Final Report Summary - DIVINOCELL (Exploiting Gram-negative cell division targets in the test tube to obtain anti-microbial compounds)
Executive Summary:
To obtain new antibiotics, DIVINOCELL exploited existing understanding and obtained new knowledge on the molecular biology of the cell division machinery (the divisome) of Gram-negative bacteria. Using the divisome, essential for bacterial survival, as a source of inhibitable targets we identified eight new compounds to block the proliferation of Gram-negative pathogens. We also generated new technology to facilitate the validation and the improvement of the properties of antibiotic hits and to help in discovering new antimicrobials. Gram-negative bacteria are encased in a complex envelope formed by two flexible membranes sandwiching a rigid peptidoglycan support layer that together maintain the integrity and the shape of the cell. Due to this complexity they are less susceptible to antibiotics than bacteria that do not contain a second membrane covering the peptidoglycan layer. For most of our work we used Escherichia coli as a model because it is by far the best understood Gram-negative bacterium and we have poweful techniques to study and handle it. Moreover, some strains of E. coli are pathogenic and cause deadly disease outbreaks.
The novel DIVINOCELL antimicrobials are designed to block the function of divisome proteins, such as FtsZ, FtsA or ZipA. They should be effective to counteract infections and be largely innocuous to humans and animals because their cells do not contain these proteins. Using models for the active centre of FtsZ, in silico procedures designed by BIOMOL have identified BIZ2013-1, BIZ2013-2 and BIZ2013-3: three molecules that inhibit the activity of the protein and are active against E. coli. From a VICHEM collection of kinase inhibitors, new inhibitors of in vitro FtsZ polymerization, VCC599460:13 and VCC342365:01, have been identified that also induce the misplacement of divisome components. One additional compound, VCC404021:01, also modifies the interaction of FtsZ with ZipA. Evolva has found compounds to disrupt the interaction between FtsZ and FtsA. One of them, GCI-512, is active against Burkholderia thailandensis, a multidrug-resistant bacterium. From a Streptomyces strain extract Demuris obtained DEM30543, one new broad spectrum antimicrobial. These compounds, in different phases of IP protection, are now part of hit to lead improvement programmes.
Several procedures to discover antibiotics have been designed by DIVINOCELL. UNEW formulated an assay that was used to screen Demuris libraries to find inhibitors of cell wall synthesis. A positive whole-cell assay based on properties of E. coli peptidoglycan hydrolase mutants, developed by UNEW, helped to screen the library of compounds from VICHEM. Strains depleted for FtsZ or MreB devised by CSIC and CNRS can detect antimicrobials at low concentrations. Using this sensitive test CSIC identified compounds affecting bacterial growth among those synthesised by UCHILE.
CSIC has developed new procedures to incorporate divisome proteins, such as ZipA, into nanodiscs. These small membrane plugs surrounded by a protein belt can be used as soluble reagents. In vivo photo cross-linking assays have been optimized by VU to test new targets in the interaction between three additional divisome proteins: FtsQ, FtsB and FtsL. Detection of protein - protein interactions using a technique known as FRET has been developed at UvA. UNEW constructed a Bacterial-Two-Hybrid assay to screen for chemical compounds that abrogate the interaction of FtsZ with MreB, a critical component to maintain the architecture of E. coli. Synthesis of the rigid peptidoglycan layer of the bacterial envelop is effected by peptidoglycan synthases, including MtgA, PBP3 and PBP1B, and DIVINOCELL has designed assays to measure their activity. Crucial for this development was the production by CSIC and UNEW of tools in the form of small-sized precursors (Lipid-II and UDP-muramyl pentapeptide), a reporter labelled precursor molecule and peptidoglycan fragments. MRC-LMB studied FtsW, a protein needed to translocate these precursors to the outer face of the membrane.
Besides the primary societal impact that the newly discovered antimicrobials will have on public health and individual well-being, DIVINOCELL participants have published over 63 papers in top ranking scientific journals, have defended 4 doctoral thesis and have participated in over 80 dissemination activities directed to the general public. DIVINOCELL has also helped to strengthen scientific collaboration between the participants and to generate future new projects to implement it.
Project Context and Objectives:
The work of DIVINOCELL aimed at identifying antibiotics active in Gram-negative bacteria. These are bacteria with a complex envelope formed by two flexible membranes sandwiching a rigid layer that together maintain the integrity and the shape of the cell. Due to the complexity of their envelop these bacteria are less susceptible to antibiotics than those that do not contain a second membrane covering the rigid support layer. The discovery of new antibiotics to treat infections is important for individuals and for society as most of those currently used have lost their efficacy due to the resistances acquired by many of the pathogens, Gram-negatives in particular. As bacteria cannot proliferate when any of the proteins required for their growth or for their multiplication is inactivated, we sought antibiotics to inhibit targets in the process of cell division or envelop synthesis.
In the process of bacterial division several proteins assemble at midcell to form a division ring, also called divisome. The action of the divisome is to separate the cell into two equal daughters by means of a septum. Once separated each daugther resumes growth and both divide independently from each other. For most of this work we used Escherichia coli as a model because it is a Gram-negative in which extensive knowledge on its biology together with a plethora of techniques are available for its study and handling in the laboratory. Moreover, some strains of E. coli are pathogenic and cause deathly disease outbreaks (Karch et al., 2012. EMBO Mol. Med. 4: 1-8). Division, as described in E. coli, is phylogenetically conserved, it has peculiarities present in Gram-negatives, and has been shown to be druggable. Among the E. coli proteins that form the division ring we have focused on those called FtsA, FtsB, FtsL FtsQ, FtsW, FtsZ and ZipA. In addition we have studied proteins that synthesize peptidoglycan, the rigid layer of the envelope. Among them FtsI, also a divisome protein, is specifically needed for the synthesis of this layer at the septum and belongs to a set of dedicated enzymes, collectively designated as penicillin-binding proteins (PBPs) because they can bind to penicillin. For this reason FtsI is also known as PBP3. It is well known that when any of these components of the E. coli divisome is missing or inactivated, cell division stops and the bacterium eventually dies.
The primary OBJECTIVE of DIVINOCELL was thus to extensively use the essential bacterial division machinery (divisome) and its product (septum) for antimicrobial discovery in Gram-negatives. The existing and new knowledge obtained from the Escherichia coli division machinery (the divisome) was planned to be used to: identify novel inhibitable targets in the activity and interactions of the divisome proteins and the septum of Gram-negative bacteria; formulate primary assays and in vivo assays by combining the properties of these targets and the application of front-line technology; use these assays to screen unbiased and biased compound collections to obtain and validate hits; and finally to select the most promising hits as lead compounds to perform lead optimisation as a prerequisite for drug candidate identification. In addition to achieving the objectives of this call the DIVINOCELL project planned to advance the most promising leads into initial programs which are proprietary to the industrial participants in order to improve their medicinal properties.
The DIVINOCELL research was based on several innovative approaches:
EXPLOITATION OF AN ESSENTIAL FUNCTION: To curb the proliferation and the viability of Gram-negative pathogens the properties of a basic essential function of bacteria, cell division, was exploited.
FRONT-LINE TECHNOLOGY: To advance on the available state of the art to formulate in vitro and cell assays, we have applied novel tools (analytical, bioinformatic, structural and imaging) and incorporated all the available knowledge of the in vivo properties of the division process. We have finally obtained and applied new knowledge on the biophysical and biochemical details of the process to obtain assays that work in the test tube under conditions that replicate those found in the cell cytoplasm and periplasm.
NOVEL TARGETS & ASSAYS: We have concentrated our efforts in the study of the specific components of the Gram-negative divisome and septum. Although many of them are phylogenetically conserved they include elements and interactions specific for the survival of Gram-negatives. Crucial for this objective has been the exploitation of the activities of three proteins, FtsZ, FtsA and ZipA and their assembly into a proto-ring. ZipA, required together with FtsA to anchor FtsZ to the division site, is specifically present in the relevant groups of Gram-negative pathogens, Enterobacteriacea and Pseudomonadacea among them, but not in other taxa. We identified and used new targets in a promising and unexploited element of the division machinery (FtsQ), in the divisome elements that participate in the synthesis of the septum (FtsI together with FtsW), as well as in the switch from elongation to septation (MreB).
CELL SECONDARY ASSAYS: We have used several recently-discovered genetic properties of cell division process and of the physiological consequences of its arrest to generate in vivo and positive cell assays for hit validation to select those having the optimum properties to yield leads.
CHOICE OF SPECIFIC COMPOUNDS: To maximise the possibilities of success some DIVINOCELL screens included a pre-selection of the compound collections and included synthetic and natural products. This was achieved by virtual in silico filtering, by the use of collections already biased to include nucleotide-binding site inhibitors and by genetic chemistry to evolve more efficient inhibitors from natural products.
CHOICE OF SCAFFOLDS: We obtained scaffolds that are not so widely present or are even absent from Nature by making extensive use of protein interactions in addition to protein activities. We also used potent in silico capabilities to help in the design of scaffolds. For these reasons, pathogens are less likely to possess a response mechanism to counter the action of the compounds derived from the project.
Virtual filtering to pre-select inhibitors of proteins with known structure.
Identification of inhibitors done by Biomol-Informatics started by virtual prefiltering to reduce the number of potential PI and PPI inhibitors to allow a more realistic wet screening approach. We estimate that the amount of compounds initially present in a collection could be reduced to just 10%. The procedure can easily pre-filter libraries containing 1 million “druggable” compounds by first obtaining two-dimensional information from commercial catalogs of ligand libraries or specialized databases (ZINC database; Irwin and Shoichet. 2005. J. Chem. Inf. Model. 45: 177–182). The molecules were then converted to 3D structures using CORINA (Sadowski et al., 1994. J. Chem. Inf. Comput. Sci., 34: 1000-1008) calculating the different conformers and assigning partial charges and consistent atomic radii with the AMBER force field (a family of force fields for molecular dynamics of biomolecules). The conformers were fed to the docking program Autodock 4 (Huey et al., 2007. J. Comput. Chem. 28: 1145-1152) to perform a fast size-dependent pre-screening to eliminate molecules with low probability to be active in the chosen surface sites, selecting a set of more favourable molecules in terms of geometry and energy of the complexes. The finally selected candidates were then studied through detailed Molecular Dynamics simulation and energetically analyzed to obtain a final selection of molecules that were synthetised and assayed experimentally.
Evolutionary genetic chemistry to evolve inhibitors of protein-protein interactions.
Developing small molecules to inhibit protein-protein interactions (PPIs) poses challenges regarding the size and nature of the interaction surface, which are often not resolved by conventional compound libraries. Natural compounds often contain unique structural features with utility in protein inhibition beyond combinatorial or synthetic medicinal chemistry. Natural structures have a strong track record as pharmaceuticals with 61% of the 877 new chemical entities that reached the market over the last 20 years having their origins in Nature (Newman and Cragg, 2007, J. Nat. Prod. 70: 361-477). EVOLVA selected PPI inhibitors based on the exploitation of the extraordinary biodiversity of active molecules provided by Nature. For this Evolva used their Watchmaker® technology platform to isolate and evolve small molecule inhibitors of the polymerization and interactions of the proto-ring components FtsA, FtsZ and ZipA. Watchmaker® is an evolutionary genetic chemistry technology that evolves small molecules as an alternative to, and synergistic with traditional synthetic-medicinal approaches. It is based on the generation of genetic chemistry libraries prepared by specifically mixing genes from relevant sources with a toolbox of specific biosynthetic pathway genes. The genes are collected on enhanced yeast artificial chromosomes (EYACS) and introduced into yeast strains harbouring a relevant built-in survival screening assay based on the reverse yeast two-hybrid system (Vidal et al., 1996. Proc. Natl. Acad. Sci. USA 93: 10315-10320) to apply selective pressure on yeast to evolve efficient inhibitors of the PPIs. This approach predisposes yeast to produce structurally-diverse novel small molecules and by continuously selecting for high-performing yeasts, highly functional compounds optimized for the selected target are isolated. Some yeast strains in which the interactions between the proto-ring elements remain functional had already been obtained (Yim et al., 2000. J. Bacteriol. 182: 6366-6373). A similar genetic system had already been used successfully to select for small compounds that interfere with protein-protein interactions of the human calcium channel subunits (Young et al., 1998. Nature Biotechnol. 16: 946-950).
Biased compound collections directed specifically to nucleotide-binding sites.
This was the approach followed by VICHEM. The Nested Chemical Library™(NCL) technology (Kéri et al., 2005 Assay Drug Dev Technol. 3: 543-551) was used to find inhibitors. NCL™ is a chemically diverse collection organised around 108 core structures with small focused sub-libraries generated around each core. It contains more than 600 published kinase inhibitory compounds including preclinically or clinically-relevant leads and large series of proprietary compounds. Both FtsZ and FtsA, two of the proto-ring components, contain nucleotide binding sites.Most of the NCL compounds, a kinase inhibitory library, act on the ATP-binding site, but the library contains as well a number of allosteric-binding site inhibitors. The structures of the few published FtsZ inhibitors and related molecules was used to complement NCL™. Initial NCL hits were expected to act on the GTP or ATP binding sites of FtsZ or FtsA or to interfere with the PPIs as allosteric inhibitors.
Screening of a collection of actinomycete bacteria.
The screening assays made available to Demuris have been cell based screens for general divisome targets. To increase the probability of getting “hits” they have turned to the screening of natural product libraries instead of screening small previously screened compound libraries from typical chemical vendors. Microbial natural products and compounds derived from natural products account for roughly half of the drugs used pharmaceutically (Baltz, R.H. 2008 Curr Opin Pharmacol, 8: 557-563). Although these molecules fell from favour as sources of screening in the late 90’s, there has been a resurgence of interest recently. This has been driven by two factors: first, disappointment at the yield of effective leads from combinatorial chemistry approaches; second, the emergence of methods, especially genome sequencing, that facilitate the development of natural products (Chun et al., 2007 International J. Systematic Evolutionary Microbiol. 57:2259-2261; Li, and Vederas 2009 Science, 325: 161-165.
Demuris acquired exclusive rights to unique collections of actinomycete bacteria from Newcastle University. The first collection, of about 6-8,000 strains, was isolated over a period of about 40 years by Prof Mike Goodfellow. The drive behind isolation of this collection was taxonomic diversity, and the strains were obtained from widely diverse habitats with a range of different and innovative isolation methods. They are also supported by a unique level of annotation and documentation of their diversity, as befitting their taxonomic purpose. The second collection, of about 2,000 mainly marine actinomycetes, is important because it has only been recognised that actinomycetes inhabit marine environments relatively recently. Therefore, this large group of organisms have been largely overlooked in classical screens for antibiotics done by major pharma. There is an argument that organisms living in marine environments need to generate very potent molecules because of the very dilute environment they inhabit, and that they are prolific producers of antimicrobials. Unlike other collections this one has been extensively “dereplicated” so that its diversity is equivalent to that of much larger strain collections. The majority of the stains screened are putatively Streptomyces or Micromonospora species these species are well known to produce compounds and give the best chance of identifying novel hits to Gram negative bacteria.
