Biomineralized Cascade Enzyme-Encapsulated ZIF-8 Nanoparticles Combined with Antisense Oligonucleotides for Drug-Resistant Bacteria Treatment.

The unrestrained use of antibiotics accelerates the development of drug-resistant bacteria and leads to an increasing threat to human health. Therefore, there is an urgent need to explore novel and effective strategies for the treatment of bacterial infections. Herein, zeolite imidazole framework-8 (ZIF-8) material was utilized to construct biomineralized nanomaterial (GOx&HRP@ZIF-8/ASO) by encapsulating biological cascade enzymes and combining with antisense oligonucleotides (ASOs), which achieved effective and synergistic antidrug-resistant bacteria therapy. Various in vitro assays confirmed that GOx&HRP@ZIF-8/ASO exhibited excellent antibacterial properties against Escherichia coli, Staphylococcus aureus, methicillin-resistant S. aureus (MRSA) during catalysis of glucose (Glu), especially the minimum inhibitory concentration (MIC) against MRSA was only 16 µg/mL. Compared with simple ZIF-8 (32.85%) and ftsZ ASO (58.65%), GOx&HRP@ZIF-8/ASO+Glu exhibited superb biofilm destruction ability, and the bacteria removal efficiency of the MRSA biofilm could be as high as 88.2%, indicating that the reactive oxygen species (ROS) produced by the cascade enzyme reaction imparted the main synergistic antibacterial capability, and simultaneously, ftsZ ASO significantly enhanced the antibacterial effect by inhibiting the expression of the ftsZ gene. In vivo anti-infection treatment experiments revealed that GOx&HRP@ZIF-8/ASO exhibited the best wound repairing performance and excellent biocompatibility in the presence of Glu. These findings suggested that GOx&HRP@ZIF-8/ASO has favorably realized high-efficiency treatment of MRSA infection and filled the gap in the antibacterial application of biological enzymes.

Permeant fluorescent probes visualize the activation of SARM1 and uncover an anti-neurodegenerative drug candidate.

SARM1 regulates axonal degeneration through its NAD-metabolizing activity and is a drug target for neurodegenerative disorders. We designed and synthesized fluorescent conjugates of styryl derivative with pyridine to serve as substrates of SARM1, which exhibited large red shifts after conversion. With the conjugates, SARM1 activation was visualized in live cells following elevation of endogenous NMN or treatment with a cell-permeant NMN-analog. In neurons, imaging documented mouse SARM1 activation preceded vincristine-induced axonal degeneration by hours. Library screening identified a derivative of nisoldipine (NSDP) as a covalent inhibitor of SARM1 that reacted with the cysteines, especially Cys311 in its ARM domain and blocked its NMN-activation, protecting axons from degeneration. The Cryo-EM structure showed that SARM1 was locked into an inactive conformation by the inhibitor, uncovering a potential neuroprotective mechanism of dihydropyridines.

Identification of the first noncompetitive SARM1 inhibitors.

Sterile Alpha and Toll Interleukin Receptor Motif-containing protein 1 (SARM1) is a key therapeutic target for diseases that exhibit Wallerian-like degeneration; Wallerian degeneration is characterized by degeneration of the axon distal to the site of injury. These diseases include traumatic brain injury, peripheral neuropathy, and neurodegenerative diseases. SARM1 promotes neurodegeneration by catalyzing the hydrolysis of NAD+ to form a mixture of ADPR and cADPR. Notably, SARM1 knockdown prevents degeneration, indicating that SARM1 inhibitors will likely be efficacious in treating these diseases. Consistent with this hypothesis is the observation that NAD+ supplementation is axoprotective. To identify compounds that block the NAD+ hydrolase activity of SARM1, we developed and performed a high-throughput screen (HTS). This HTS assay exploits an NAD+ analog, etheno-NAD+ (ENAD) that fluoresces upon cleavage of the nicotinamide moiety. From this screen, we identified berberine chloride and zinc chloride as the first noncompetitive inhibitors of SARM1. Though modest in potency, the noncompetitive mode of inhibition, suggests the presence of an allosteric binding pocket on SARM1 that can be targeted for future therapeutic development. Additionally, zinc inhibition and site-directed mutagenesis reveals that cysteines 629 and 635 are critical for SARM1 catalysis, highlighting these sites for the design of inhibitors targeting SARM1.

