DIVINOCELL

DIVINOCELL Second Periodic Report

2012-09-18

Second Report Publishable summary.

DIVINOCELL summary and objectives.

DIVINOCELL aims at identifying antimicrobial compounds 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 this complexity these bacteria are less susceptible to antibiotics than those that do not contain a second membrane covering the rigid support layer. The antimicrobials that we seek are those that will inhibit bacterial proliferation by blocking already known and novel targets in the process of cell division and envelop synthesis.

The strategy of DIVINOCELL is both to apply the existing knowledge on the molecular biology of the cell division machinery in Gram-negatives and to generate new knowledge on this process to discover new targets. Application of this background to the screening of selected compound collections will yield a set of hits active in preventing bacterial proliferation. Hits will then be validated and improved to convert them into lead compounds that will serve to formulate new antibiotics. The strategy is complemented by the development of new tools that allow to apply the
structure of the known and novel targets and their interactions for screening, validation and improvement of inhibitors in the test tube.

Cell division is an essential process with excellent properties to yield new inhibitors to attack infection by blocking the proliferation of pathogens. 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 inhibitors will be based on the structures of synthetic scaffolds, not frequently present in Nature, they will be less likely than the present antibiotics to favour the acquisition of resistances by the pathogens.

We use Escherichia coli as a model bacterium 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. Proliferation of E. coli involves a process, called division, in which several proteins assemble at midcell to form a division ring. In the absence of any of them, division is blocked and the bacterium does not divide and eventually dies. Among these proteins we focus our research on those called FtsA, FtsB, FtsL FtsQ, FtsW, FtsZ and ZipA. In addition we study proteins that synthesize peptidoglycan, the rigid layer of the envelope. Among them FtsI that is specific for the synthesis of this layer within the division ring and belongs to a set of dedicated enzymes, collectively designated as penicillin-binding proteins because they can bind to a specific class of antibiotics like penicillin called betalactams. FtsI is also designated as PBP3 to indicate that it is a penicillin-binding protein.

The assembly of all these proteins forms a division ring structure, also called the divisome, and the result of the action of this divisome is the production of the septum that separates the cell into two equal daughters that reinitiate the growth and division cycle independently from each other. All these proteins are needed for bacterial division and if they are absent or inactivated bacteria cannot proliferate.

Description of the work performed by DIVINOCELL since the beginning of the project.

TARGET DISCOVERY, ASSAY DESIGN.

DIVISOME. Novel inhibitable targets have been discovered in the interactions between divisome proteins, for example FtsZ and ZipA, FtsQ with either FtsB or FtsL, and FtsA with itself. The interaction between FtsZ and FtsA was tested in vitro using vesicles made from the natural E. coli membrane. We have progressed in defining these targets using in silico procedures to yield chemical scaffolds that fit into the FtsZ active site. Chemical synthesis has provided analogs of DAPI a molecule able to bind to DNA and also to FtsZ. Soluble and potent DAPI derivatives have been tested as inhibitors.

SEPTUM. MtgA, PBP3 and PBP1B, are proteins that work to synthesise peptidoglycan. The wild type enzyme and variants with a single activity are available. We have obtained specific substrates to quantify their activities on peptidoglycan synthesis. Particularly important are the small-sized precursors, lipid II and UDP-muramyl pentapeptide. We have purified FtsW, a protein that transfers these precursors to the periplasm, the space in between the two membranes where peptidoglycan is produced. Novel targets in FtsW will be revealed upon its crystallization and elucidation of its structure. Apart from the divisome proteins, MreB is an essential protein that functions in shifting the direction of peptidoglycan growth. From our results we know that MreB is needed in the initiation of cell division and can be used as a target.

SCREENING

Screening. The industrial participant Evolva has found compounds that may be able to 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. These compounds show inhibitory effects on bacterial survival at concentrations that make them suitable hits to merit further development. The screening performed by Demuris has identified one broad spectrum compound with desirable antimicrobial activity
properties, working at sufficiently low concentrations, to make it a good candidate for lead development. The production of this natural compound, obtained from a Streptomyces strain, is being scaled to obtain sufficient amounts for testing. Screening of a selected collection of 197 compounds, belonging to a collection of kinase inhibitors supplied by Vichem, has been performed using three different growth assays. A few show a positive result in the three assays and their properties are being investigated.

Cell and secondary assays. Protocols to assist in the process of lead development that are based on strains depleted from FtsZ or MreB have been devised. They have been applied to assay the DAPI analogs described above. Positive assays, those in which the inhibition of division results in the avoidance of cell lysis, have been used to screen the Vichem collection.
Assays to detect protein - protein interactions using a technique known as FRET have been developed. They detect the interaction between two fluorescently labelled proteins. Our FRET system even detects interactions between the peptidoglycan synthesizing enzymes that work in the periplasm.

NEW TECHNOLOGY

We have developed new procedures to use the divisome proteins in convenient assays, among them vesicles that can mimic the cell interior. 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. Together with Molecular Dynamics to model chemical activities in silico, these technologies open new options to study microbial proliferation and to discover antibiotics to cure infection.

Expected final results, potential impact and use.
Using the divisome and the septum, both essential for bacterial survival, as inhibitable targets, the project has identified new compounds that block the proliferation of Gram-negative pathogens. Besides further investigating these compounds, other collections of possible inhibitors are being screened. In addition new simplified and more potent assay tools have been constructed to facilitate the progress from these initial hits into leads that can be improved to obtain new antibiotics. 