Project Results:
DIVINOCELL has exploited the existing knowledge on the molecular biology of the cell division machinery in Gram-negatives and has also generated new knowledge on this process to discover new targets. The application of this background to the screening of selected compound collections has identified several hits active in preventing bacterial proliferation. Exploitation of these new hits proceeds now to improve and convert them into lead compounds to formulate new antibiotics. Inhibitors directed against bacterial division targets will be effective to cure infections and will be largely innocuous to humans and animals because their cells either do not contain these proteins altogether or at the most they have greatly divergent variants of some of them. Moreover, as some of the identified inhibitors are based on the structures of synthetic scaffolds, not frequently present in Nature, they are less likely than the present antibiotics to favour the acquisition of resistances by the pathogens. The strategy of DIVINOCELL has been finally complemented by the development of new tools for screening, validation and improvement of inhibitors in the test tube and in whole cells. These tools are largely derived from our discoveries on the structure and interactions of the known and novel targets and in the physiology of bacteria defective in division.
SCREENING
Evolva has found compounds that may disrupt the interaction between FtsZ and FtsA. They have been isolated using the proprietary Watchmaker strategy in which compounds that block interactions between proteins are generated within engineered yeast strains and are revealed as able to rescue the toxicity of a suicide system equally engineered into them. Six novel compounds, i.e. unknown when compared to all known chemical structures using the SciFinder algorithm, show inhibitory effects on bacterial survival at concentrations that make them suitable hits to merit further development. One of them, GCI-512, is active in a Burkholderia thailandensis growth assay with an MIC of 28 g/ml.
The screening performed by Demuris has identified one broad spectrum compound, DEM30543, obtained from a Streptomyces strain extract, with desirable antimicrobial activity properties. The extract is active against Methicillin-resistant Staphylococcus aureus (MRSA). MIC values for E. coli are 5 g/ml. DEM30543 has demonstrated little toxicity towards human cells. Production of this natural compound, has been scaled to obtain sufficient amounts for additional testing by the UNEW.
Screening of 197 compounds, belonging to a collection of kinase inhibitors supplied by Vichem, has been performed at CSIC, UvA and UNEW using both in vivo and in vitro assays. Among those able to inhibit growth in vivo at concentrations that make them good choices to use in hit improvement, two new inhibitors of in vitro FtsZ polymerization, VCC599460:13 and VCC342365:01, have been identified that also induce the misplacement of divisome components. In addition, compound VCC404021:01 modifies the interaction of FtsZ with another component of the E. coli proto-ring.
Chemical synthesis by the UCHILE has provided eight analogs of DAPI, a molecule able to bind to DNA and also to FtsZ. Soluble and potent DAPI derivatives also inhibit bacterial proliferation. Inhibition of growth by these compounds occurs in strains in which the concentration of FtsZ is limiting.
In silico procedures designed by BIOMOL have provided high-quality models for the active centre of FtsZ in the FtsZ-FtsZ interface, both in the FtsZ dimer and in short polymers (Mendieta et al. 2009. J. Mol. Biol 390: 17-25; Martin-Garcia et al., 2012. FEBS Letters 586: 1236-1239). Using this interface model and a proprietary system of “in silico” combinatorial chemistry and docking (BIOMOL-HTCM; BIOMOL-High Throughput Combinatorial Modelling), three molecules, BIZ2013-1, BIZ2013-2 and BIZ2013-3, that bind specifically to the active centre of the FtsZ polymer have been designed. They inhibit the activity of the protein, are active against E. coli and are currently being improved in a hit-to-lead phase.
DISCOVERY OF NEW TARGETS IN BACTERIAL DIVISION
Polymerization of FtsZ on lipid surfaces is sensitive to the composition of the membrane, as proven by CSIC. The structure and the dynamics of individual FtsZ filaments in the presence of GTP, visualized by Atomic Force Microscopy on surfaces, showed curved filaments that depolymerize from the ends. Further quantitative analysis and theoretical modeling indicated that all monomer-monomer interfaces are equivalent and that the monomer at the ends diffuse away from the filament more easily (Mateos-Gil et al., 2012. Proc. Nat. Acad. Sci. 109: 8133-8138). Novel inhibitable targets have been discovered by CSIC in the interactions between divisome proteins, for example FtsZ with ZipA, FtsQ with either FtsB or FtsL, and FtsA with itself. A new function of ZipA in the stabilization of FtsZ (Pazos et al., 2013. J. Biol. Chem. 288: 3219-3226) and new interactions between ZipA and two proteins, ZapA and ZapB, that assist FtsZ in the divisome were discovered (Pazos et al., 2013. Environmental Microbiol. DOI: 10.1111/1462-2920.12227 ). The interaction surface between FtsZ and ZapA has been determined by UvA. To study the possible targets in the FtsA protein it has been obtained by CSIC from Streptococcus pneumoniae, as this protein is easier to handle when obtained from this Gram-positive pathogen. Its C-terminus is as a molecular regulator that affects FtsA polymerization in the cytoplasm and facilitates this process when the protein is attached to lipid membranes. Other regions as the 1C and the S12-13 domains also play significant roles in polymerization and together with the C-terminus define three novel inhibitable targets (Krupka et al., 2012. J. Biol. Chem. 287: 7756–7765). That the polymers formed by some FtsA proteins resemble actin filaments was then shown by MRC-LMB (Szwedziak et al., 2012. EMBO J. 31, 2249-2260).
An interaction hotspot for FtsB and an interaction region for both FtsB and FtsL were identified by VU and UvA on the surface of FtsQ. The hotspot is highly conserved among the class of gammaproteobacteria (Glas et al., 2013. J. Biol. Chem. 288: 24340-24350).
FtsW is a protein that transfers peptidoglycan precursors to the periplasm, the space in between the two membranes where this rigid polymer is produced. It has been purified and crystallized by MRC-LMB. Further improvement of the crystallization setups are being tested to obtain a high resolution dataset to solve the structure of FtsW thus supplying novel inhibitable targets in this protein.
As shown by UNEW, FtsZ and MreB of E. coli interact and the interaction is required for cell division and therefore is essential. The interaction functions to deliver to the FtsZ ring cell wall synthesising enzymes that are essential to synthesise the septum and/or pre-septal peptidoglycan.
Results from CNRS show that mreB expression is regulated by non-coding RNAs, this regulatory mechanism can also be used as a source for new inhibitable targets.
ASSAY DESIGN AND NEW TECHNOLOGY
Protocols to assist in the process of lead development that are based on strains depleted from FtsZ or MreB have been devised by CSIC and CNRS. They have been applied to assay the DAPI analogs from UCHILE. The CNRS found that an hfq minus strain, which allows to simultaneously deregulate the expression of different cell division proteins, shows a significant increase for Aztreonam and Piperacillin sensitivity. UNEW constructed a Bacterial-Two-Hybrid (BTH) assay that can be used to screen for chemical compounds that abrogate the FtsZ - MreB interaction and an assay that was used to screen Demuris compound libraries for inhibitors of cell wall synthesis in E. coli.
A positive whole-cell assay making use of the unique properties of E. coli peptidoglycan hydrolase mutants to identify cell division inhibitors, developed by UNEW, was used to screen the library of compounds from VICHEM.
Various types of in vitro peptidoglycan synthesis assays to study the activities of peptidoglycan synthases have been generated. The design includes the use of MtgA, PBP3 and PBP1B, all of them proteins that work to synthesise peptidoglycan. The wild type enzyme and variants with a single activity are available. Small-sized precursors of peptidoglycan synthesis, as Lipid-II and UDP-muramyl pentapeptide, are used as substrates to quantify the enzymatic activities. They have been obtained by CSIC and UNEW. Using immobilized vancomycin as a matrix, affinity chromatography was used to purify UDP-muramyl pentapeptide whereas apolar affinity chromatography was chosen to purify Lipid-II. In addition, suitable (spread on glass, mica and plastic, and unilamellar) PG-fragments, and an adequate reporter molecule labeled precursor were produced.
CSIC has developed new procedures to apply the divisome proteins in easy to use screening assays, among them vesicles that can mimic the cell interior and nanodiscs. The interaction between FtsZ and FtsA and between FtsZ and ZipA was tested in vitro using natural or artificial vesicles respectively. We have discovered that within natural membrane vesicles the FtsA protein does not anchor to the membrane at sufficient strength to withstand the pull exerted by polymerizing FtsZ (Jiménez et al., 2011 J. Biol. Chem. 286: 11236-11241). ZipA may provide a stronger anchoring for FtsZ polymers as it contains a membrane domain. Permeable vesicles in which it has been artificially incorporated collapse upon FtsZ polymerization (Cabré et al., 2013. J. Biol. Chem. 288: 26625-26634).
Nanodiscs are small membrane plugs surrounded by a protein belt. Proteins that are integrated in the membrane, as ZipA, can be used when inserted in nanodiscs as soluble reagents. This assay, developed by CSIC, was used to measure the strength of the interaction between the ZipA bound to nanodiscs and the soluble FtsZ (Hernández-Rocamora et al., 2012. J. Biol. Chem. 287: 30097-30104).
An assay initially designed by CSIC to measure the binding of FtsA to lipid-coated glass microbeads (Martos et al., 2012, PLoS ONE 7, e39829) has been adapted to test the interaction between FtsZ and ZipA. A simple assay to measure FtsZ assembly in solution using fluorescence (Reija et al., 2011. Anal. Biochem. 418: 89–96) has also been formulated.
In vivo photo cross-linking has been optimized by VU as an assay to test a potential target for novel antimicrobials based on the interaction between divisome proteins FtsQ and FtsB and FtsL (Glas et al., 2013. J. Biol. Chem. 288: 24340-24350).
Detection of protein - protein interactions using a technique known as FRET has been developed at UvA. The assay detects the interaction between two fluorescently labelled proteins and can measure simultaneously whether an antibiotic is able to pass the bacterial envelope and identify the protein complex that it targets. Using the assay it has been proven that the loss of activity of the proteins in the complex involved in cell elongation results in a dissociation of the complex.
DESCRIPTION OF RESULTS FOR EACH PARTICIPANT
CSIC-CNB probed protein-protein interactions (PPIs) between ZipA and FtsZ by testing the effect of ZipA overproduction on FtsZ degradation. Our results show that in the nucleoid-free maxicells (Sancar et al. (1979) J Bacteriol 137: 692-693) FtsZ is present at low levels due to the specific degradation of FtsZ by the housekeeping protease system ClpXP and to the absence of de novo synthesis (Pazos et al. (2013) J Biol Chem 288: 3219-3226). The overproduction of ZipA in maxicells reduces the degradation of FtsZ in maxicells (Pazos et al (2013) J Biol Chem 288: 3219-3226). To check which domains of ZipA are involved in FtsZ protection, different ZipA deletion mutants were constructed and probed for FtsZ protection (Figure 1). Overproduction of ZipA mutants in maxicells shows that the ZipA mutant ZipA4 (lacking part of the FZB domain from Pro-273 to Ala-328) and the FZB domain were able to protect FtsZ from degradation as observed for the full length ZipA (Pazos et al (2013) J Biol Chem 288: 3219-3226). The protection of FtsZ by ZipA is not affected by the simultaneous presence of either FtsA or FtsA* (Figure 3) (Pazos et al (2013) J Biol Chem 288: 3219-3226).
CSIC-CNB used Bimolecular Fluorescence Complementation (BiFC) to detect and localize protein-protein interactions (PPI) during proto-ring formation within the cell. The assay is based on the irreversible reconstitution of the fluorescence signal upon association of two non-fluorescent fragments of the green fluorescent protein or its derivatives once they are brought into close proximity (Kerppola (2008) Annu Rev Biophys 37: 465-487). Using BiFC, we confirmed already described interactions (FtsZ-ZipA, FtsZ-ZapB, ZapB-ZapB or ZapB-ZapA) and obtained two novel and unexpected interactions (ZapB-ZipA and ZipA-ZapA) (Pazos et al. (2013) Environ Microbiol DOI:10.1111/1462-2920.12225).
Given the difficulties in the biochemical handling of the FtsA protein form E. coli, we have studied the polymerization of FtsA from Streptococcus penumoniae, a relevant pathogen although Gram positive. Two different truncations, involving either the 1C domain or the simultaneous absence of the S12-13 β-strands of the FtsA protein located at opposite terminal sides in the molecular structure, are essential for ATP-dependent polymerization. These two truncated proteins are not able to polymerize themselves but can be incorporated to some extent into the FtsA+ polymers during the assembling process. Consequently, they block the growth of the FtsA+ polymers and slow down the polymerization rate. The combined action of the two truncated proteins produces an additive effect on the inhibition of FtsA+ polymerization, indicating that each truncation affects a different interaction site within the FtsA molecule.
CSIC-CNB performed a set of in vivo assays to test the effect of compounds supplied by VICHEM on the proliferation, viability and division defects of E. coli strain JW0451 (as BW25113, ∆acrB). JW0451 is an acrB null mutant that does not export molecules outside the cell, as the AcrB protein functions as part of the AcrAB-TolC multidrug efflux system (Baba et al. (2006) Mol Syst Biol 2: 2006 0008; Kawabe, et al. (2000) J. Biochem. 128 195-200). The absence of multi-drug efflux system allows that the compounds supplied by VICHEM could be used at lower dosages. Using JW0451 we have performed compound susceptibility tests to determine the growth on agar plates and the Minimal Inhibitory Concentration (MIC) in a resazurin assay. We finally tested the effects of selected compounds on cell division by examining their effect on the morphology of the cells (filamentation or lysis) and on the localization of FtsZ and ZipA.
CSIC-CNB has exploited observations on the differential gene expression levels found in cells undergoing cell division arrest by deprivation of division ring proteins, namely FtsZ. These results have been used to construct plasmids reporting the absence of division as a fluorescent signal. For this purpose, specific promoters from the regulatory regions of the differentially expressed genes have been fused to the structural genes coding for naturally fluorescent proteins, as GFP. Strains deprived of proto-ring elements have been used to obtain assays to detect the effect of low antimicrobial concentrations.
At CSIC-CIB the mechanistic biochemistry of FtsZ, the prokaryotic homolog of tubulin, was studied to complete the understanding of its functional activities and interactions with ZipA and FtsA, the two elements of the division machinery that anchor FtsZ to the membrane forming the first molecular complex of the divisome, the proto-ring. We have exploited new reconstitution technologies to test the properties of elements of the bacterial cell division machinery and their interactions, in well-defined lipid environments (nanodiscs, microbeads, vesicles) under conditions that reproduce those found in the cell cytoplasm, to gain insights into their precise functions. This knowledge has been used to generate fluorescence-based assays to screen for inhibitors of specific proto-ring protein-protein interactions.
