IC50 of 58 nM against Escherichia coli DNA gyrase and displayed MIC of 14 µg/mL against E. coli ΔtolC strain. Only minor improvements in the antibacterial activities against wild-type E. coli in low-iron conditions were seen for DNA gyrase inhibitor - siderophore mimic conjugates.

Composition and Antibacterial Effect of Mint Flavorings in Candies and Food Supplements.

Mint flavorings are widely used in confections, beverages, and dairy products. For the first time, mint flavoring composition of mint candies and food supplements (n = 45), originating from 16 countries, as well as their antibacterial properties, was analyzed. The flavorings were isolated by Marcusson's type micro-apparatus and analyzed by GC-MS. The total content of the mint flavoring hydrodistilled extracts was in the range of 0.01 - 0.9%. The most abundant compounds identified in the extracts were limonene, 1,8-cineole, menthone, menthofuran, isomenthone, menthol and its isomers, menthyl acetate. The antimicrobial activity of 13 reference substances and 10 selected mint flavoring hydrodistilled extracts was tested on Escherichia coli and Staphylococcus aureus by broth dilution method. Linalool acetate and (-)-carvone, as most active against both bacteria, had the lowest MIC90 values. (+)-Menthyl acetate, (-)-menthyl acetate, and limonene showed no antimicrobial activity. Three of the tested extracts had antimicrobial activity against E. coli and 8 extracts against S. aureus. Their summary antimicrobial activity was not always in concordance with the activities of respective reference substances.

2-Aminopyridine Analogs Inhibit Both Enzymes of the Glyoxylate Shunt in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen responsible for many hospital-acquired infections. P. aeruginosa can thrive in diverse infection scenarios by rewiring its central metabolism. An example of this is the production of biomass from C2 nutrient sources such as acetate via the glyoxylate shunt when glucose is not available. The glyoxylate shunt is comprised of two enzymes, isocitrate lyase (ICL) and malate synthase G (MS), and flux through the shunt is essential for the survival of the organism in mammalian systems. In this study, we characterized the mode of action and cytotoxicity of structural analogs of 2-aminopyridines, which have been identified by earlier work as being inhibitory to both shunt enzymes. Two of these analogs were able to inhibit ICL and MS in vitro and prevented growth of P. aeruginosa on acetate (indicating cell permeability). Moreover, the compounds exerted negligible cytotoxicity against three human cell lines and showed promising in vitro drug metabolism and safety profiles. Isothermal titration calorimetry was used to confirm binding of one of the analogs to ICL and MS, and the mode of enzyme inhibition was determined. Our data suggest that these 2-aminopyridine analogs have potential as anti-pseudomonal agents.

Targeting Quorum Sensing: High-Throughput Screening to Identify Novel LsrK Inhibitors.

Since quorum sensing (QS) is linked to the establishment of bacterial infection, its inactivation represents one of the newest strategies to fight bacterial pathogens. LsrK is a kinase playing a key role in the processing of autoinducer-2 (AI-2), a quorum-sensing mediator in gut enteric bacteria. Inhibition of LsrK might thus impair the quorum-sensing cascade and consequently reduce bacterial pathogenicity. Aiming for the development of a target-based assay for the discovery of LsrK inhibitors, we evaluated different assay set-ups based on ATP detection and optimized an automation-compatible method for the high-throughput screening of chemical libraries. The assay was then used to perform the screening of a 2000-compound library, which provided 12 active compounds with an IC50 ≤ 10 µM confirming the effectiveness and sensitivity of our assay. Follow-up studies on the positive hits led to the identification of two compounds, harpagoside and rosolic acid, active in a cell-based AI-2 QS interference assay, which are at the moment the most promising candidates for the development of a new class of antivirulence agents based on LsrK inhibition.

Identification and Characterization of Approved Drugs and Drug-Like Compounds as Covalent Escherichia coli ClpP Inhibitors.