Inhibitory activity of traditional plants against Mycobacterium smegmatis and their action on Filamenting temperature sensitive mutant Z (FtsZ)-A cell division protein.

The study was designed to assess whether plant extracts / phytochemical (D-Pinitol) synergistically combine with antituberculosis drugs and act on Mycobacterium smegmatis (M. smegmatis) as well as assess their mode of action on Mycobacterium tuberculosis (M.tb) Filamenting temperature sensitive mutant Z (FtsZ) protein. Resazurin microtitre plate assay (Checker board) was performed to analyze the activity of plant extracts against M. smegmatis. Synergistic behaviour of plant extracts / D-Pinitol with Isoniazid (INH) and Rifampicin (RIF) were determined by time-kill and checker board assays. Elongation of M. smegmatis cells due to this treatment was determined by light microscopy. The effect of Hexane methanol extract (HXM) plant extracts on cell viability was determined using PI/SYTO9 dual dye reporter Live/Dead assay. Action of HXM plant extracts / D-Pinitol on inhibition of FtsZ protein was done using Guanosine triphosphatase (GTPase) light scattering assay and quantitative Polymerase Chain Reaction (qPCR). The Hexane-methanolic plant extract of Acacia nilotica, Aegle marmelos and Glycyrrhiza glabra showed antimycobacterial activity at 1.56 ± 0.03, 1.32 ± 0.02 and 1.25 ± 0.03 mg/mL respectively and that of INH and RIF were 4.00 ± 0.06 µg/mL and 2.00 ± 0.04 µg/mL respectively. These plant extracts and major phytochemical exudate D-Pinitol was found to act synergistically with antimycobacterial drugs INH and RIF with an FIC index ~ 0.20. Time-Kill kinetics studies indicate that, these plant extracts were bacteriostatic in nature. D-Pinitol in conjunction with INH and RIF exhibited a 2 Log reduction in the growth of viable cells compared to untreated. Attempt to elucidate their mode of action through phenotypic analysis indicated that these plant extracts and D-Pinitol was found to interfere in cell division there by leading to an abnormal elongated cellular morphology. HXM extracts and D-Pinitol synergistically combined with the first line tuberculosis drugs, INH and RIF, to act on M. smegmatis. The increase in the length of M. smegmatis cells on treatment with D-Pinitol and HXM extract of the plants indicated that they hinder the cell division mechanism thereby leading to a filamentous phenotype, and finally leading to cell death. In addition, the integrity of the bacterial cell membrane is also altered causing cell death. Further gene expression analysis showed that these plant extracts and D-Pinitol hampers with function of FtsZ protein which was confirmed through in vitro inhibition of FtsZ-GTPase enzymatic activity.

Target Based Virtual Screening of New Leads Inhibitor against Bacterial Cell Division Protein FtsZ for the Discovery of Antibacterial Agents.

BACKGROUND: Staphylococus epidermidis coagulase negative and gram positive streptococci have emerged as major nosocomial pathogens associated with the infection of implanted medical devices and dandruff on human scalp. S. epidermidis filamenting temperature-sensitive mutant Z (FtsZ) gene encoded FtsZ protein that assembles at future bacterial cell division site that forms Z-ring structure. FtsZ is a tubulin homolog protein with low sequence similarity; this makes it possible to inhibit bacterial FtsZ protein without affecting the eukaryote cell division. OBJECTIVE: In the present study, phytochemicals of Cinnamomum zeylanicum, Punica granatum and Glycyrrhiza glabra were virtually screened for their antibacterial activity against Staphylococcus epidermidis cell division protein, FtsZ. METHODS: Molecular docking method was used to investigate new lead inhibitor against bacterial cell division protein FtsZ. SwissADME and ProTox tool were used to evaluate the toxicity of the lead molecule. RESULTS: Molecular docking based screening confirmed that among 122 phytochemicals, ß- sitosterol and glabrol showed the highest inhibitory activity against FtsZ. SwissADME tool showed ß-sitosterol and glabrol as the ideal antibacterial agents. CONCLUSION: Structure based drug design strategy has been broadly used to optimize antimicrobial activity of small molecule/ligand against large protein receptor of disease, causing pathogens which gives a major breakthrough in pharmaceuticals industries. The molecular docking and SwissADME tool showed that ß-sitosterol and glabrol may be developed to be potential topical and sublingual antibacterial agents, respectively.