The assembly and disassembly cycle of FtsZ, linked to GTP hydrolysis, is thought to be one of the essential functional activities of FtsZ in bacterial division. We have completed the characterization of the Mg2+-linked FtsZ self-assembly in the presence of GTP or a GTP analogue using a set of biophysical tools (sedimentation velocity, concentration-gradient static light scattering, fluorescence correlation spectroscopy and dynamic light scattering). The combined analysis of these measurements has revealed that FtsZ polymerization involves the concerted formation of a narrow distribution of oligomeric species (around a hundred FtsZ subunits for GTP fibres) (Monterroso et al., 2012 Biochemistry 51:4541-4550). The size of the narrowly distributed GTP-FtsZ polymers decreases upon lowering potassium and is independent of GTPase activity. Potassium oppositely controls the size distribution of GTP-FtsZ polymers and the abundance of GDP oligomers (Ahijado-Guzmán et al. 2013 J. Biol. Chem. doi:10.1074/jbc.M113.482943). In parallel, a homogeneous fluorescence anisotropy assay to measure FtsZ assembly in dilute and crowded solutions has been developed in cooperation with CSIC-CNB. This assay allows measuring the effect of osmolytes, divalent cations and high concentrations of unrelated macromolecules (acting as crowding agents) on the critical concentration of FtsZ assembly, the degree of polymer formation and the dynamics of disassembly of these polymers. The assay is sensitive, low sample consuming, rapid, and easily adapted to systematic screening assays (Reija et al., 2011 Anal. Biochem. 418: 89-96).
We have also optimized a procedure to obtain milligram amounts of the recalcitrant FtsA division protein from E. coli that allows measuring the lipid-binding properties of FtsA relevant for its functional activity. The interaction between soluble FtsA and lipids has been found to be of moderate affinity as measured by biochemical assays using micron-size beads coated with either natural or artificial membranes (Martos et al., 2012 PloS One. 7(6):e39829).
We have designed assays to measure the binding of FtsZ to other proto-ring elements and to screen for inhibitors of proto-ring protein-protein interactions, which may be exploited in the screening assays for new antimicrobial agents targeting the proto-ring. Together with CSIC-CNB, the dynamic interaction between FtsZ and ZipA has been evidenced in nanodiscs - structures formed by a membrane scaffold protein encircling a lipid mixture. The strength of the interaction has been measured and found to be of similar moderate affinity for the FtsZ polymers formed with GTP and for the GDP forms (Hernández-Rocamora et al., 2012 J. Biol. Chem. 287: 30097-30104; Hernández-Rocamora et al., 2012 J. Struct. Biol. 180: 531-538). A fluorescence-based assay to screen for inhibitors of the interaction of FtsZ and ZipA incorporated in lipid bilayer nanodiscs has been implemented. Peptides containing partial sequences of the C-terminus of FtsZ compete with FtsZ polymers for binding to ZipA. The assay can proceed in solution and is therefore suitable for systematic assays. In addition, microbeads encoded with gold sensor nanoparticles prior to membrane coating have been used to design assays to monitor the interaction of FtsZ with other proto-ring elements (Ahijado-Guzmán et al., 2012 ACS Nano 6: 7514-7520). In addition, assays to measure the binding of soluble proto-ring elements to other elements of the divisome reconstituted in microbeads (coated with proteo-lipids or inner membranes) by differential centrifugation and fluorescence spectroscopy have been designed. This procedure is suitable for the screening of inhibitors of these interactions. Finally, a turbidity assay to detect and measure the binding of soluble proto-ring proteins (FtsZ) to other elements of the divisome reconstituted in lipid vesicles has been developed (Martos et al., 2012 Biochemistry 49: 10780-10787).
To define more precise conditions to reconstruct, with a minimal set of elements, functional divisome assemblies in cell-like containers, we have studied the assembly properties of FtsZ in minimal reconstructions of the proto-ring structured as giant vesicles. These studies will help to complete our understanding of the initial steps of bacterial division, leading to applications in antimicrobial drug discovery. Collaborating with CSIC-CNB, E. coli proto-ring elements (FtsZ and FtsA) have been reconstituted inside giant unilamellar vesicles obtained from bacterial inner membranes; their spatial organization have shown that during polymerization in the presence of GTP the interaction between FtsZ and FtsA is stronger than the binding strength of FtsA to the membrane (Jiménez et al., 2011 J. Biol. Chem. 286: 11236-11241). More recently, procedures to efficiently incorporate FtsZ inside permeable giant vesicles, formed by reverse emulsion, with sZipA artificially incorporated at the inner membrane face have been optimized. These vesicles allow controlling the polymerization of encapsulated FtsZ by externally added GTP and Mg2+. Dynamic FtsZ polymers shrink sZipA-containing vesicles. Shrinkage, resembling the constriction of the cytoplasmic membrane, occurs at sZipA densities higher than those found in the cell, and it is modulated by the FtsZ polymerization dynamics. These results, obtained in cooperation with CSIC-CNB, show that defined bacterial elements can reproduce division functions when assembled in vitro (Cabré et al., J. Biol. Chem. 288: 26625-266342013).
CSIC-ICP have studied the initial stages of the formation of the septal ring looking at the interactions of FtsZ with ZipA on the lipid membrane. Two surface characterization techniques were mainly used: atomic force microscopy (AFM) and quartz crystal microbalance (QCM). Both operate in solution and allow to characterize, respectively, the structure and dynamic behaviour of single filaments and to quantify the interactions between soluble ligand and modified surfaces. The procedures allowed measuring in vitro, on solid surfaces and with the proteins properly oriented (both conditions which resemble those found in the cell membrane), the binding and unbinding of FtsZ to ZipA oriented on solid lipid surfaces of different composition. They also allowed to follow the structure and dynamic formation of FtsZ polymers on lipid membranes. The results provide data on the interactions between FtsZ monomers and polymers. They show that lateral interaction between filaments participate in the formation of aggregates on surfaces. Single filaments on mica are curved in the presence of GTP and depolymerize from the ends. Quantitative analysis and theoretical modelling indicated that all monomer-monomer interfaces are equivalent and that monomer from the ends diffuse away from the filament more easily. The shape and dynamics of FtsZ filament aggregates attached through oriented ZipA on lipid surfaces is dependent on the type of lipid present. On phosphatidylcholine lipids filament aggregates are straight and closely attached to the surface whereas on E. coli lipids they are curved and held above the surface. The nucleotide content affects the binding of FtsZ to ZipA as it binds with higher affinity in the presence of a phosphorylated nucleotide. Reversibility of the binding is dependent on both the nucleotide content and on the type of lipid present on the surface
The work at CSIC-CBMSO has been to develop a PG-biosynthesis assay for high throughput screening, i.e. an array-like device with immobilized cell wall fragments, including as many of the assay components, proteins, lipids, substrate ligands, as possible. Our role has been to procure appropriate PG synthesis precursors, UDP-muramyl pentapeptide purified by means of affinity chromatography on immobilized vancomycin, Lipid-II purified by means of nonpolar affinity chromatography on immobilized vancomycin. Also we have produced suitable (spread on glass, mica and plastic, and unilamellar) PG-fragments, and an adequate reporter molecule labelled precursor.
Biomol-Informatics have obtained, using Molecular Dynamics techniques, an excellent model for the FtsZ-FtsZ interface, including the role of monovalent cations and water molecules in the maintenance of the dimer structure and GTPase activity (Mendieta et al. 2009. J. Mol. Biol 390, 17; Martin-Garcia et al. 2012. FEBS Letters 586, 1236). Using this interface model and a system of in silico combinatorial chemistry and docking, they have designed 3 molecules able to bind specifically the active centre of FtsZ polymer in vitro and to inhibit bacterial division in vivo. The three compounds are currently being improved in a hit-to-lead phase as new antimicrobials.
CNRS was involved in Imaging and sRNA Technologies. CNRS identified a new way used by the cell to regulate the expression of some division proteins and used this antisense strategy to sensitise E. coli strains to antibiotics. In parallel, CNRS also set up a protocol to observe the alterations of divisome architecture caused by inhibitors on whole cells.
In vivo yeast assays for screening for small molecule inhibitors of the Divisome FtsA-FtsZ and the FtsA:MreB protein-protein interactions were constructed by EVOLVA. Fourteen “Genetic Chemistry” libraries were constructed and used to screen in yeast for the production of protein-protein interaction inhibitors of the Divisome FtsA-FtsZ protein-protein interaction. Each library expresses random cDNA from a certain organism known to produce antibacterial principles or a number of genes together constituting a pathway to biosynthesize basic chemical structures. More than 7 billion individual library yeast cell were screened in the primary survival screen, and 8000 primary yeast “hit” colonies were subjected to the secondary reporter screen. Of these 1397 were confirmed as true “hits” (CEYs). 300 of these CEYs were tested in an OD600-based bacterial survival assay. More than 68% of the CEYs tested had significant antibacterial effects using this assay. Of the positives from the antibacterial screen 20 CEYs were subjected to chemical de-replication, based on differential analysis between an (inactive) control yeast strain and the (active) CEYs. This analysis gave rise to 8 compounds which were dependent on expression of the “Genetic Chemistry” of the Watchmaker libraries. Of these 8 compounds 6 were novel, i.e. unknown when compared to all known chemical structures using the SciFinder algorithm. One of the 8 compounds, GCI-512, was found to be active in a Burkholderia thailandensis OD600 assay with an MIC of 28 g/ml. Though serious efforts were undertaken to produce enough of these compounds to attempt glycosylation to possibly improve ADME properties this was not successful. As an alternative it was attempted to glycosylate a compound (DEM30355) isolated by one of the other consortium partners (Demuris) but this particular compound proved chemically unstable under the assay conditions.
The results of MRC-LMB have centred around FtsW, the lipid II flippase of the divisome. The molecule is of considerable interest because its inhibition is anticipated to be both more effective and less difficult than intracellular targets since it is located in the inner membrane. The protein is the stuff of legends. When we started, we could only obtain around 10 g from 10 litres of E. coli culture. After much optimisation and organism screening that has now improved to several tens of pure, detergent solubilised FtsW protein. The time it took to get to this point has meant that both obtaining crystals and performing lipid II transport assays have been delayed and will not be completed, unfortunately. Nevertheless, we are optimistic that this can now be achieved after it has been demonstrated that FtsW is a manageable protein for structural and biochemical studies and we will continue this avenue until the desired results are obtained, after so much investment in time and resource.
Most antibiotics are produced by microorganisms and bacteria from the order Actinomycetales are especially prolific producers of antibiotics. These bacteria produce the majority of currently known classes of antibiotic. During the DIVINOCELL project Demuris screened a unique collection of actinomycete bacteria which was isolated over a period of 40 years by leading taxonomic academics. The drive behind isolation of this collection was taxonomic diversity, and the strains were obtained from widely diverse habitats with a range of different and innovative isolation methods. They are also supported by a unique level of annotation and documentation of their diversity, as befitting their taxonomic purpose. Unlike other collections this one has been extensively “dereplicated” so that its diversity is equivalent to that of much larger strain collections.
Demuris developed and implemented Standard Operating Procedures (SOP’s) to provide the high quality extracts needed for antibiotic screening. This has involved optimising conditions for revival and growth of the cultures, preparation of extracts, arraying into formats ready for screening, and storage. The company has also put in place methods for purification of active molecules by HPLC, identification by mass spectrometry and structural determination by NMR. So far over 36 different genera have been identified from the isolates screened, indicating that the Demuris actinomycete collection is extremely taxonomically diverse. At present, 6297 dereplicated isolates have been resurrected and tested for antimicrobial activity. Screening has revealed over 1571 “hits” with activity towards Gram-positive bacteria, and over 122 “hits” have activity against Gram-negative bacteria. These data confirm that actinomycete bacteria are prolific producers of antibiotics and provide a platform that Demuris can use for antibiotic discovery.
Demuris has incorporated assays developed by consortium partners into all screening activities. These include a strain with a β-lactam inducible reporter gene. This strain can be used in a simple disk diffusion assay and several β -lactam antibiotics have been used to validate its use such as the penicillin’s (Ampicillin) and cephalosporin’s (Cefotaxime). From this screening over 20 different isolates were identified as producing compounds which are indicative of cell wall synthesis inhibition. During this project several of these strains were prioritised.
The strain DEM30543 was chosen for further analysis due to its strong inhibitory activity towards Gram-positive bacteria and Gram-negative bacteria. In addition, this strain produces a compound which activates the E. coli cell wall reporter and an independent Bacillus cell wall reporter, whilst no inhibitory activity was observed towards the eukaryotic microorganisms. In order to produce sufficient material for further analysis, scale up from 5L to 500L was performed at the National Industrial Biotechnology Facility (NIBF). The antibiotic compound DEM30543 was purified from crude extract via ion exchange, reverse phase and size exclusion chromatography. After these steps a single peak was observed by HPLC analysis indicating that the compound was pure. Mass spectrometry analysis of the purified compound indicated that the mass was novel. The activity spectrum was investigated and several pathogenic strains of bacteria were screened against the antibiotic. All Gram positive isolates were found to be susceptible to the antibiotic, and most Gram negative strains tested, with the exception of Acinetobacter baumannii and Burkholderia cepacia. Both of these pathogens have been widely reported to be resistant to many clinically used antibiotics and are especially difficult to treat. To evaluate the cytotoxicity, a human cell line was tested with increasing concentrations of the antibiotic DEM30543. Cell integrity was evaluated using two independent assays (LDH and ATP). The results from these assays indicated that DEM30543 demonstrated little toxicity towards human cells. This compound is undergoing further evaluation for clinical development.
In addition, several compounds targeting cell wall/division targets were identified during this project. These compounds are currently at different stages of development and will be evaluated in due course.
UCHILE has synthesized 20 structural analogues of 4′6-diamidino-2-phenylindole (DAPI) DAPI as scaffold for FtsZ inhibitors. In order to determine the preferential binding of these compounds either by the monomer or the polymer of FtsZ, the Wayman function was used. The critical concentrations at different concentrations of FtsZ in the presence of different concentrations of the compounds were determined. By bioinformatics the free energy for the binding of the compounds to FtsZ was determined using Molecular Docking computer programs. Five binding sites were found, four for the monomer and one exclusive site for the dimer. The preferential binding for the polymer is compatible with the binding of one mole of compound per two mole of FtsZ monomer. Then the exclusive binding site formed in the dimer should correspond to the binding sites for the compounds in the polymer, which has higher affinity than the sites in the monomer.
FtsQ interactions with FtsB and FtsL were investigated by UCHILE. Two oligomeric models for the periplasmic region of the FtsB/FtsL/FtsQ E. coli complex were obtained from bioinformatics analysis. The FtsB/FtsL subcomplex was modelled as a coiled-coil based on sequence information and several stoichiometric possibilities. The crystallographic structure of FtsQ was added to this complex, through protein-protein docking. Two final structurally-stable models, one trimeric and one hexameric, were obtained. The nature of the protein-protein contacts is energetically favourable in both models and the overall structures are in agreement with the experimental evidence reported (Villanelo et al., 2011 BMC Struct Biol. 11, 28).