The serine protease Caseinolytic protease subunit P (ClpP) plays an important role for protein homeostasis in bacteria and contributes to various developmental processes, as well as virulence. Therefore, ClpP is considered as a potential drug target in Gram-positive and Gram-negative bacteria. In this study, we utilized a biochemical assay to screen several small molecule libraries of approved and investigational drugs for Escherichia coli ClpP inhibitors. The approved drugs bortezomib, cefmetazole, cisplatin, as well as the investigational drug cDPCP, and the protease inhibitor 3,4-dichloroisocoumarin (3,4-DIC) emerged as ClpP inhibitors with IC50 values ranging between 0.04 and 31 µM. Compound profiling of the inhibitors revealed cefmetazole and cisplatin not to inhibit the serine protease bovine α-chymotrypsin, and for cefmetazole no cytotoxicity against three human cell lines was detected. Surface plasmon resonance studies demonstrated all novel ClpP inhibitors to bind covalently to ClpP. Investigation of the potential binding mode for cefmetazole using molecular docking suggested a dual covalent binding to Ser97 and Thr168. While only the antibiotic cefmetazole demonstrated an intrinsic antibacterial effect, cDPCP clearly delayed the bacterial growth recovery time upon chemically induced nitric oxide stress in a ClpP-dependent manner.

Analyzing user-generated online content for drug discovery: development and use of MedCrawler.

Motivation: Ethnopharmacology, or the scientific validation of traditional medicine, is a respected starting point in drug discovery. Home remedies and traditional use of plants are still widespread, also in Western societies. Instead of perusing ancient pharmacopeias, we developed MedCrawler, which we used to analyze blog posts for mentions of home remedies and their applications. This method is free and accessible from the office computer. Results: We developed MedCrawler, a data mining tool for analyzing user-generated blog posts aiming to find modern 'traditional' medicine or home remedies. It searches user-generated blog posts and analyzes them for correlations between medically relevant terms. We also present examples and show that this method is capable of delivering both scientifically validated uses as well as not so well documented applications, which might serve as a starting point for follow-up research. Availability and Implementation: Source code is available on GitHub at {{ https://github.com/a-hel/medcrawler }}. Contact: paivi.tammela@helsinki.fi. Supplementary information: Supplementary data are available at Bioinformatics online.

Defining conditions for biofilm inhibition and eradication assays for Gram-positive clinical reference strains.

BACKGROUND: Biofilms are formed by a complex bacterial community encapsulated by a polymeric matrix, with strong adherent properties and persistent phenotype. Biofilms are considered one of the most challenging areas of modern medicine. Existing antibiotics have been developed against free-floating bacterial cells, and thus, many treatments of biofilm-related infection fail. In this study, we compared the effects of different media on biofilm growth of clinical reference strains of Staphylococci and Enterococci, including multi-drug resistant representatives. Further, we optimized the resazurin-based assay for determining the minimal biofilm inhibitory concentration (MBIC) of standard antibiotics, and evaluated its use for the determination of minimal biofilm eradication concentration (MBEC). RESULTS: We showed that tryptic soy broth supplemented with 1% glucose was an optimal media for maximum biofilm growth of all strains tested, with an extended incubation time for Enterococci. A range of parameters were tested for the resazurin assay, including concentration, temperature and time of incubation. Using quality parameters to analyze the assay's performance, the conditions for the resazurin assay were set as follows: 4 µg/mL and 8 µg/mL, with incubation at 25 °C for 20 min and 40 min for Staphylococci and Enterococci, respectively. CONCLUSIONS: In summary, we defined conditions for optimal biofilm growth and for standardized resazurin assay for MBIC determination against six Gram-positive clinical reference strains. We also observed that MBEC determination by the resazurin-based assay is limited due to the poor detection limit of the assay. Complementary cell counting data is needed for precise determination of MBEC.

Marine Microalgae: Promising Source for New Bioactive Compounds.