Competitive Fitness of Essential Gene Knockdowns Reveals a Broad-Spectrum Antibacterial Inhibitor of the Cell Division Protein FtsZ.

To streamline the elucidation of antibacterial compounds' mechanism of action, comprehensive high-throughput assays interrogating multiple putative targets are necessary. However, current chemogenomic approaches for antibiotic target identification have not fully utilized the multiplexing potential of next-generation sequencing. Here, we used Illumina sequencing of transposon insertions to track the competitive fitness of a Burkholderia cenocepacia library containing essential gene knockdowns. Using this method, we characterized a novel benzothiadiazole derivative, 10126109 (C109), with antibacterial activity against B. cenocepacia, for which whole-genome sequencing of low-frequency spontaneous drug-resistant mutants had failed to identify the drug target. By combining the identification of hypersusceptible mutants and morphology screening, we show that C109 targets cell division. Furthermore, fluorescence microscopy of bacteria harboring green fluorescent protein (GFP) cell division protein fusions revealed that C109 prevents divisome formation by altering the localization of the essential cell division protein FtsZ. In agreement with this, C109 inhibited both the GTPase and polymerization activities of purified B. cenocepacia FtsZ. C109 displayed antibacterial activity against Gram-positive and Gram-negative cystic fibrosis pathogens, including Mycobacterium abscessus C109 effectively cleared B. cenocepacia infection in the Caenorhabditis elegans model and exhibited additive interactions with clinically relevant antibiotics. Hence, C109 is an enticing candidate for further drug development.

New antimicrobial compounds produced by endophytic Penicillium janthinellum isolated from Panax notoginseng as potential inhibitors of FtsZ.

A total of 180 fungal isolates, belonging to 20 genera and 47 species, were obtained from the roots, stems and leaves of Panax notoginseng. One isolate, the endophytic fungus Penicillium janthinellum SYPF 7899, displayed the strongest antibacterial activity and was studied for its production of secondary metabolites. In total, three new compounds, including rotational isomers 1a, 1b and 2 were isolated from the solid cultures of P. janthinellum, as well as eight known compounds (3-10). These structures were determined on the basis of 1D, 2D NMR and electronic circular dichroism (ECD) spectroscopic analyses as well as theoretical calculations. Compound 1 exhibited significant inhibitory activities against Bacillus subtilis and Staphylococcus aureus with MIC values of 15 and 18 µg/ml, respectively. The other compounds showed moderate or weak activities. In addition, morphological observation showed the rod-shaped cells of B. subtilis growing into long filaments, which reached 1.5- to 2-fold of the length of the original cells after treatment with compound 1. The coccoid cells of S. aureus exhibited a similar response and swelled to a 2-fold volume after treatment with compound 1. In silico molecular docking was explored to study the binding interactions between the compounds and the active sites of filamentous temperature-sensitive protein Z (FtsZ) from B. subtilis and S. aureus. Compound 1a, 1b and 2 showed high binding energies, strong H-bond interactions and hydrophobic interactions with FtsZ. Based on the antimicrobial activities, cellular phenotype observation and docking studies, compound 1 is considered to be a promising antimicrobial inhibitor of FtsZ.

An endophytic Fungi of Ginkgo biloba L. produces antimicrobial metabolites as potential inhibitors of FtsZ of Staphylococcus aureus.