UCHILE also studied the proto-ring FtsA protein as target. FtsA with a histidine tag has been purified through Ni affinity column and SDS-PAGE electrophoresis.
UNEW has shown for the first time that FtsZ and MreB of E. coli interact, that the interaction is required for cell division and therefore is essential. The interaction functions to deliver to the FtsZ ring cell wall synthesising enzymes that are essential to synthesise the septum and/or pre-septal PG. We have constructed a Bacterial-Two-Hybrid (BTH) assay that can be used to screen for chemical compounds that abrogate the FtsZ - MreB interaction. We have also constructed an assay that was used to screening compound libraries for inhibitors of cell wall synthesis in E. coli.
Further work at UNEW has developed a positive whole-cell assay making use of the unique properties of E. coli peptidoglycan hydrolase mutants to identify cell division inhibitors, and have used this assay to screen a library of compounds from the VICHEM. We have developed and used various types of in vitro peptidoglycan synthesis assays to study the activities of peptidoglycan synthases. These assays can be used to screen for inhibitors. We have purified relevant proteins and enzymes of the E. coli division machinery as well as substrates for activity assays. We have developed several in vitro peptidoglycan synthesis assays and used them to characterize the major peptidoglycan synthases (Penicillin-binding-proteins 1B and 3) together with their interacting proteins. A major achievement was the establishment of peptidoglycan synthesis in lipid vesicles (phospholipid liposomes). We used these assays to characterize both activities, glycosyltransferase and transpeptidase, of peptidoglycan synthases using native, radioactive or fluorescently labelled substrates. UNEW has also participated in multiple collaborations on bacterial cell wall topics with partners inside or outside DIVINOCELL, providing our experience in peptidoglycan structural analysis. These collaborations resulted in novel insights into the regulation of peptidoglycan synthesis during cell elongation and cell division in E. coli and Caulobacter crescentus, and into the degradation of peptidoglycan by antibacterial enzymes in a variety of bacterial species. Moreover, the collaboration with the UCHILE provided detailed knowledge on the lateral protofilament interactions of FtsZ.
UvA developed an assay based on Förster resonance Energy transfer (FRET) that is able to measure protein-protein interactions in living bacterial cells. We showed that this assay is able to measure the loss of interactions between proteins due to the action of antibiotics. The assay is useful because it can assay simultaneously whether the antibiotic is able to pass the bacterial envelope and which proteins complex it targets. We also showed that the loss of activity of the proteins complex involved in cell elongation, results in a dissociation of the complex. This FRET assay will be very useful and cost effective for the screening of new antibiotics and could be applied by anybody for any protein interaction that could be inhibitable. The new antibiotics that inhibit FtsZ can be used as leads for further optimization of the antibiotics towards clinical application. Work at UvA has also established the interaction surface between FtsZ and ZapA and between FtsB, FtsL and FtsQ. These proteins are part of the cell division machinery and knowledge on their interaction surfaces provides information that assists drug design and computer assisted drug screening. Using classical in vitro assays 6 new inhibitors of FtsZ polymerization in vitro and that have a reasonable EC50 in vivo have been identified in the VICHEM collection.
In the DIVINOCELL project VICHEM designed and obtained a new class of antimicrobial compounds to block bacterial division. Inhibitors directed towards bacterial division targets, which are not present in eukaryotic cells, like FtsZ, can be both effective and harmless to humans. We gained hits from a compound collection with enzymatic assays and selected the most promising hits as lead compounds to perform lead optimization - such as selectivity analysis - as a prerequisite for drug candidate identification.
VICHEM established a compound collection with more than 1700 compounds which were selected from the Nested Chemical Library™and adapted a GDP-based FtsZ screening assay which was used to find structures which bound to the protein and inhibit its function. With this assay we found two hit molecules. Inhibitory effect of these structures was in the same range as the published FtsZ inhibitors. Based on the structure of these inhibitors a new collection of 197 compounds were selected for further investigation, such as bactericide tests. 74 out of these molecules had effect in the first round. During the validation process some of them had promising but not convincing effect in different bacterial tests, and some of them were validated in FtsZ polymerization assays. The later group of compounds was effective in further bacterial assays, as well. A small library was synthesized around these compounds by VICHEM. To minimise the low solubility problem that lead to compound precipitation in some assays, the water solubility of the compounds was increased by preparing the salt forms. Selectivity for bacterial targets was investigated with determination of effect on eukaryotic counterparts. Selective compounds, which are harmless to humans, from the group of effective ones were chosen as potential antibiotic candidates. These results were done in cooperation with CSIC, UNEW and UvA.
The DIVINOCELL project identified promising structures as potential inhibitors that block the proliferation of pathogens. Although these compounds will require additional in vitro investigations and chemical development to develop them into usable drugs, VICHEM results provided drug candidates that are new potential inhibitors to attack infection. The project resulted in compounds that could step into the next phase of drug development process [medicinal properties, e.g. ADMET analysis (to describe the fate of substances in a living organism) and optimization, in vivo tests], and provided early lead compounds. This process demands further chemical development. During the project we acquired chemical experience in preparation in this field of chemistry and explored the chemical behaviour of the structures that possess activity against essential bacterial targets.
The in vivo interaction between divisome proteins FtsQ and FtsB and FtsL, studied by VU, is a potential target for novel antimicrobials. For directed design of compounds targeting this interaction detailed knowledge at the molecular level is needed. To this end an assay for in vivo photo cross-linking has been tested and optimized. By thus probing the surface of FtsQ for contact with the other two proteins an interaction hotspot for FtsB and an interaction region for both FtsB and FtsL were identified. The identification of contact sites in the FtsQBL complex will guide future development of interaction inhibitors that block cell division. Moreover, the region of the interaction hotspot is highly conserved among the class of gammaproteobacteria. An antibiotic targeting this site can therefore be expected to be effective against a range of pathogenic Gram-negative bacteria. These findings have been published in the Journal of Biological Chemistry (11 July 2013, 288 [34], 24340-24350).
Potential Impact:
Socio-economic impact, societal implications, dissemination and exploitation.
Using the divisome and the septum, both essential for bacterial survival, as inhibitable targets, DIVINOCELL has identified eight new antimicrobial compounds that block the proliferation of Gram-negative pathogens. In addition the project has generated new technology to discover antibiotics and to help to improve the properties of the already discovered hits.
Compound GCI-512, identified by EVOLVA, will be a welcomed addition to obtain antibiotics towards serious bacterial threats such as those of the Burkholderia genus. Burkholderia-induced melioidosis is feared as a biological warfare agent and as these bacteria are resistant towards most antibiotics an efficient drug could be highly interesting. It should also be kept in mind that certain Burkholderia species are used in agriculture (e.g. for biocontrol) and so it may be feared that the future could bring problems with e.g. hospital-acquired infections with these bacteria. Filing of IP on this compound is possible, allowing then DIVINOCELL partners other than EVOLVA to continue working with it. The compound will be synthesized now in larger amounts to be tested in a range of divisome and general antibacterials assays before a hit molecule for pre-clinical development proper is in hand. The three chemical entities selected by BIOMOL as having an antibacterial effect, BIZ2013-1, BIZ2013-2 and BIZ2013-3, have been adopted into a hit to lead program for development of new drugs (ENABLE) integrated into the IMI initiative. The collaborative work of CSIC, UNEW and UvA within DIVINOCELL identified at least three promising compounds in the VICHEM collection, VCC599460:13, VCC342365:01 and VCC404021:01, as being potential inhibitors to block the proliferation of pathogens. These compounds are now ready to step into the next phase of drug development process. To advance them into valuable compounds for clinical use additional research is required to obtain early lead compounds. This includes the improvement of their medicinal properties, e.g. ADMET analysis (to describe the fate of substances in a living organism) and optimization, and in vivo tests. This process demands further chemical development. During the project the VICHEM staff acquired valuable experience in the production of these compounds and explored the chemical behaviour of the structures that possess activity against targets in the divisome.
Demuris is currently collaborating with Cubist Pharmaceuticals (USA), a bio-pharmaceutical company who focuses on research, commercialisation and creation of pharmaceutical products that address unmet medical needs in the acute care environment, who are providing a service of screening and compound characterisation and will take promising leads emerging from the project, as compound DEM30543, forward for pre-clinical testing. Throughout the project the company has undertaken a process of frequent internal IP searches to ensure that there is strong protection for all of the drug candidates discovered within the 4.5 year period. In keeping with industry standard practice Demuris has evaluated all of its inventions, such as compound structures and their analogues, targets and assays and continues to review its IP assets. Demuris has been engaging with the biotech and pharmaceutical market to establish current trends for the timing of licensing deals for antibiotic related products. Several important collaborations both academically (with experts in divisome structure and function) and commercially (with various chemistry and drug development companies) have derived from the Demuris participation. These collaborations and partnerships have led to the sharing of scientific knowledge and expertise including a few in related fields as agrochemicals, cosmeceuticals and cancer research. These collaborations will produce further grant funding and commercial deals.
The general social and economic benefits of developing new antibiotics are the lower rates of nosocomial infections, shorter time in hospital stays, lower number of readmissions and mortality, and increased confidence in healthcare systems. Benefits to society include increased productivity, life expectancy and lower costs of public health services. Infectious diseases count as the second leading cause of death worldwide and “even now, in the antibiotic era, common infectious diseases are major contributors to morbidity and mortality world-wide, especially in the developing world, but also in the developed world (World Health Organization, 1996). In developing countries infectious diseases cause over 60% of total deaths, many of them caused by bacterial pathogens. They are the third leading cause of death in Europe, mostly in elderly and debilitated populations, and, despite existing antibiotic therapies and vaccines, they remain the leading cause of mortality and morbidity worldwide. A similar question can be raised concerning those patients that, for diverse medical reasons, are immunocompromised. Their numbers, as medical procedures are technically being perfected, are likely to rise creating another segment of the population with an increased risk of succumbing to infections. In developed countries, nosocomial infections, occurring in 5-7% of patients hospitalised for other reasons, increase the hospital stay by an average of four days with an increased cost per day of nearly 500 €. If patients are in an Intensive Care Unit both the risk and the cost are more than double; their additional stay can extend up to fifteen days and have a concomitant higher mortality rate, often associated with antibacterial therapeutic failure” (Vicente et al., 2006. FEMS Microbiol. Rev. 30: 841-852). Among the pathogens, Gram-negatives are more resistant to commonly used antibiotics because of their complex envelope and at the same time they cause many serious community and hospital acquired infections.
Although there are over 100 antibiotics commercially available, bacterial infections still represent an area of acute unmet medical need as a result of the continuous development of bacterial resistance, increased global longevity and travel, and side effects from existing products. The number of antibiotic resistant pathogens is rising and some microbes are now resistant to most of the antibiotics currently used to treat infections. According to the US Centers for Disease Control, up to 70% of hospital-acquired bacterial infections are resistant to at least one antibiotic, and up to 40% are resistant to three. At the same time “the efforts in new antimicrobial development have focused largely on the modification and improvement of agents already discovered, while research on new classes of antibiotics has lagged behind” (Álvarez and Vicente, 2007. Expert Opin. Ther. Patents. 17: 667-674, and corrigendum). Hardly any new antibiotic has been introduced during the last decade to treat infections, and little more than 20 products are in the late stages of development, according to EP Vantage. The British Society for Antimicrobial Chemotherapy Initiative notes that ‘the pipeline is at its lowest ebb since the 1940s when penicillin was discovered’. There is therefore urgent need for novel antibacterial therapies. There is good evidence that new classes of antibiotic can capture a significant market share.
Besides the primary societal impact that the newly discovered antimicrobials will have on public health and individual well-being, DIVINOCELL participants have published over 63 papers in top ranking scientific journals, have defended 4 doctoral thesis and have participated in over 80 dissemination activities directed to the general public. A brief summary of some of them is below:
CSIC DIVINOCELL researchers organised a scientific meeting “Reconstructing the essential bacterial cell cycle machinery” within the prestigious EMBO workshops in 2012 at La Granja de San Ildefonso, Spain. The workshop (http://events.embo.org/12-bacteria-cell-cycle/index.html) discussed advances on the construction of essential bacterial machineries in the absence of cells and offered a three day conference programme in which a total of 90 leading world scientists in the field of bacterial division and young scientists participated. The Coordinator of DIVINOCELL, being the main organiser of the workshop, has also edited a special issue of Environmental Microbiology now in press.
Prof. Octavio Monasterio, from UCHILE, appeared in the CNN Chile program "Cápsula científica" where he talked about function and structure of the proteins, and his investigations in the project (http://www.youtube.com/watch?v=LO-dDlwHeXA#t=13).
VICHEM participated at the Open Information Day on FP7 Heath Research. Brussels, 9 June 2011 (http://webcast.ec.europa.eu/eutv/portal/res/_v_fl_300_es/player/index_player.html?id=12102&pId=12082#)
A summary of the DIVINOCELL project was presented on October 1, 2010 by Tanneke den Blaauwen (UvA) at the European Inter-network meeting on antibiotic resistance. Representatives of the Aeropath, AntiPathoGN, Divinocell, EU-INTAFAR, Nabativi, PAR, Pneumopath and UC BACWAN were present. The meeting was organized by the Centre of Protein Engineering and the Doctoral School of Sciences (EDT: Structure and Function of Biological Macromolecules, Modelling and Bioinformatics) of the university of Liege and was held in the Chateau de Harze in Belgium.
Piotr Szwedziak (from Jan Lowe's team, at MRC-LMB), was awarded the prize sponsored by Molecular Microbiology for his poster "Biochemical and structural studies of the FtsZ/FtsA complex" presented at the 2010 Gordon Conference on Bacterial Cell Surfaces (New London, NH, USA. June 27- July 2, 2010). These results were obtained within the framework of DIVINOCELL.
Finally, DIVINOCELL has trained a sizable number of next generation European microbiologists, from student to Postdoctoral level, in the area of bacterial cell wall synthesis, cell division, antibiotic inhibition of these processes and assay development. The project has also helped to strengthen scientific collaboration between the participants and to generate future new projects to implement it.
List of Websites:
Project public website.
http://www.cnb.csic.es/~divinocell/
Contact information.