The study of marine natural products for their bioactive potential has gained strength in recent years. Oceans harbor a vast variety of organisms that offer a biological and chemical diversity with metabolic abilities unrivalled in terrestrial systems, which makes them an attractive target for bioprospecting as an almost untapped resource of biotechnological applications. Among them, there is no doubt that microalgae could become genuine "cell factories" for the biological synthesis of bioactive substances. Thus, in the course of inter-laboratory collaboration sponsored by the European Union (7th FP) into the MAREX Project focused on the discovery of novel bioactive compounds of marine origin for the European industry, a bioprospecting study on 33 microalgae strains was carried out. The strains were cultured at laboratory scale. Two extracts were prepared for each one (biomass and cell free culture medium) and, thus, screened to provide information on the antimicrobial, the anti-proliferative, and the apoptotic potential of the studied extracts. The outcome of this study provides additional scientific data for the selection of Alexadrium tamarensis WE, Gambierdiscus australes, Prorocentrum arenarium, Prorocentrum hoffmannianum, and Prorocentrum reticulatum (Pr-3) for further investigation and offers support for the continued research of new potential drugs for human therapeutics from cultured microalgae.

Synthesis and Antiproliferative Activity of Marine Bromotyrosine Purpurealidin I and Its Derivatives.

The first total synthesis of the marine bromotyrosine purpurealidin I (1) using trifluoroacetoxy protection group and its dimethylated analog (29) is reported along with 16 simplified bromotyrosine derivatives lacking the tyramine moiety. Their cytotoxicity was evaluated against the human malignant melanoma cell line (A-375) and normal skin fibroblast cells (Hs27) together with 33 purpurealidin-inspired simplified amides, and the structure⁻activity relationships were investigated. The synthesized simplified analogs without the tyramine part retained the cytotoxic activity. Purpurealidin I (1) showed no selectivity but its simplified pyridin-2-yl derivative (36) had the best improvement in selectivity (Selectivity index 4.1). This shows that the marine bromotyrosines are promising scaffolds for developing cytotoxic agents and the full understanding of the elements of their SAR and improving the selectivity requires further optimization of simplified bromotyrosine derivatives.

Pumilol, a Diterpenoid with a Rare Strobane Skeleton from Pinus pumila (Pinaceae).

Pumilol (1), a strobane diterpenoid, reported herein for the first time, was isolated from the bark of Pinus pumila (Pall.) Regel (Siberian Dwarf Pine or Japanese Stone Pine), along with 17 known compounds including serratane triterpenoids, not previously reported from this species, and four ferulate derivatives. The absolute configuration of pumilol was established using HR-ESI-MS, NMR, the DP4+ probabilities and by comparison of the experimental and calculated electronic circular dichroism (ECD) spectra. Labda-8(17),13-dien-15-oic acid (4), bornyl (E)-4-hydroxycinnamate (14) and bornyl (E)-ferulate (15) showed activity against S. aureus and/or E. faecalis with MIC90 values 12.5 - 50 µm.

Design, synthesis, and biological evaluation of 1-ethyl-3-(thiazol-2-yl)urea derivatives as Escherichia coli DNA gyrase inhibitors.

Discovery of novel DNA gyrase B inhibitors remains an attractive field in the search for new antibacterial drugs to overcome the known bacterial resistance mechanisms. In the present study, we designed and synthesized novel ethylurea derivatives of 4,5,6,7-tetrahydrobenzo[1,2-d]thiazole-2,6-diamine, 2-(2-aminothiazol-4-yl)acetic acid, and benzo[1,2-d]thiazole-2,6-diamine and evaluated their Escherichia coli DNA gyrase inhibition. The most potent DNA gyrase inhibitors in the prepared library of compounds were benzo[1,2-d]thiazoles 32-34, 36, and 37 with IC50 values in the low micromolar range. The most promising inhibitors identified were evaluated against selected Gram-positive and Gram-negative bacterial strains. Compound 33 showed a MIC of 50 µM against an E. coli efflux pump-defective strain, which suggests that efflux decreases the on-target concentrations of these compounds.

A Versatile Strategy for the Synthesis of 4,5-Dihydroxy-2,3-Pentanedione (DPD) and Related Compounds as Potential Modulators of Bacterial Quorum Sensing.