A total of 58 fungal isolates, belonging to 24 genera, were obtained from the leaves, stems and roots of Ginkgo biloba L.. Among them, one endophytic fungal strain, Penicillium cataractum SYPF 7131, displayed the strongest antibacterial activity. Four new compounds (1-4) were isolated from the strain fermentation broth together with four known compounds (5-8). These structures were determined on the basis of 1D and 2D NMR and [Rh2(OCOCF3)4]-induced electronic circular dichroism (ECD) spectroscopic analyses. All the isolated compounds were screened for their in vitro antimicrobial activities. Compound 3 and 4 showed moderate inhibitory activity against Staphylococcus aureus. Compound 7 exhibited significant inhibitory activity against S. aureus with MIC value of 10 µg/mL. Further, the in silico molecular docking studies of the active compounds was used to explore the binding interactions with the active site of filamentous temperature-sensitive protein Z (FtsZ) from Staphylococcus aureus. The docking results revealed that compounds 3, 4 and 7 showed high binding energies, strong H-bond interactions and hydrophobic interactions with FtsZ from S. aureus validating the observed antimicrobial activity. Based on antimicrobial activities and docking studies, compounds 3, 4 and 7 were identified as promising antimicrobial lead molecules.

Antibacterial activities of viriditoxin congeners and synthetic analogues against fish pathogens.

Viriditoxin is a fungal secondary metabolite of the fungus Paecilomyces variotii derived from the inner tissues of the giant jellyfish Nemopilema nomurai. Viriditoxin exhibits antibacterial activity against Streptococcus iniae and Streptococcus parauberis, which are major pathogens of aqua cultured fish. Viriditoxin induced abnormal cell morphologies in the fish pathogens S. iniae and S. parauberis, presumably by inhibiting FtsZ polymerization as was previously observed in Escherichia coli. Synthetic analogues of viriditoxin, designed based on docking simulation results to FtsZ of Staphylococcus aureus, were prepared and compared with viriditoxin for antibacterial activity. Reconstitution of free hydroxyl or carboxyl groups of the methoxyl or methyl ester groups of viriditoxin led to significant reduction of antibacterial activity, implying that the natural molecule is optimized for antibacterial activity to deter bacteria potentially harmful to Paecilomyces.

Pocket detection and interaction-weighted ligand-similarity search yields novel high-affinity binders for Myocilin-OLF, a protein implicated in glaucoma.

Traditional structure and ligand based virtual screening approaches rely on the availability of structural and ligand binding information. To overcome this limitation, hybrid approaches were developed that relied on extraction of ligand binding information from proteins sharing similar folds and hence, evolutionarily relationship. However, they cannot target a chosen pocket in a protein. To address this, a pocket centric virtual ligand screening approach is required. Here, we employ a new, iterative implementation of a pocket and ligand-similarity based approach to virtual ligand screening to predict small molecule binders for the olfactomedin domain of human myocilin implicated in glaucoma. Small-molecule binders of the protein might prevent the aggregation of the protein, commonly seen during glaucoma. First round experimental assessment of the predictions using differential scanning fluorimetry with myoc-OLF yielded 7 hits with a success rate of 12.7%; the best hit had an apparent dissociation constant of 99nM. By matching to the key functional groups of the best ligand that were likely involved in binding, the affinity of the best hit was improved by almost 10,000 fold from the high nanomolar to the low picomolar range. Thus, this study provides preliminary validation of the methodology on a medically important glaucoma associated protein.

Identification of Novel Ezrin Inhibitors Targeting Metastatic Osteosarcoma by Screening Open Access Malaria Box.

Ezrin is a member of the ERM (ezrin, radixin, moesin) family of proteins and functions as a linker between the plasma membrane and the actin cytoskeleton. Ezrin is a key driver of tumor progression and metastatic spread of osteosarcoma. We discovered a quinoline-based small molecule, NSC305787, that directly binds to ezrin and inhibits its functions in promoting invasive phenotype. NSC305787 possesses a very close structural similarity to commonly used quinoline-containing antimalarial drugs. On the basis of this similarity and of recent findings that ezrin has a likely role in the pathogenesis of malaria infection, we screened antimalarial compounds in an attempt to identify novel ezrin inhibitors with better efficacy and drug properties. Screening of Medicines for Malaria Venture (MMV) Malaria Box compounds for their ability to bind to recombinant ezrin protein yielded 12 primary hits with high selective binding activity. The specificity of the hits on ezrin function was confirmed by inhibition of the ezrin-mediated cell motility of osteosarcoma cells. Compounds were further tested for phenocopying the morphologic defects associated with ezrin suppression in zebrafish embryos as well as for inhibiting the lung metastasis of high ezrin-expressing osteosarcoma cells. The compound MMV667492 exhibited potent anti-ezrin activity in all biologic assays and had better physicochemical properties for drug-likeness than NSC305787. The drug-like compounds MMV020549 and MMV666069 also showed promising activities in functional assays. Thus, our study suggests further evaluation of antimalarial compounds as a novel class of antimetastatic agents for the treatment of metastatic osteosarcoma.