Scientific coordinator:
Miguel Vicente
Centro Nacional de Biotecnología
C/ Darwin nº 3, 28049, Madrid, Spain
mvicente@cnb.csic.es
http://www.cnb.csic.es/~mvicente/
Project Manager:
Moira Torrent
Centro Nacional de Biotecnología
C/ Darwin nº 3, 28049 Madrid, Spain
mtorrent@cnb.csic.es
CSIC institutional contact:
José Ramón Naranjo
C/ Serrano 11, 28006 Madrid, Spain
sgaae@csic.es
http://www.csic.es/web/guest/home
To obtain new antibiotics, DIVINOCELL exploited existing understanding and obtained new knowledge on the molecular biology of the cell division machinery (the divisome) of Gram-negative bacteria. Using the divisome, essential for bacterial survival, as a source of inhibitable targets we identified eight new compounds to block the proliferation of Gram-negative pathogens. We also generated new technology to facilitate the validation and the improvement of the properties of antibiotic hits and to help in discovering new antimicrobials. Gram-negative bacteria are encased in a complex envelope formed by two flexible membranes sandwiching a rigid peptidoglycan support layer that together maintain the integrity and the shape of the cell. Due to this complexity they are less susceptible to antibiotics than bacteria that do not contain a second membrane covering the peptidoglycan layer. For most of our work we used Escherichia coli as a model because it is by far the best understood Gram-negative bacterium and we have poweful techniques to study and handle it. Moreover, some strains of E. coli are pathogenic and cause deadly disease outbreaks.
The novel DIVINOCELL antimicrobials are designed to block the function of divisome proteins, such as FtsZ, FtsA or ZipA. They should be effective to counteract infections and be largely innocuous to humans and animals because their cells do not contain these proteins. Using models for the active centre of FtsZ, in silico procedures designed by BIOMOL have identified BIZ2013-1, BIZ2013-2 and BIZ2013-3: three molecules that inhibit the activity of the protein and are active against E. coli. From a VICHEM collection of kinase inhibitors, new inhibitors of in vitro FtsZ polymerization, VCC599460:13 and VCC342365:01, have been identified that also induce the misplacement of divisome components. One additional compound, VCC404021:01, also modifies the interaction of FtsZ with ZipA. Evolva has found compounds to disrupt the interaction between FtsZ and FtsA. One of them, GCI-512, is active against Burkholderia thailandensis, a multidrug-resistant bacterium. From a Streptomyces strain extract Demuris obtained DEM30543, one new broad spectrum antimicrobial. These compounds, in different phases of IP protection, are now part of hit to lead improvement programmes.
Several procedures to discover antibiotics have been designed by DIVINOCELL. UNEW formulated an assay that was used to screen Demuris libraries to find inhibitors of cell wall synthesis. A positive whole-cell assay based on properties of E. coli peptidoglycan hydrolase mutants, developed by UNEW, helped to screen the library of compounds from VICHEM. Strains depleted for FtsZ or MreB devised by CSIC and CNRS can detect antimicrobials at low concentrations. Using this sensitive test CSIC identified compounds affecting bacterial growth among those synthesised by UCHILE.
CSIC has developed new procedures to incorporate divisome proteins, such as ZipA, into nanodiscs. These small membrane plugs surrounded by a protein belt can be used as soluble reagents. In vivo photo cross-linking assays have been optimized by VU to test new targets in the interaction between three additional divisome proteins: FtsQ, FtsB and FtsL. Detection of protein - protein interactions using a technique known as FRET has been developed at UvA. UNEW constructed a Bacterial-Two-Hybrid assay to screen for chemical compounds that abrogate the interaction of FtsZ with MreB, a critical component to maintain the architecture of E. coli. Synthesis of the rigid peptidoglycan layer of the bacterial envelop is effected by peptidoglycan synthases, including MtgA, PBP3 and PBP1B, and DIVINOCELL has designed assays to measure their activity. Crucial for this development was the production by CSIC and UNEW of tools in the form of small-sized precursors (Lipid-II and UDP-muramyl pentapeptide), a reporter labelled precursor molecule and peptidoglycan fragments. MRC-LMB studied FtsW, a protein needed to translocate these precursors to the outer face of the membrane.
Besides the primary societal impact that the newly discovered antimicrobials will have on public health and individual well-being, DIVINOCELL participants have published over 63 papers in top ranking scientific journals, have defended 4 doctoral thesis and have participated in over 80 dissemination activities directed to the general public. DIVINOCELL has also helped to strengthen scientific collaboration between the participants and to generate future new projects to implement it.
Project Context and Objectives:
The work of DIVINOCELL aimed at identifying antibiotics active in Gram-negative bacteria. These are bacteria with a complex envelope formed by two flexible membranes sandwiching a rigid layer that together maintain the integrity and the shape of the cell. Due to the complexity of their envelop these bacteria are less susceptible to antibiotics than those that do not contain a second membrane covering the rigid support layer. The discovery of new antibiotics to treat infections is important for individuals and for society as most of those currently used have lost their efficacy due to the resistances acquired by many of the pathogens, Gram-negatives in particular. As bacteria cannot proliferate when any of the proteins required for their growth or for their multiplication is inactivated, we sought antibiotics to inhibit targets in the process of cell division or envelop synthesis.
In the process of bacterial division several proteins assemble at midcell to form a division ring, also called divisome. The action of the divisome is to separate the cell into two equal daughters by means of a septum. Once separated each daugther resumes growth and both divide independently from each other. For most of this work we used Escherichia coli as a model because it is a Gram-negative in which extensive knowledge on its biology together with a plethora of techniques are available for its study and handling in the laboratory. Moreover, some strains of E. coli are pathogenic and cause deathly disease outbreaks (Karch et al., 2012. EMBO Mol. Med. 4: 1-8). Division, as described in E. coli, is phylogenetically conserved, it has peculiarities present in Gram-negatives, and has been shown to be druggable. Among the E. coli proteins that form the division ring we have focused on those called FtsA, FtsB, FtsL FtsQ, FtsW, FtsZ and ZipA. In addition we have studied proteins that synthesize peptidoglycan, the rigid layer of the envelope. Among them FtsI, also a divisome protein, is specifically needed for the synthesis of this layer at the septum and belongs to a set of dedicated enzymes, collectively designated as penicillin-binding proteins (PBPs) because they can bind to penicillin. For this reason FtsI is also known as PBP3. It is well known that when any of these components of the E. coli divisome is missing or inactivated, cell division stops and the bacterium eventually dies.
The primary OBJECTIVE of DIVINOCELL was thus to extensively use the essential bacterial division machinery (divisome) and its product (septum) for antimicrobial discovery in Gram-negatives. The existing and new knowledge obtained from the Escherichia coli division machinery (the divisome) was planned to be used to: identify novel inhibitable targets in the activity and interactions of the divisome proteins and the septum of Gram-negative bacteria; formulate primary assays and in vivo assays by combining the properties of these targets and the application of front-line technology; use these assays to screen unbiased and biased compound collections to obtain and validate hits; and finally to select the most promising hits as lead compounds to perform lead optimisation as a prerequisite for drug candidate identification. In addition to achieving the objectives of this call the DIVINOCELL project planned to advance the most promising leads into initial programs which are proprietary to the industrial participants in order to improve their medicinal properties.
The DIVINOCELL research was based on several innovative approaches:
EXPLOITATION OF AN ESSENTIAL FUNCTION: To curb the proliferation and the viability of Gram-negative pathogens the properties of a basic essential function of bacteria, cell division, was exploited.
FRONT-LINE TECHNOLOGY: To advance on the available state of the art to formulate in vitro and cell assays, we have applied novel tools (analytical, bioinformatic, structural and imaging) and incorporated all the available knowledge of the in vivo properties of the division process. We have finally obtained and applied new knowledge on the biophysical and biochemical details of the process to obtain assays that work in the test tube under conditions that replicate those found in the cell cytoplasm and periplasm.
NOVEL TARGETS & ASSAYS: We have concentrated our efforts in the study of the specific components of the Gram-negative divisome and septum. Although many of them are phylogenetically conserved they include elements and interactions specific for the survival of Gram-negatives. Crucial for this objective has been the exploitation of the activities of three proteins, FtsZ, FtsA and ZipA and their assembly into a proto-ring. ZipA, required together with FtsA to anchor FtsZ to the division site, is specifically present in the relevant groups of Gram-negative pathogens, Enterobacteriacea and Pseudomonadacea among them, but not in other taxa. We identified and used new targets in a promising and unexploited element of the division machinery (FtsQ), in the divisome elements that participate in the synthesis of the septum (FtsI together with FtsW), as well as in the switch from elongation to septation (MreB).
CELL SECONDARY ASSAYS: We have used several recently-discovered genetic properties of cell division process and of the physiological consequences of its arrest to generate in vivo and positive cell assays for hit validation to select those having the optimum properties to yield leads.
CHOICE OF SPECIFIC COMPOUNDS: To maximise the possibilities of success some DIVINOCELL screens included a pre-selection of the compound collections and included synthetic and natural products. This was achieved by virtual in silico filtering, by the use of collections already biased to include nucleotide-binding site inhibitors and by genetic chemistry to evolve more efficient inhibitors from natural products.
CHOICE OF SCAFFOLDS: We obtained scaffolds that are not so widely present or are even absent from Nature by making extensive use of protein interactions in addition to protein activities. We also used potent in silico capabilities to help in the design of scaffolds. For these reasons, pathogens are less likely to possess a response mechanism to counter the action of the compounds derived from the project.
Virtual filtering to pre-select inhibitors of proteins with known structure.
Identification of inhibitors done by Biomol-Informatics started by virtual prefiltering to reduce the number of potential PI and PPI inhibitors to allow a more realistic wet screening approach. We estimate that the amount of compounds initially present in a collection could be reduced to just 10%. The procedure can easily pre-filter libraries containing 1 million “druggable” compounds by first obtaining two-dimensional information from commercial catalogs of ligand libraries or specialized databases (ZINC database; Irwin and Shoichet. 2005. J. Chem. Inf. Model. 45: 177–182). The molecules were then converted to 3D structures using CORINA (Sadowski et al., 1994. J. Chem. Inf. Comput. Sci., 34: 1000-1008) calculating the different conformers and assigning partial charges and consistent atomic radii with the AMBER force field (a family of force fields for molecular dynamics of biomolecules). The conformers were fed to the docking program Autodock 4 (Huey et al., 2007. J. Comput. Chem. 28: 1145-1152) to perform a fast size-dependent pre-screening to eliminate molecules with low probability to be active in the chosen surface sites, selecting a set of more favourable molecules in terms of geometry and energy of the complexes. The finally selected candidates were then studied through detailed Molecular Dynamics simulation and energetically analyzed to obtain a final selection of molecules that were synthetised and assayed experimentally.
Evolutionary genetic chemistry to evolve inhibitors of protein-protein interactions.
Developing small molecules to inhibit protein-protein interactions (PPIs) poses challenges regarding the size and nature of the interaction surface, which are often not resolved by conventional compound libraries. Natural compounds often contain unique structural features with utility in protein inhibition beyond combinatorial or synthetic medicinal chemistry. Natural structures have a strong track record as pharmaceuticals with 61% of the 877 new chemical entities that reached the market over the last 20 years having their origins in Nature (Newman and Cragg, 2007, J. Nat. Prod. 70: 361-477). EVOLVA selected PPI inhibitors based on the exploitation of the extraordinary biodiversity of active molecules provided by Nature. For this Evolva used their Watchmaker® technology platform to isolate and evolve small molecule inhibitors of the polymerization and interactions of the proto-ring components FtsA, FtsZ and ZipA. Watchmaker® is an evolutionary genetic chemistry technology that evolves small molecules as an alternative to, and synergistic with traditional synthetic-medicinal approaches. It is based on the generation of genetic chemistry libraries prepared by specifically mixing genes from relevant sources with a toolbox of specific biosynthetic pathway genes. The genes are collected on enhanced yeast artificial chromosomes (EYACS) and introduced into yeast strains harbouring a relevant built-in survival screening assay based on the reverse yeast two-hybrid system (Vidal et al., 1996. Proc. Natl. Acad. Sci. USA 93: 10315-10320) to apply selective pressure on yeast to evolve efficient inhibitors of the PPIs. This approach predisposes yeast to produce structurally-diverse novel small molecules and by continuously selecting for high-performing yeasts, highly functional compounds optimized for the selected target are isolated. Some yeast strains in which the interactions between the proto-ring elements remain functional had already been obtained (Yim et al., 2000. J. Bacteriol. 182: 6366-6373). A similar genetic system had already been used successfully to select for small compounds that interfere with protein-protein interactions of the human calcium channel subunits (Young et al., 1998. Nature Biotechnol. 16: 946-950).
Biased compound collections directed specifically to nucleotide-binding sites.
This was the approach followed by VICHEM. The Nested Chemical Library™(NCL) technology (Kéri et al., 2005 Assay Drug Dev Technol. 3: 543-551) was used to find inhibitors. NCL™ is a chemically diverse collection organised around 108 core structures with small focused sub-libraries generated around each core. It contains more than 600 published kinase inhibitory compounds including preclinically or clinically-relevant leads and large series of proprietary compounds. Both FtsZ and FtsA, two of the proto-ring components, contain nucleotide binding sites.Most of the NCL compounds, a kinase inhibitory library, act on the ATP-binding site, but the library contains as well a number of allosteric-binding site inhibitors. The structures of the few published FtsZ inhibitors and related molecules was used to complement NCL™. Initial NCL hits were expected to act on the GTP or ATP binding sites of FtsZ or FtsA or to interfere with the PPIs as allosteric inhibitors.
Screening of a collection of actinomycete bacteria.
The screening assays made available to Demuris have been cell based screens for general divisome targets. To increase the probability of getting “hits” they have turned to the screening of natural product libraries instead of screening small previously screened compound libraries from typical chemical vendors. Microbial natural products and compounds derived from natural products account for roughly half of the drugs used pharmaceutically (Baltz, R.H. 2008 Curr Opin Pharmacol, 8: 557-563). Although these molecules fell from favour as sources of screening in the late 90’s, there has been a resurgence of interest recently. This has been driven by two factors: first, disappointment at the yield of effective leads from combinatorial chemistry approaches; second, the emergence of methods, especially genome sequencing, that facilitate the development of natural products (Chun et al., 2007 International J. Systematic Evolutionary Microbiol. 57:2259-2261; Li, and Vederas 2009 Science, 325: 161-165.
Demuris acquired exclusive rights to unique collections of actinomycete bacteria from Newcastle University. The first collection, of about 6-8,000 strains, was isolated over a period of about 40 years by Prof Mike Goodfellow. The drive behind isolation of this collection was taxonomic diversity, and the strains were obtained from widely diverse habitats with a range of different and innovative isolation methods. They are also supported by a unique level of annotation and documentation of their diversity, as befitting their taxonomic purpose. The second collection, of about 2,000 mainly marine actinomycetes, is important because it has only been recognised that actinomycetes inhabit marine environments relatively recently. Therefore, this large group of organisms have been largely overlooked in classical screens for antibiotics done by major pharma. There is an argument that organisms living in marine environments need to generate very potent molecules because of the very dilute environment they inhabit, and that they are prolific producers of antimicrobials. Unlike other collections this one has been extensively “dereplicated” so that its diversity is equivalent to that of much larger strain collections. The majority of the stains screened are putatively Streptomyces or Micromonospora species these species are well known to produce compounds and give the best chance of identifying novel hits to Gram negative bacteria.