Resistance to antibiotics is an increasingly serious threat to global public health and its management translates to significant health care costs. The validation of new Gram-negative antibacterial targets as sources for potential new antibiotics remains a challenge for all the scientists working in this field. The interference with bacterial Quorum Sensing (QS) mechanisms represents a potentially interesting approach to control bacterial growth and pursue the next generation of antimicrobials. In this context, our research is focused on the discovery of novel compounds structurally related to (S)-4,5-dihydroxy-2,3-pentanedione, commonly known as (S)-DPD, a small signaling molecule able to modulate bacterial QS in both Gram-negative and Gram-positive bacteria. In this study, a practical and versatile synthesis of racemic DPD is presented. Compared to previously reported syntheses, the proposed strategy is short and robust: it requires only one purification step and avoids the use of expensive or hazardous starting materials as well as the use of specific equipment. It is therefore well suited to the synthesis of derivatives for pharmaceutical research, as demonstrated by four series of novel DPD-related compounds described herein.

Inhibition of Breast Cancer Resistance Protein and Multidrug Resistance Associated Protein 2 by Natural Compounds and Their Derivatives.

The food and dietary supplements we consume contain a wide variety of plant secondary metabolites and other compounds, which, like drugs, can be absorbed, metabolized, distributed, and excreted from the body. In the intestine, these compounds can interact with transport proteins such as the multidrug resistance associated protein 2 (MRP2, ABCC2) and the breast cancer resistance protein (BCRP, ABCG2) that regulate the absorption of drugs and other compounds. Inhibition of these transporters by dietary components could lead to increased exposure and adverse effects of concomitantly administered drugs. Therefore, we screened a library of 124 natural compounds and their derivatives using the vesicular transport assay to evaluate their inhibitory potential on MRP2 and BCRP. Of the library compounds, 36% were identified as BCRP inhibitors, whereas the number was only 3.2% for MRP2. BCRP inhibitors are described by higher molecular weight, number of rings, aromaticity, and LogD7.4 than noninhibitors. IC50 values were measured for six dual inhibitors, among which three novel inhibitors, gossypin, nordihydroguaiaretic acid, and octyl gallate, were identified. Our results confirm that flavonoids are avid inhibitors of BCRP, and flavones and flavonols appear to be important subclasses of flavonoids for this inhibition. The strong inhibition of BCRP transport by some compounds suggests that their presence at high levels in the diet could cause food-drug interactions, but this seems to be a minor cause of concern for MRP2.

Design, synthesis and biological evaluation of 4,5-dibromo-N-(thiazol-2-yl)-1H-pyrrole-2-carboxamide derivatives as novel DNA gyrase inhibitors.

Development of novel DNA gyrase B inhibitors is an important field of antibacterial drug discovery whose aim is to introduce a more effective representative of this mechanistic class into the clinic. In the present study, two new series of Escherichia coli DNA gyrase inhibitors bearing the 4,5-dibromopyrrolamide moiety have been designed and synthesized. 4,5,6,7-Tetrahydrobenzo[1,2-d]thiazole-2,6-diamine derivatives inhibited E. coli DNA gyrase in the submicromolar to low micromolar range (IC50 values between 0.891 and 10.4µM). Their "ring-opened" analogues, based on the 2-(2-aminothiazol-4-yl)acetic acid scaffold, displayed weaker DNA gyrase inhibition with IC50 values between 15.9 and 169µM. Molecular docking experiments were conducted to study the binding modes of inhibitors.

Antibacterial profiling of abietane-type diterpenoids.

Abietic and dehydroabietic acid are interesting diterpenes with a highly diverse repertoire of associated bioactivities. They have, among others, shown antibacterial and antifungal activity, potentially valuable in the struggle against the increasing antimicrobial resistance and imminent antibiotic shortage. In this paper, we describe the synthesis of a set of 9 abietic and dehydroabietic acid derivatives containing amino acid side chains and their in vitro antimicrobial profiling against a panel of human pathogenic microbial strains. Furthermore, their in vitro cytotoxicity against mammalian cells was evaluated. The experimental results showed that the most promising compound was 10 [methyl N-(abiet-8,11,13-trien-18-yl)-d-serinate], with an MIC90 of 60µg/mL against Staphylococcus aureus ATCC 25923, and 8µg/mL against methicillin-resistant S. aureus, Staphylococcus epidermidis and Streptococcus mitis. The IC50 value for compound 10 against Balb/c 3T3 cells was 45µg/mL.