Virtual screening of potential inhibitor against FtsZ protein from Staphylococcus aureus.

The gram-positive bacterium Staphylococcus aureus, responsible for a wide variety of diseases in human involve all organ systems ranging from localized skin infections to life-threatening systemic infections. FtsZ, the key protein of bacterial cell division was selected as a potent anti bacterial target. In order to identify the new compounds structure based screening process was carried out. An enrichment study was performed to select a suitable scoring function and to retrieve potential candidates against FtsZ from a large chemical database. The docking score and docking energy values were compared and their atomic interaction was also evaluated. Furthermore molecular dynamics simulation were also been performed to check the stability and the amino acids interacted towards the FtsZ. Finally we selected C ID 16284, 25916, 15894, 13403 as better lead compounds. From these results, we conclude that our insilico results will provide a framework for the detailed in vitro and in vivo studies about the FtsZ protein activity in drug development process.

Biological activity of Pinus nigra terpenes--evaluation of FtsZ inhibition by selected compounds as contribution to their antimicrobial activity.

In the current work, in vitro antioxidant, antibacterial, and antifungal activites of the needle terpenes of three taxa of Pinus nigra from Serbia (ssp. nigra, ssp. pallasiana, and var. banatica) were analyzed. The black pine essential oils showed generally weak antioxidative properties tested by two methods (DPPH and ABTS scavenging assays), where the highest activity was identified in P. nigra var. banatica (IC50=25.08 mg/mL and VitC=0.67 mg (vitamin C)/g when tested with the DPPH and ABTS reagents, respectively). In the antimicrobial assays, one fungal (Aspergilus niger) and two bacterial strains (Staphylococcus aureus and Bacillus cereus) showed sensitivity against essential oils of all three P. nigra taxa. The tested oils have been shown to possess inhibitory action in the range from 20.00 to 0.62 mg/mL, where var. banatica exhibited the highest and ssp. nigra the lowest antimicrobial action. In order to determine potential compounds that are responsible for alternative mode of action, molecular docking simulations inside FtsZ (a prokaryotic homolog of tubulin) were performed. Tested compounds were the most abundant terpenoid (germacrene D-4-ol) and its structurally similar terpene (germacrene D), both present in all three essential oils. It was determined that the oxygenated form of the molecule creates stable bonds with investigated enzyme FtsZ, and that this compound, through this mechanism of action participates in the antimicrobial activity.

Targeting the Wolbachia cell division protein FtsZ as a new approach for antifilarial therapy.

The use of antibiotics targeting the obligate bacterial endosymbiont Wolbachia of filarial parasites has been validated as an approach for controlling filarial infection in animals and humans. Availability of genomic sequences for the Wolbachia (wBm) present in the human filarial parasite Brugia malayi has enabled genome-wide searching for new potential drug targets. In the present study, we investigated the cell division machinery of wBm and determined that it possesses the essential cell division gene ftsZ which was expressed in all developmental stages of B. malayi examined. FtsZ is a GTPase thereby making the protein an attractive Wolbachia drug target. We described the molecular characterization and catalytic properties of Wolbachia FtsZ. We also demonstrated that the GTPase activity was inhibited by the natural product, berberine, and small molecule inhibitors identified from a high-throughput screen. Furthermore, berberine was also effective in reducing motility and reproduction in B. malayi parasites in vitro. Our results should facilitate the discovery of selective inhibitors of FtsZ as a novel anti-symbiotic approach for controlling filarial infection. NOTE: The nucleotide sequences reported in this paper are available in GenBank™ Data Bank under the accession number wAlB-FtsZ (JN616286).

Genetic evidence for inhibition of bacterial division protein FtsZ by berberine.