Project Results:
DIVINOCELL has exploited the existing knowledge on the molecular biology of the cell division machinery in Gram-negatives and has also generated new knowledge on this process to discover new targets. The application of this background to the screening of selected compound collections has identified several hits active in preventing bacterial proliferation. Exploitation of these new hits proceeds now to improve and convert them into lead compounds to formulate new antibiotics. Inhibitors directed against bacterial division targets will be effective to cure infections and will be largely innocuous to humans and animals because their cells either do not contain these proteins altogether or at the most they have greatly divergent variants of some of them. Moreover, as some of the identified inhibitors are based on the structures of synthetic scaffolds, not frequently present in Nature, they are less likely than the present antibiotics to favour the acquisition of resistances by the pathogens. The strategy of DIVINOCELL has been finally complemented by the development of new tools for screening, validation and improvement of inhibitors in the test tube and in whole cells. These tools are largely derived from our discoveries on the structure and interactions of the known and novel targets and in the physiology of bacteria defective in division.
SCREENING
Evolva has found compounds that may disrupt the interaction between FtsZ and FtsA. They have been isolated using the proprietary Watchmaker strategy in which compounds that block interactions between proteins are generated within engineered yeast strains and are revealed as able to rescue the toxicity of a suicide system equally engineered into them. Six novel compounds, i.e. unknown when compared to all known chemical structures using the SciFinder algorithm, show inhibitory effects on bacterial survival at concentrations that make them suitable hits to merit further development. One of them, GCI-512, is active in a Burkholderia thailandensis growth assay with an MIC of 28 g/ml.
The screening performed by Demuris has identified one broad spectrum compound, DEM30543, obtained from a Streptomyces strain extract, with desirable antimicrobial activity properties. The extract is active against Methicillin-resistant Staphylococcus aureus (MRSA). MIC values for E. coli are 5 g/ml. DEM30543 has demonstrated little toxicity towards human cells. Production of this natural compound, has been scaled to obtain sufficient amounts for additional testing by the UNEW.
Screening of 197 compounds, belonging to a collection of kinase inhibitors supplied by Vichem, has been performed at CSIC, UvA and UNEW using both in vivo and in vitro assays. Among those able to inhibit growth in vivo at concentrations that make them good choices to use in hit improvement, two new inhibitors of in vitro FtsZ polymerization, VCC599460:13 and VCC342365:01, have been identified that also induce the misplacement of divisome components. In addition, compound VCC404021:01 modifies the interaction of FtsZ with another component of the E. coli proto-ring.
Chemical synthesis by the UCHILE has provided eight analogs of DAPI, a molecule able to bind to DNA and also to FtsZ. Soluble and potent DAPI derivatives also inhibit bacterial proliferation. Inhibition of growth by these compounds occurs in strains in which the concentration of FtsZ is limiting.
In silico procedures designed by BIOMOL have provided high-quality models for the active centre of FtsZ in the FtsZ-FtsZ interface, both in the FtsZ dimer and in short polymers (Mendieta et al. 2009. J. Mol. Biol 390: 17-25; Martin-Garcia et al., 2012. FEBS Letters 586: 1236-1239). Using this interface model and a proprietary system of “in silico” combinatorial chemistry and docking (BIOMOL-HTCM; BIOMOL-High Throughput Combinatorial Modelling), three molecules, BIZ2013-1, BIZ2013-2 and BIZ2013-3, that bind specifically to the active centre of the FtsZ polymer have been designed. They inhibit the activity of the protein, are active against E. coli and are currently being improved in a hit-to-lead phase.
DISCOVERY OF NEW TARGETS IN BACTERIAL DIVISION
Polymerization of FtsZ on lipid surfaces is sensitive to the composition of the membrane, as proven by CSIC. The structure and the dynamics of individual FtsZ filaments in the presence of GTP, visualized by Atomic Force Microscopy on surfaces, showed curved filaments that depolymerize from the ends. Further quantitative analysis and theoretical modeling indicated that all monomer-monomer interfaces are equivalent and that the monomer at the ends diffuse away from the filament more easily (Mateos-Gil et al., 2012. Proc. Nat. Acad. Sci. 109: 8133-8138). Novel inhibitable targets have been discovered by CSIC in the interactions between divisome proteins, for example FtsZ with ZipA, FtsQ with either FtsB or FtsL, and FtsA with itself. A new function of ZipA in the stabilization of FtsZ (Pazos et al., 2013. J. Biol. Chem. 288: 3219-3226) and new interactions between ZipA and two proteins, ZapA and ZapB, that assist FtsZ in the divisome were discovered (Pazos et al., 2013. Environmental Microbiol. DOI: 10.1111/1462-2920.12227 ). The interaction surface between FtsZ and ZapA has been determined by UvA. To study the possible targets in the FtsA protein it has been obtained by CSIC from Streptococcus pneumoniae, as this protein is easier to handle when obtained from this Gram-positive pathogen. Its C-terminus is as a molecular regulator that affects FtsA polymerization in the cytoplasm and facilitates this process when the protein is attached to lipid membranes. Other regions as the 1C and the S12-13 domains also play significant roles in polymerization and together with the C-terminus define three novel inhibitable targets (Krupka et al., 2012. J. Biol. Chem. 287: 7756–7765). That the polymers formed by some FtsA proteins resemble actin filaments was then shown by MRC-LMB (Szwedziak et al., 2012. EMBO J. 31, 2249-2260).
An interaction hotspot for FtsB and an interaction region for both FtsB and FtsL were identified by VU and UvA on the surface of FtsQ. The hotspot is highly conserved among the class of gammaproteobacteria (Glas et al., 2013. J. Biol. Chem. 288: 24340-24350).
FtsW is a protein that transfers peptidoglycan precursors to the periplasm, the space in between the two membranes where this rigid polymer is produced. It has been purified and crystallized by MRC-LMB. Further improvement of the crystallization setups are being tested to obtain a high resolution dataset to solve the structure of FtsW thus supplying novel inhibitable targets in this protein.
As shown by UNEW, FtsZ and MreB of E. coli interact and the interaction is required for cell division and therefore is essential. The interaction functions to deliver to the FtsZ ring cell wall synthesising enzymes that are essential to synthesise the septum and/or pre-septal peptidoglycan.
Results from CNRS show that mreB expression is regulated by non-coding RNAs, this regulatory mechanism can also be used as a source for new inhibitable targets.
ASSAY DESIGN AND NEW TECHNOLOGY
Protocols to assist in the process of lead development that are based on strains depleted from FtsZ or MreB have been devised by CSIC and CNRS. They have been applied to assay the DAPI analogs from UCHILE. The CNRS found that an hfq minus strain, which allows to simultaneously deregulate the expression of different cell division proteins, shows a significant increase for Aztreonam and Piperacillin sensitivity. UNEW constructed a Bacterial-Two-Hybrid (BTH) assay that can be used to screen for chemical compounds that abrogate the FtsZ - MreB interaction and an assay that was used to screen Demuris compound libraries for inhibitors of cell wall synthesis in E. coli.
A positive whole-cell assay making use of the unique properties of E. coli peptidoglycan hydrolase mutants to identify cell division inhibitors, developed by UNEW, was used to screen the library of compounds from VICHEM.
Various types of in vitro peptidoglycan synthesis assays to study the activities of peptidoglycan synthases have been generated. The design includes the use of MtgA, PBP3 and PBP1B, all of them proteins that work to synthesise peptidoglycan. The wild type enzyme and variants with a single activity are available. Small-sized precursors of peptidoglycan synthesis, as Lipid-II and UDP-muramyl pentapeptide, are used as substrates to quantify the enzymatic activities. They have been obtained by CSIC and UNEW. Using immobilized vancomycin as a matrix, affinity chromatography was used to purify UDP-muramyl pentapeptide whereas apolar affinity chromatography was chosen to purify Lipid-II. In addition, suitable (spread on glass, mica and plastic, and unilamellar) PG-fragments, and an adequate reporter molecule labeled precursor were produced.
CSIC has developed new procedures to apply the divisome proteins in easy to use screening assays, among them vesicles that can mimic the cell interior and nanodiscs. The interaction between FtsZ and FtsA and between FtsZ and ZipA was tested in vitro using natural or artificial vesicles respectively. We have discovered that within natural membrane vesicles the FtsA protein does not anchor to the membrane at sufficient strength to withstand the pull exerted by polymerizing FtsZ (Jiménez et al., 2011 J. Biol. Chem. 286: 11236-11241). ZipA may provide a stronger anchoring for FtsZ polymers as it contains a membrane domain. Permeable vesicles in which it has been artificially incorporated collapse upon FtsZ polymerization (Cabré et al., 2013. J. Biol. Chem. 288: 26625-26634).
Nanodiscs are small membrane plugs surrounded by a protein belt. Proteins that are integrated in the membrane, as ZipA, can be used when inserted in nanodiscs as soluble reagents. This assay, developed by CSIC, was used to measure the strength of the interaction between the ZipA bound to nanodiscs and the soluble FtsZ (Hernández-Rocamora et al., 2012. J. Biol. Chem. 287: 30097-30104).
An assay initially designed by CSIC to measure the binding of FtsA to lipid-coated glass microbeads (Martos et al., 2012, PLoS ONE 7, e39829) has been adapted to test the interaction between FtsZ and ZipA. A simple assay to measure FtsZ assembly in solution using fluorescence (Reija et al., 2011. Anal. Biochem. 418: 89–96) has also been formulated.
In vivo photo cross-linking has been optimized by VU as an assay to test a potential target for novel antimicrobials based on the interaction between divisome proteins FtsQ and FtsB and FtsL (Glas et al., 2013. J. Biol. Chem. 288: 24340-24350).
Detection of protein - protein interactions using a technique known as FRET has been developed at UvA. The assay detects the interaction between two fluorescently labelled proteins and can measure simultaneously whether an antibiotic is able to pass the bacterial envelope and identify the protein complex that it targets. Using the assay it has been proven that the loss of activity of the proteins in the complex involved in cell elongation results in a dissociation of the complex.
DESCRIPTION OF RESULTS FOR EACH PARTICIPANT
CSIC-CNB probed protein-protein interactions (PPIs) between ZipA and FtsZ by testing the effect of ZipA overproduction on FtsZ degradation. Our results show that in the nucleoid-free maxicells (Sancar et al. (1979) J Bacteriol 137: 692-693) FtsZ is present at low levels due to the specific degradation of FtsZ by the housekeeping protease system ClpXP and to the absence of de novo synthesis (Pazos et al. (2013) J Biol Chem 288: 3219-3226). The overproduction of ZipA in maxicells reduces the degradation of FtsZ in maxicells (Pazos et al (2013) J Biol Chem 288: 3219-3226). To check which domains of ZipA are involved in FtsZ protection, different ZipA deletion mutants were constructed and probed for FtsZ protection (Figure 1). Overproduction of ZipA mutants in maxicells shows that the ZipA mutant ZipA4 (lacking part of the FZB domain from Pro-273 to Ala-328) and the FZB domain were able to protect FtsZ from degradation as observed for the full length ZipA (Pazos et al (2013) J Biol Chem 288: 3219-3226). The protection of FtsZ by ZipA is not affected by the simultaneous presence of either FtsA or FtsA* (Figure 3) (Pazos et al (2013) J Biol Chem 288: 3219-3226).
CSIC-CNB used Bimolecular Fluorescence Complementation (BiFC) to detect and localize protein-protein interactions (PPI) during proto-ring formation within the cell. The assay is based on the irreversible reconstitution of the fluorescence signal upon association of two non-fluorescent fragments of the green fluorescent protein or its derivatives once they are brought into close proximity (Kerppola (2008) Annu Rev Biophys 37: 465-487). Using BiFC, we confirmed already described interactions (FtsZ-ZipA, FtsZ-ZapB, ZapB-ZapB or ZapB-ZapA) and obtained two novel and unexpected interactions (ZapB-ZipA and ZipA-ZapA) (Pazos et al. (2013) Environ Microbiol DOI:10.1111/1462-2920.12225).
Given the difficulties in the biochemical handling of the FtsA protein form E. coli, we have studied the polymerization of FtsA from Streptococcus penumoniae, a relevant pathogen although Gram positive. Two different truncations, involving either the 1C domain or the simultaneous absence of the S12-13 β-strands of the FtsA protein located at opposite terminal sides in the molecular structure, are essential for ATP-dependent polymerization. These two truncated proteins are not able to polymerize themselves but can be incorporated to some extent into the FtsA+ polymers during the assembling process. Consequently, they block the growth of the FtsA+ polymers and slow down the polymerization rate. The combined action of the two truncated proteins produces an additive effect on the inhibition of FtsA+ polymerization, indicating that each truncation affects a different interaction site within the FtsA molecule.
CSIC-CNB performed a set of in vivo assays to test the effect of compounds supplied by VICHEM on the proliferation, viability and division defects of E. coli strain JW0451 (as BW25113, ∆acrB). JW0451 is an acrB null mutant that does not export molecules outside the cell, as the AcrB protein functions as part of the AcrAB-TolC multidrug efflux system (Baba et al. (2006) Mol Syst Biol 2: 2006 0008; Kawabe, et al. (2000) J. Biochem. 128 195-200). The absence of multi-drug efflux system allows that the compounds supplied by VICHEM could be used at lower dosages. Using JW0451 we have performed compound susceptibility tests to determine the growth on agar plates and the Minimal Inhibitory Concentration (MIC) in a resazurin assay. We finally tested the effects of selected compounds on cell division by examining their effect on the morphology of the cells (filamentation or lysis) and on the localization of FtsZ and ZipA.
CSIC-CNB has exploited observations on the differential gene expression levels found in cells undergoing cell division arrest by deprivation of division ring proteins, namely FtsZ. These results have been used to construct plasmids reporting the absence of division as a fluorescent signal. For this purpose, specific promoters from the regulatory regions of the differentially expressed genes have been fused to the structural genes coding for naturally fluorescent proteins, as GFP. Strains deprived of proto-ring elements have been used to obtain assays to detect the effect of low antimicrobial concentrations.
At CSIC-CIB the mechanistic biochemistry of FtsZ, the prokaryotic homolog of tubulin, was studied to complete the understanding of its functional activities and interactions with ZipA and FtsA, the two elements of the division machinery that anchor FtsZ to the membrane forming the first molecular complex of the divisome, the proto-ring. We have exploited new reconstitution technologies to test the properties of elements of the bacterial cell division machinery and their interactions, in well-defined lipid environments (nanodiscs, microbeads, vesicles) under conditions that reproduce those found in the cell cytoplasm, to gain insights into their precise functions. This knowledge has been used to generate fluorescence-based assays to screen for inhibitors of specific proto-ring protein-protein interactions.