BACKGROUND: Berberine is a plant alkaloid that is widely used as an anti-infective in traditional medicine. Escherichia coli exposed to berberine form filaments, suggesting an antibacterial mechanism that involves inhibition of cell division. Berberine is a DNA ligand and may induce filamentation through induction of the SOS response. Also, there is biochemical evidence for berberine inhibition of the cell division protein FtsZ. Here we aimed to assess possible berberine mechanism(s) of action in growing bacteria using genetics tools. METHODOLOGY/PRINCIPAL FINDINGS: First, we tested whether berberine inhibits bacterial growth through DNA damage and induction of the SOS response. The SOS response induced by berberine was much lower compared to that induced by mitomycin C in an SOS response reporter strain. Also, cell filamentation was observed in an SOS-negative E. coli strain. To test whether berberine inhibits FtsZ, we assessed its effects on formation of the cell division Z-rings, and observed a dramatic reduction in Z-rings in the presence of berberine. We next used two different strategies for RNA silencing of ftsZ and both resulted in sensitisation of bacteria to berberine, visible as a drop in the Minimum Inhibitory Concentration (MIC). Furthermore, Fractional Inhibitory Concentration Indices (FICIs) showed a high level of synergy between ftsZ silencing and berberine treatment (FICI values of 0.23 and 0.25 for peptide nucleic acid- and expressed antisense RNA-based silencing of ftsZ, respectively). Finally, over-expression of ftsZ led to a mild rescue effect in berberine-treated cells. CONCLUSIONS: The results argue against DNA binding as the primary mechanism of action of berberine and support the hypothesis that its antibacterial properties are due to inhibition of the cell division protein FtsZ. In addition, the genetic approach used here provides a means to rapidly test the activity of other putative FtsZ inhibitors.

Sorafenib blocks tumour growth, angiogenesis and metastatic potential in preclinical models of osteosarcoma through a mechanism potentially involving the inhibition of ERK1/2, MCL-1 and ezrin pathways.

BACKGROUND: Osteosarcoma (OS) is the most common primary bone tumour in children and young adults. Despite improved prognosis, metastatic or relapsed OS remains largely incurable and no significant improvement has been observed in the last 20 years. Therefore, the search for alternative agents in OS is mandatory. RESULTS: We investigated phospho-ERK 1/2, MCL-1, and phospho-Ezrin/Radixin/Moesin (P-ERM) as potential therapeutic targets in OS. Activation of these pathways was shown by immunohistochemistry in about 70% of cases and in all OS cell lines analyzed. Mutational analysis revealed no activating mutations in KRAS whereas BRAF gene was found to be mutated in 4/30 OS samples from patients. Based on these results we tested the multi-kinase inhibitor sorafenib (BAY 43-9006) in preclinical models of OS. Sorafenib inhibited OS cell line proliferation, induced apoptosis and downregulated P-ERK1/2, MCL-1, and P-ERM in a dose-dependent manner. The dephosphorylation of ERM was not due to ERK inhibition. The downregulation of MCL-1 led to an increase in apoptosis in OS cell lines. In chick embryo chorioallantoic membranes, OS supernatants induced angiogenesis, which was blocked by sorafenib and it was also shown that sorafenib reduced VEGF and MMP2 production. In addition, sorafenib treatment dramatically reduced tumour volume of OS xenografts and lung metastasis in SCID mice. CONCLUSION: In conclusion, ERK1/2, MCL-1 and ERM pathways are shown to be active in OS. Sorafenib is able to inhibit their signal transduction, both in vitro and in vivo, displaying anti-tumoural activity, anti-angiogenic effects, and reducing metastatic colony formation in lungs. These data support the testing of sorafenib as a potential therapeutic option in metastatic or relapsed OS patients unresponsive to standard treatments.

The pharmacological properties of a novel MCH1 receptor antagonist isolated from combinatorial libraries.