The assembly and disassembly cycle of FtsZ, linked to GTP hydrolysis, is thought to be one of the essential functional activities of FtsZ in bacterial division. We have completed the characterization of the Mg2+-linked FtsZ self-assembly in the presence of GTP or a GTP analogue using a set of biophysical tools (sedimentation velocity, concentration-gradient static light scattering, fluorescence correlation spectroscopy and dynamic light scattering). The combined analysis of these measurements has revealed that FtsZ polymerization involves the concerted formation of a narrow distribution of oligomeric species (around a hundred FtsZ subunits for GTP fibres) (Monterroso et al., 2012 Biochemistry 51:4541-4550). The size of the narrowly distributed GTP-FtsZ polymers decreases upon lowering potassium and is independent of GTPase activity. Potassium oppositely controls the size distribution of GTP-FtsZ polymers and the abundance of GDP oligomers (Ahijado-Guzmán et al. 2013 J. Biol. Chem. doi:10.1074/jbc.M113.482943). In parallel, a homogeneous fluorescence anisotropy assay to measure FtsZ assembly in dilute and crowded solutions has been developed in cooperation with CSIC-CNB. This assay allows measuring the effect of osmolytes, divalent cations and high concentrations of unrelated macromolecules (acting as crowding agents) on the critical concentration of FtsZ assembly, the degree of polymer formation and the dynamics of disassembly of these polymers. The assay is sensitive, low sample consuming, rapid, and easily adapted to systematic screening assays (Reija et al., 2011 Anal. Biochem. 418: 89-96).
We have also optimized a procedure to obtain milligram amounts of the recalcitrant FtsA division protein from E. coli that allows measuring the lipid-binding properties of FtsA relevant for its functional activity. The interaction between soluble FtsA and lipids has been found to be of moderate affinity as measured by biochemical assays using micron-size beads coated with either natural or artificial membranes (Martos et al., 2012 PloS One. 7(6):e39829).
We have designed assays to measure the binding of FtsZ to other proto-ring elements and to screen for inhibitors of proto-ring protein-protein interactions, which may be exploited in the screening assays for new antimicrobial agents targeting the proto-ring. Together with CSIC-CNB, the dynamic interaction between FtsZ and ZipA has been evidenced in nanodiscs - structures formed by a membrane scaffold protein encircling a lipid mixture. The strength of the interaction has been measured and found to be of similar moderate affinity for the FtsZ polymers formed with GTP and for the GDP forms (Hernández-Rocamora et al., 2012 J. Biol. Chem. 287: 30097-30104; Hernández-Rocamora et al., 2012 J. Struct. Biol. 180: 531-538). A fluorescence-based assay to screen for inhibitors of the interaction of FtsZ and ZipA incorporated in lipid bilayer nanodiscs has been implemented. Peptides containing partial sequences of the C-terminus of FtsZ compete with FtsZ polymers for binding to ZipA. The assay can proceed in solution and is therefore suitable for systematic assays. In addition, microbeads encoded with gold sensor nanoparticles prior to membrane coating have been used to design assays to monitor the interaction of FtsZ with other proto-ring elements (Ahijado-Guzmán et al., 2012 ACS Nano 6: 7514-7520). In addition, assays to measure the binding of soluble proto-ring elements to other elements of the divisome reconstituted in microbeads (coated with proteo-lipids or inner membranes) by differential centrifugation and fluorescence spectroscopy have been designed. This procedure is suitable for the screening of inhibitors of these interactions. Finally, a turbidity assay to detect and measure the binding of soluble proto-ring proteins (FtsZ) to other elements of the divisome reconstituted in lipid vesicles has been developed (Martos et al., 2012 Biochemistry 49: 10780-10787).
To define more precise conditions to reconstruct, with a minimal set of elements, functional divisome assemblies in cell-like containers, we have studied the assembly properties of FtsZ in minimal reconstructions of the proto-ring structured as giant vesicles. These studies will help to complete our understanding of the initial steps of bacterial division, leading to applications in antimicrobial drug discovery. Collaborating with CSIC-CNB, E. coli proto-ring elements (FtsZ and FtsA) have been reconstituted inside giant unilamellar vesicles obtained from bacterial inner membranes; their spatial organization have shown that during polymerization in the presence of GTP the interaction between FtsZ and FtsA is stronger than the binding strength of FtsA to the membrane (Jiménez et al., 2011 J. Biol. Chem. 286: 11236-11241). More recently, procedures to efficiently incorporate FtsZ inside permeable giant vesicles, formed by reverse emulsion, with sZipA artificially incorporated at the inner membrane face have been optimized. These vesicles allow controlling the polymerization of encapsulated FtsZ by externally added GTP and Mg2+. Dynamic FtsZ polymers shrink sZipA-containing vesicles. Shrinkage, resembling the constriction of the cytoplasmic membrane, occurs at sZipA densities higher than those found in the cell, and it is modulated by the FtsZ polymerization dynamics. These results, obtained in cooperation with CSIC-CNB, show that defined bacterial elements can reproduce division functions when assembled in vitro (Cabré et al., J. Biol. Chem. 288: 26625-266342013).
CSIC-ICP have studied the initial stages of the formation of the septal ring looking at the interactions of FtsZ with ZipA on the lipid membrane. Two surface characterization techniques were mainly used: atomic force microscopy (AFM) and quartz crystal microbalance (QCM). Both operate in solution and allow to characterize, respectively, the structure and dynamic behaviour of single filaments and to quantify the interactions between soluble ligand and modified surfaces. The procedures allowed measuring in vitro, on solid surfaces and with the proteins properly oriented (both conditions which resemble those found in the cell membrane), the binding and unbinding of FtsZ to ZipA oriented on solid lipid surfaces of different composition. They also allowed to follow the structure and dynamic formation of FtsZ polymers on lipid membranes. The results provide data on the interactions between FtsZ monomers and polymers. They show that lateral interaction between filaments participate in the formation of aggregates on surfaces. Single filaments on mica are curved in the presence of GTP and depolymerize from the ends. Quantitative analysis and theoretical modelling indicated that all monomer-monomer interfaces are equivalent and that monomer from the ends diffuse away from the filament more easily. The shape and dynamics of FtsZ filament aggregates attached through oriented ZipA on lipid surfaces is dependent on the type of lipid present. On phosphatidylcholine lipids filament aggregates are straight and closely attached to the surface whereas on E. coli lipids they are curved and held above the surface. The nucleotide content affects the binding of FtsZ to ZipA as it binds with higher affinity in the presence of a phosphorylated nucleotide. Reversibility of the binding is dependent on both the nucleotide content and on the type of lipid present on the surface
The work at CSIC-CBMSO has been to develop a PG-biosynthesis assay for high throughput screening, i.e. an array-like device with immobilized cell wall fragments, including as many of the assay components, proteins, lipids, substrate ligands, as possible. Our role has been to procure appropriate PG synthesis precursors, UDP-muramyl pentapeptide purified by means of affinity chromatography on immobilized vancomycin, Lipid-II purified by means of nonpolar affinity chromatography on immobilized vancomycin. Also we have produced suitable (spread on glass, mica and plastic, and unilamellar) PG-fragments, and an adequate reporter molecule labelled precursor.
Biomol-Informatics have obtained, using Molecular Dynamics techniques, an excellent model for the FtsZ-FtsZ interface, including the role of monovalent cations and water molecules in the maintenance of the dimer structure and GTPase activity (Mendieta et al. 2009. J. Mol. Biol 390, 17; Martin-Garcia et al. 2012. FEBS Letters 586, 1236). Using this interface model and a system of in silico combinatorial chemistry and docking, they have designed 3 molecules able to bind specifically the active centre of FtsZ polymer in vitro and to inhibit bacterial division in vivo. The three compounds are currently being improved in a hit-to-lead phase as new antimicrobials.
CNRS was involved in Imaging and sRNA Technologies. CNRS identified a new way used by the cell to regulate the expression of some division proteins and used this antisense strategy to sensitise E. coli strains to antibiotics. In parallel, CNRS also set up a protocol to observe the alterations of divisome architecture caused by inhibitors on whole cells.
In vivo yeast assays for screening for small molecule inhibitors of the Divisome FtsA-FtsZ and the FtsA:MreB protein-protein interactions were constructed by EVOLVA. Fourteen “Genetic Chemistry” libraries were constructed and used to screen in yeast for the production of protein-protein interaction inhibitors of the Divisome FtsA-FtsZ protein-protein interaction. Each library expresses random cDNA from a certain organism known to produce antibacterial principles or a number of genes together constituting a pathway to biosynthesize basic chemical structures. More than 7 billion individual library yeast cell were screened in the primary survival screen, and 8000 primary yeast “hit” colonies were subjected to the secondary reporter screen. Of these 1397 were confirmed as true “hits” (CEYs). 300 of these CEYs were tested in an OD600-based bacterial survival assay. More than 68% of the CEYs tested had significant antibacterial effects using this assay. Of the positives from the antibacterial screen 20 CEYs were subjected to chemical de-replication, based on differential analysis between an (inactive) control yeast strain and the (active) CEYs. This analysis gave rise to 8 compounds which were dependent on expression of the “Genetic Chemistry” of the Watchmaker libraries. Of these 8 compounds 6 were novel, i.e. unknown when compared to all known chemical structures using the SciFinder algorithm. One of the 8 compounds, GCI-512, was found to be active in a Burkholderia thailandensis OD600 assay with an MIC of 28 g/ml. Though serious efforts were undertaken to produce enough of these compounds to attempt glycosylation to possibly improve ADME properties this was not successful. As an alternative it was attempted to glycosylate a compound (DEM30355) isolated by one of the other consortium partners (Demuris) but this particular compound proved chemically unstable under the assay conditions.
The results of MRC-LMB have centred around FtsW, the lipid II flippase of the divisome. The molecule is of considerable interest because its inhibition is anticipated to be both more effective and less difficult than intracellular targets since it is located in the inner membrane. The protein is the stuff of legends. When we started, we could only obtain around 10 g from 10 litres of E. coli culture. After much optimisation and organism screening that has now improved to several tens of pure, detergent solubilised FtsW protein. The time it took to get to this point has meant that both obtaining crystals and performing lipid II transport assays have been delayed and will not be completed, unfortunately. Nevertheless, we are optimistic that this can now be achieved after it has been demonstrated that FtsW is a manageable protein for structural and biochemical studies and we will continue this avenue until the desired results are obtained, after so much investment in time and resource.
Most antibiotics are produced by microorganisms and bacteria from the order Actinomycetales are especially prolific producers of antibiotics. These bacteria produce the majority of currently known classes of antibiotic. During the DIVINOCELL project Demuris screened a unique collection of actinomycete bacteria which was isolated over a period of 40 years by leading taxonomic academics. The drive behind isolation of this collection was taxonomic diversity, and the strains were obtained from widely diverse habitats with a range of different and innovative isolation methods. They are also supported by a unique level of annotation and documentation of their diversity, as befitting their taxonomic purpose. Unlike other collections this one has been extensively “dereplicated” so that its diversity is equivalent to that of much larger strain collections.
Demuris developed and implemented Standard Operating Procedures (SOP’s) to provide the high quality extracts needed for antibiotic screening. This has involved optimising conditions for revival and growth of the cultures, preparation of extracts, arraying into formats ready for screening, and storage. The company has also put in place methods for purification of active molecules by HPLC, identification by mass spectrometry and structural determination by NMR. So far over 36 different genera have been identified from the isolates screened, indicating that the Demuris actinomycete collection is extremely taxonomically diverse. At present, 6297 dereplicated isolates have been resurrected and tested for antimicrobial activity. Screening has revealed over 1571 “hits” with activity towards Gram-positive bacteria, and over 122 “hits” have activity against Gram-negative bacteria. These data confirm that actinomycete bacteria are prolific producers of antibiotics and provide a platform that Demuris can use for antibiotic discovery.
Demuris has incorporated assays developed by consortium partners into all screening activities. These include a strain with a β-lactam inducible reporter gene. This strain can be used in a simple disk diffusion assay and several β -lactam antibiotics have been used to validate its use such as the penicillin’s (Ampicillin) and cephalosporin’s (Cefotaxime). From this screening over 20 different isolates were identified as producing compounds which are indicative of cell wall synthesis inhibition. During this project several of these strains were prioritised.
The strain DEM30543 was chosen for further analysis due to its strong inhibitory activity towards Gram-positive bacteria and Gram-negative bacteria. In addition, this strain produces a compound which activates the E. coli cell wall reporter and an independent Bacillus cell wall reporter, whilst no inhibitory activity was observed towards the eukaryotic microorganisms. In order to produce sufficient material for further analysis, scale up from 5L to 500L was performed at the National Industrial Biotechnology Facility (NIBF). The antibiotic compound DEM30543 was purified from crude extract via ion exchange, reverse phase and size exclusion chromatography. After these steps a single peak was observed by HPLC analysis indicating that the compound was pure. Mass spectrometry analysis of the purified compound indicated that the mass was novel. The activity spectrum was investigated and several pathogenic strains of bacteria were screened against the antibiotic. All Gram positive isolates were found to be susceptible to the antibiotic, and most Gram negative strains tested, with the exception of Acinetobacter baumannii and Burkholderia cepacia. Both of these pathogens have been widely reported to be resistant to many clinically used antibiotics and are especially difficult to treat. To evaluate the cytotoxicity, a human cell line was tested with increasing concentrations of the antibiotic DEM30543. Cell integrity was evaluated using two independent assays (LDH and ATP). The results from these assays indicated that DEM30543 demonstrated little toxicity towards human cells. This compound is undergoing further evaluation for clinical development.
In addition, several compounds targeting cell wall/division targets were identified during this project. These compounds are currently at different stages of development and will be evaluated in due course.
UCHILE has synthesized 20 structural analogues of 4′6-diamidino-2-phenylindole (DAPI) DAPI as scaffold for FtsZ inhibitors. In order to determine the preferential binding of these compounds either by the monomer or the polymer of FtsZ, the Wayman function was used. The critical concentrations at different concentrations of FtsZ in the presence of different concentrations of the compounds were determined. By bioinformatics the free energy for the binding of the compounds to FtsZ was determined using Molecular Docking computer programs. Five binding sites were found, four for the monomer and one exclusive site for the dimer. The preferential binding for the polymer is compatible with the binding of one mole of compound per two mole of FtsZ monomer. Then the exclusive binding site formed in the dimer should correspond to the binding sites for the compounds in the polymer, which has higher affinity than the sites in the monomer.
FtsQ interactions with FtsB and FtsL were investigated by UCHILE. Two oligomeric models for the periplasmic region of the FtsB/FtsL/FtsQ E. coli complex were obtained from bioinformatics analysis. The FtsB/FtsL subcomplex was modelled as a coiled-coil based on sequence information and several stoichiometric possibilities. The crystallographic structure of FtsQ was added to this complex, through protein-protein docking. Two final structurally-stable models, one trimeric and one hexameric, were obtained. The nature of the protein-protein contacts is energetically favourable in both models and the overall structures are in agreement with the experimental evidence reported (Villanelo et al., 2011 BMC Struct Biol. 11, 28).
UCHILE also studied the proto-ring FtsA protein as target. FtsA with a histidine tag has been purified through Ni affinity column and SDS-PAGE electrophoresis.
UNEW has shown for the first time that FtsZ and MreB of E. coli interact, that the interaction is required for cell division and therefore is essential. The interaction functions to deliver to the FtsZ ring cell wall synthesising enzymes that are essential to synthesise the septum and/or pre-septal PG. We have constructed a Bacterial-Two-Hybrid (BTH) assay that can be used to screen for chemical compounds that abrogate the FtsZ - MreB interaction. We have also constructed an assay that was used to screening compound libraries for inhibitors of cell wall synthesis in E. coli.
Further work at UNEW has developed a positive whole-cell assay making use of the unique properties of E. coli peptidoglycan hydrolase mutants to identify cell division inhibitors, and have used this assay to screen a library of compounds from the VICHEM. We have developed and used various types of in vitro peptidoglycan synthesis assays to study the activities of peptidoglycan synthases. These assays can be used to screen for inhibitors. We have purified relevant proteins and enzymes of the E. coli division machinery as well as substrates for activity assays. We have developed several in vitro peptidoglycan synthesis assays and used them to characterize the major peptidoglycan synthases (Penicillin-binding-proteins 1B and 3) together with their interacting proteins. A major achievement was the establishment of peptidoglycan synthesis in lipid vesicles (phospholipid liposomes). We used these assays to characterize both activities, glycosyltransferase and transpeptidase, of peptidoglycan synthases using native, radioactive or fluorescently labelled substrates. UNEW has also participated in multiple collaborations on bacterial cell wall topics with partners inside or outside DIVINOCELL, providing our experience in peptidoglycan structural analysis. These collaborations resulted in novel insights into the regulation of peptidoglycan synthesis during cell elongation and cell division in E. coli and Caulobacter crescentus, and into the degradation of peptidoglycan by antibacterial enzymes in a variety of bacterial species. Moreover, the collaboration with the UCHILE provided detailed knowledge on the lateral protofilament interactions of FtsZ.
UvA developed an assay based on Förster resonance Energy transfer (FRET) that is able to measure protein-protein interactions in living bacterial cells. We showed that this assay is able to measure the loss of interactions between proteins due to the action of antibiotics. The assay is useful because it can assay simultaneously whether the antibiotic is able to pass the bacterial envelope and which proteins complex it targets. We also showed that the loss of activity of the proteins complex involved in cell elongation, results in a dissociation of the complex. This FRET assay will be very useful and cost effective for the screening of new antibiotics and could be applied by anybody for any protein interaction that could be inhibitable. The new antibiotics that inhibit FtsZ can be used as leads for further optimization of the antibiotics towards clinical application. Work at UvA has also established the interaction surface between FtsZ and ZapA and between FtsB, FtsL and FtsQ. These proteins are part of the cell division machinery and knowledge on their interaction surfaces provides information that assists drug design and computer assisted drug screening. Using classical in vitro assays 6 new inhibitors of FtsZ polymerization in vitro and that have a reasonable EC50 in vivo have been identified in the VICHEM collection.
In the DIVINOCELL project VICHEM designed and obtained a new class of antimicrobial compounds to block bacterial division. Inhibitors directed towards bacterial division targets, which are not present in eukaryotic cells, like FtsZ, can be both effective and harmless to humans. We gained hits from a compound collection with enzymatic assays and selected the most promising hits as lead compounds to perform lead optimization - such as selectivity analysis - as a prerequisite for drug candidate identification.
VICHEM established a compound collection with more than 1700 compounds which were selected from the Nested Chemical Library™and adapted a GDP-based FtsZ screening assay which was used to find structures which bound to the protein and inhibit its function. With this assay we found two hit molecules. Inhibitory effect of these structures was in the same range as the published FtsZ inhibitors. Based on the structure of these inhibitors a new collection of 197 compounds were selected for further investigation, such as bactericide tests. 74 out of these molecules had effect in the first round. During the validation process some of them had promising but not convincing effect in different bacterial tests, and some of them were validated in FtsZ polymerization assays. The later group of compounds was effective in further bacterial assays, as well. A small library was synthesized around these compounds by VICHEM. To minimise the low solubility problem that lead to compound precipitation in some assays, the water solubility of the compounds was increased by preparing the salt forms. Selectivity for bacterial targets was investigated with determination of effect on eukaryotic counterparts. Selective compounds, which are harmless to humans, from the group of effective ones were chosen as potential antibiotic candidates. These results were done in cooperation with CSIC, UNEW and UvA.
The DIVINOCELL project identified promising structures as potential inhibitors that block the proliferation of pathogens. Although these compounds will require additional in vitro investigations and chemical development to develop them into usable drugs, VICHEM results provided drug candidates that are new potential inhibitors to attack infection. The project resulted in compounds that could step into the next phase of drug development process [medicinal properties, e.g. ADMET analysis (to describe the fate of substances in a living organism) and optimization, in vivo tests], and provided early lead compounds. This process demands further chemical development. During the project we acquired chemical experience in preparation in this field of chemistry and explored the chemical behaviour of the structures that possess activity against essential bacterial targets.
The in vivo interaction between divisome proteins FtsQ and FtsB and FtsL, studied by VU, is a potential target for novel antimicrobials. For directed design of compounds targeting this interaction detailed knowledge at the molecular level is needed. To this end an assay for in vivo photo cross-linking has been tested and optimized. By thus probing the surface of FtsQ for contact with the other two proteins an interaction hotspot for FtsB and an interaction region for both FtsB and FtsL were identified. The identification of contact sites in the FtsQBL complex will guide future development of interaction inhibitors that block cell division. Moreover, the region of the interaction hotspot is highly conserved among the class of gammaproteobacteria. An antibiotic targeting this site can therefore be expected to be effective against a range of pathogenic Gram-negative bacteria. These findings have been published in the Journal of Biological Chemistry (11 July 2013, 288 [34], 24340-24350).
Potential Impact:
Socio-economic impact, societal implications, dissemination and exploitation.
Using the divisome and the septum, both essential for bacterial survival, as inhibitable targets, DIVINOCELL has identified eight new antimicrobial compounds that block the proliferation of Gram-negative pathogens. In addition the project has generated new technology to discover antibiotics and to help to improve the properties of the already discovered hits.
Compound GCI-512, identified by EVOLVA, will be a welcomed addition to obtain antibiotics towards serious bacterial threats such as those of the Burkholderia genus. Burkholderia-induced melioidosis is feared as a biological warfare agent and as these bacteria are resistant towards most antibiotics an efficient drug could be highly interesting. It should also be kept in mind that certain Burkholderia species are used in agriculture (e.g. for biocontrol) and so it may be feared that the future could bring problems with e.g. hospital-acquired infections with these bacteria. Filing of IP on this compound is possible, allowing then DIVINOCELL partners other than EVOLVA to continue working with it. The compound will be synthesized now in larger amounts to be tested in a range of divisome and general antibacterials assays before a hit molecule for pre-clinical development proper is in hand. The three chemical entities selected by BIOMOL as having an antibacterial effect, BIZ2013-1, BIZ2013-2 and BIZ2013-3, have been adopted into a hit to lead program for development of new drugs (ENABLE) integrated into the IMI initiative. The collaborative work of CSIC, UNEW and UvA within DIVINOCELL identified at least three promising compounds in the VICHEM collection, VCC599460:13, VCC342365:01 and VCC404021:01, as being potential inhibitors to block the proliferation of pathogens. These compounds are now ready to step into the next phase of drug development process. To advance them into valuable compounds for clinical use additional research is required to obtain early lead compounds. This includes the improvement of their medicinal properties, e.g. ADMET analysis (to describe the fate of substances in a living organism) and optimization, and in vivo tests. This process demands further chemical development. During the project the VICHEM staff acquired valuable experience in the production of these compounds and explored the chemical behaviour of the structures that possess activity against targets in the divisome.
Demuris is currently collaborating with Cubist Pharmaceuticals (USA), a bio-pharmaceutical company who focuses on research, commercialisation and creation of pharmaceutical products that address unmet medical needs in the acute care environment, who are providing a service of screening and compound characterisation and will take promising leads emerging from the project, as compound DEM30543, forward for pre-clinical testing. Throughout the project the company has undertaken a process of frequent internal IP searches to ensure that there is strong protection for all of the drug candidates discovered within the 4.5 year period. In keeping with industry standard practice Demuris has evaluated all of its inventions, such as compound structures and their analogues, targets and assays and continues to review its IP assets. Demuris has been engaging with the biotech and pharmaceutical market to establish current trends for the timing of licensing deals for antibiotic related products. Several important collaborations both academically (with experts in divisome structure and function) and commercially (with various chemistry and drug development companies) have derived from the Demuris participation. These collaborations and partnerships have led to the sharing of scientific knowledge and expertise including a few in related fields as agrochemicals, cosmeceuticals and cancer research. These collaborations will produce further grant funding and commercial deals.
The general social and economic benefits of developing new antibiotics are the lower rates of nosocomial infections, shorter time in hospital stays, lower number of readmissions and mortality, and increased confidence in healthcare systems. Benefits to society include increased productivity, life expectancy and lower costs of public health services. Infectious diseases count as the second leading cause of death worldwide and “even now, in the antibiotic era, common infectious diseases are major contributors to morbidity and mortality world-wide, especially in the developing world, but also in the developed world (World Health Organization, 1996). In developing countries infectious diseases cause over 60% of total deaths, many of them caused by bacterial pathogens. They are the third leading cause of death in Europe, mostly in elderly and debilitated populations, and, despite existing antibiotic therapies and vaccines, they remain the leading cause of mortality and morbidity worldwide. A similar question can be raised concerning those patients that, for diverse medical reasons, are immunocompromised. Their numbers, as medical procedures are technically being perfected, are likely to rise creating another segment of the population with an increased risk of succumbing to infections. In developed countries, nosocomial infections, occurring in 5-7% of patients hospitalised for other reasons, increase the hospital stay by an average of four days with an increased cost per day of nearly 500 €. If patients are in an Intensive Care Unit both the risk and the cost are more than double; their additional stay can extend up to fifteen days and have a concomitant higher mortality rate, often associated with antibacterial therapeutic failure” (Vicente et al., 2006. FEMS Microbiol. Rev. 30: 841-852). Among the pathogens, Gram-negatives are more resistant to commonly used antibiotics because of their complex envelope and at the same time they cause many serious community and hospital acquired infections.
Although there are over 100 antibiotics commercially available, bacterial infections still represent an area of acute unmet medical need as a result of the continuous development of bacterial resistance, increased global longevity and travel, and side effects from existing products. The number of antibiotic resistant pathogens is rising and some microbes are now resistant to most of the antibiotics currently used to treat infections. According to the US Centers for Disease Control, up to 70% of hospital-acquired bacterial infections are resistant to at least one antibiotic, and up to 40% are resistant to three. At the same time “the efforts in new antimicrobial development have focused largely on the modification and improvement of agents already discovered, while research on new classes of antibiotics has lagged behind” (Álvarez and Vicente, 2007. Expert Opin. Ther. Patents. 17: 667-674, and corrigendum). Hardly any new antibiotic has been introduced during the last decade to treat infections, and little more than 20 products are in the late stages of development, according to EP Vantage. The British Society for Antimicrobial Chemotherapy Initiative notes that ‘the pipeline is at its lowest ebb since the 1940s when penicillin was discovered’. There is therefore urgent need for novel antibacterial therapies. There is good evidence that new classes of antibiotic can capture a significant market share.
Besides the primary societal impact that the newly discovered antimicrobials will have on public health and individual well-being, DIVINOCELL participants have published over 63 papers in top ranking scientific journals, have defended 4 doctoral thesis and have participated in over 80 dissemination activities directed to the general public. A brief summary of some of them is below:
CSIC DIVINOCELL researchers organised a scientific meeting “Reconstructing the essential bacterial cell cycle machinery” within the prestigious EMBO workshops in 2012 at La Granja de San Ildefonso, Spain. The workshop (http://events.embo.org/12-bacteria-cell-cycle/index.html) discussed advances on the construction of essential bacterial machineries in the absence of cells and offered a three day conference programme in which a total of 90 leading world scientists in the field of bacterial division and young scientists participated. The Coordinator of DIVINOCELL, being the main organiser of the workshop, has also edited a special issue of Environmental Microbiology now in press.
Prof. Octavio Monasterio, from UCHILE, appeared in the CNN Chile program "Cápsula científica" where he talked about function and structure of the proteins, and his investigations in the project (http://www.youtube.com/watch?v=LO-dDlwHeXA#t=13).
VICHEM participated at the Open Information Day on FP7 Heath Research. Brussels, 9 June 2011 (http://webcast.ec.europa.eu/eutv/portal/res/_v_fl_300_es/player/index_player.html?id=12102&pId=12082#)
A summary of the DIVINOCELL project was presented on October 1, 2010 by Tanneke den Blaauwen (UvA) at the European Inter-network meeting on antibiotic resistance. Representatives of the Aeropath, AntiPathoGN, Divinocell, EU-INTAFAR, Nabativi, PAR, Pneumopath and UC BACWAN were present. The meeting was organized by the Centre of Protein Engineering and the Doctoral School of Sciences (EDT: Structure and Function of Biological Macromolecules, Modelling and Bioinformatics) of the university of Liege and was held in the Chateau de Harze in Belgium.
Piotr Szwedziak (from Jan Lowe's team, at MRC-LMB), was awarded the prize sponsored by Molecular Microbiology for his poster "Biochemical and structural studies of the FtsZ/FtsA complex" presented at the 2010 Gordon Conference on Bacterial Cell Surfaces (New London, NH, USA. June 27- July 2, 2010). These results were obtained within the framework of DIVINOCELL.
Finally, DIVINOCELL has trained a sizable number of next generation European microbiologists, from student to Postdoctoral level, in the area of bacterial cell wall synthesis, cell division, antibiotic inhibition of these processes and assay development. The project has also helped to strengthen scientific collaboration between the participants and to generate future new projects to implement it.
List of Websites:
Project public website.
http://www.cnb.csic.es/~divinocell/
Contact information.
Scientific coordinator:
Miguel Vicente
Centro Nacional de Biotecnología
C/ Darwin nº 3, 28049, Madrid, Spain
mvicente@cnb.csic.es
http://www.cnb.csic.es/~mvicente/
Project Manager:
Moira Torrent
Centro Nacional de Biotecnología
C/ Darwin nº 3, 28049 Madrid, Spain
mtorrent@cnb.csic.es
CSIC institutional contact:
José Ramón Naranjo
C/ Serrano 11, 28006 Madrid, Spain
sgaae@csic.es
http://www.csic.es/web/guest/home