Melanin-concentrating hormone (MCH) is a neuropeptide that exhibits potent orexigenic activity. In rodents, it exerts its actions by interacting with one receptor, MCH(1) receptor which is expressed in many parts of the central nervous system (CNS). To study the physiological implications of the MCH system, we need to be able to block it locally and acutely. This necessitates the use of MCH(1) receptor antagonists. While MCH(1) receptor antagonists have been previously reported, they are mainly not accessible to academic research. We apply here a strategy that leads to the isolation of a high affinity and selective MCH(1) receptor antagonist amenable to in vivo analyses without further chemical modifications. This antagonist, TPI 1361-17, was identified through the screening of multiple non-peptide positional scanning synthetic combinatorial libraries (PS-SCL) totaling more than eight hundred thousand compounds in conditions that allow for the identification of only high-affinity compounds. TPI 1361-17 exhibited an IC(50) value of 6.1 nM for inhibition of 1 nM MCH-induced Ca(2+) mobilization and completely displaced the binding of [(125)I] MCH to rat MCH(1) receptor. TPI 1361-17 was found specific, having no affinity for a variety of other G-protein coupled receptors and channels. TPI 1361-17 was found active in vivo since it blocked MCH-induced food intake by 75%. Our results indicate that TPI 1361-17 is a novel and selective MCH(1) receptor antagonist and is an effective tool to study the physiological functions of the MCH system. These results also illustrate the successful application of combinatorial library screening to identify specific surrogate antagonists in an academic setting.

Targeting cell division: small-molecule inhibitors of FtsZ GTPase perturb cytokinetic ring assembly and induce bacterial lethality.

FtsZ, the ancestral homolog of eukaryotic tubulins, is a GTPase that assembles into a cytokinetic ring structure essential for cell division in prokaryotic cells. Similar to tubulin, purified FtsZ polymerizes into dynamic protofilaments in the presence of GTP; polymer assembly is accompanied by GTP hydrolysis. We used a high-throughput protein-based chemical screen to identify small molecules that target assembly-dependent GTPase activity of FtsZ. Here, we report the identification of five structurally diverse compounds, named Zantrins, which inhibit FtsZ GTPase either by destabilizing the FtsZ protofilaments or by inducing filament hyperstability through increased lateral association. These two classes of FtsZ inhibitors are reminiscent of the antitubulin drugs colchicine and Taxol, respectively. We also show that Zantrins perturb FtsZ ring assembly in Escherichia coli cells and cause lethality to a variety of bacteria in broth cultures, indicating that FtsZ antagonists may serve as chemical leads for the development of new broad-spectrum antibacterial agents. Our results illustrate the utility of small-molecule chemical probes to study FtsZ polymerization dynamics and the feasibility of FtsZ as a novel therapeutic target.

Inhibition of beta-catenin/Tcf activity by white tea, green tea, and epigallocatechin-3-gallate (EGCG): minor contribution of H(2)O(2) at physiologically relevant EGCG concentrations.

Epigallocatechin-3-gallate (EGCG) is the major polyphenol present in white tea and green tea. Recently, it was reported that the addition of EGCG and other tea polyphenols to cell culture media, minus cells, generated significant levels of H(2)O(2), with the corollary that this might represent an "artifact" in cell culture studies which seek to examine the chemopreventive mechanisms of tea. We show here that in cell growth media with and without serum, and in growth media containing human embryonic kidney 293 (HEK293) cells plus serum, physiologically relevant concentrations of EGCG (< or =25 microM) generated H(2)O(2) with a peak concentration of the order of 10-12 microM. However, addition of 20 microM H(2)O(2) directly to HEK293 cells transiently transfected with wild-type or mutant beta-catenin constructs and TCF-4 had no significant effect on beta-catenin/TCF-4 reporter activity or beta-catenin expression levels. In contrast, 2-25 microM EGCG inhibited beta-catenin/TCF-4 reporter activity in a concentration-dependent fashion and there was a concomitant reduction in beta-catenin protein levels in the cell lysates without changes in TCF-4 expression. The inhibition of reporter activity was recapitulated by white tea and green tea, each tested at a 25 microM EGCG equivalent concentration in the assay, and this was unaffected by the addition of exogenous catalase. The results indicate that physiologically relevant concentrations of tea and EGCG inhibit beta-catenin/TCF-4 reporter activity in HEK293 cells due to reduced expression of beta-catenin and that this is unlikely to be an artifact of H(2)O(2) generation under the assay conditions used here. These data are consistent with the findings from in vivo studies, showing the suppression of intestinal polyps by tea, via an apparent down-regulation of beta-catenin and Wnt target genes.