The number of antibiotic resistant pathogens is rising and some microbes are now resistant to most of the antibiotics currently used to treat infections. Among the pathogens, Gram-negatives are particularly dangerous because they have a complex envelope, making them more resistant to commonly used antibiotics. These bacteria are responsible for many serious community and hospital acquired infections. Division produces a septum that separates the two daughter cells by first assembling at midcell a complex ring of proteins called the divisome. Microbes are not able to proliferate when any of the divisome proteins is absent or inactive. The division of Gram-negatives has some peculiarities. Among them, three divisome proteins, FtsZ, FtsA and ZipA have to assemble together in a proto-ring. We have used specific properties as this one to discover the new compounds. In addition, as some of them derive from artificial scaffolds they are less likely to induce an immediate resistance mechanism, as pathogens may have never evolved in their presence.

To put our research in proper focus it can be stressed, as it is described elsewhere, that “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). This means that hardly any new antibiotic has been introduced during the last decade to treat infections. On the other hand, “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 to 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).

DIVINOCELL First Periodic Report

2011-01-21

DIVINOCELL, HEALTH-F3-2009-223431 First periodic report.
Executive overview (publishable)

DIVINOCELL objectives

DIVINOCELL aims at identifying novel targets and antimicrobial compounds active in Gram-negative bacteria by exploiting the components of the divisome, their activities and interactions. It includes both the design of selective assays for screening, cell based secondary assays and the selection of a new class of antimicrobials to block bacterial division.


DIVINOCELL is applying existing and new knowledge on the molecular biology of Gram-negative cell division and developing novel tools to exploit in the test tube the structures and interactions of targets in the divisome and the septum. They include the use of analytical (nanodiscs), bioinformatic (molecular dynamics and docking), structural (membrane protein crystals) and imaging (lanthanide staining) techniques. DIVINOCELL will then apply potent systematic screening assays to select inhibitors of the division of Gram-negatives (not precluding broad spectrum ones). Secondary activity and cell assays, based on the properties of bacterial division, are being generated to validate hits and advance them to leads. Finally the medicinal properties of selected leads will be improved.
Cell division is an essential and still underexploited process with excellent properties to yield new inhibitors to attack infection by blocking the proliferation of pathogens. Inhibitors directed against bacterial division targets, that are not present in eukaryotic cells, will be both effective and innocuous to humans and animals. In addition, as many of their structures will be based on interaction domains and synthetic scaffolds, they will generate resistance at levels lower than the present antibiotics.

Description of the work performed by DIVINOCELL during the first reporting period.

TARGET DISCOVERY AND ASSAY DESIGN

DIVISOME targets. Cell division is initiated by the assembly of the divisome, a multiprotein ring at midcell. The DIVINOCELL results have yielded a detailed description of the active site of the essential FtsZ protein, one of the ring proteins and a highly promising target to attack Gram-negatives. They show important details on the activity and polymerization properties of the protein, among them the most crucial one is the strict requirement for potassium and the detrimental effects of sodium on GTP hydrolysis. This is helping to formulate adequate conditions for the screening assays to identify inhibitors. Even if the three dimensional structure of the protein from Escherichia coli (the most widely used Gram negative model organism) is not yet available, in silico procedures have advanced a valuable deliverable on the chemical scaffolds tailored to fit into the active site that can be used to generate inhibitors. Chemical synthesis has provided a few compounds, analogs to the DAPI molecule, that retain the ability to bind to the FtsZ protein. One of them shows the desired property of binding more efficiently to FtsZ than to the eukaryotic equivalent Tubulin, and is therefore a promising new potential division inhibitor.
The E. coli proto-ring components (the essential proteins FtsZ, FtsA and ZipA) have been purified, tagged with suitable fluorescent dyes and used to probe their interactions. Important new hotspots have been identified in the ZipA protein that help to determine its interaction map with FtsZ. In addition a strict dependence on the chemical composition of the phospholipids for the functional integration of ZipA into lipid bilayers has been evidenced. FtsA modifies the hydrolytic activity of FtsZ, while on the other hand its location in vesicles obtained from natural E. coli membranes is sensitive to the polymerization of FtsZ. The interaction between molecules of FtsA is esential for division in E. coli. To help in the design of protein-protein interaction assays the FtsA protein has been also obtained from Streptococcus pneumoniae, a Gram positive microbe, as it is more amenable to biochemical analysis than its E. coli counterpart. Variants of this streptococcal protein are helping to determine the regions of the protein involved in interactions and polymerization.
FtsQ, a protein assembling into the division ring at a later stage than the proto-ring elements, is being probed by in vivo photo crosslinking assays to determine its interaction partners and map the interacting regions. Compounds that may interfere with the interactions with FtsB and FtsL, two other elements that assemble into the division ring together with FtsQ have been identified in a pharmacophore screen and tested for the inhibition of cell division. Although a few of them inhibit bacterial growth, none seems to be a specific inhibitor of septation.

SEPTUM targets. Assembly of proteins at a division ring has the effect of constricting the membrane and producing a cell wall septum. The synthesis of a rigid peptidoglycan septum able to withstand the turgor pressure of the highly crowded bacterial cytoplasm involves a set of dedicated enzymes, collectively designated as penicillin-binding proteins due to their ability to bind betalactam antibiotics. DIVINOCELL has achieved the purification of several of these enzymes together with a good number of substrates to assist in the set up of screening assays. Among the proteins, PBP3, a bifunctional protein showing transglycosylase and transpeptidase activities, is available both as the wild type enzyme and as a variant with no transpeptidase activity. Another enzyme, PBP1B, is available as a bifunctional protein and also as the two variants containing one single activity. Purified monofunctional glycosyltransferase MtgA has also been obtained. In addition to some commercially available compounds, DIVINOCELL has obtained some highly specific substrates to assist in measuring peptidoglycan synthesis. They include sacculi, as the largest available structure, fragmented peptidoglycan and smaller-sized precursors as lipid II and UDP-muramyl pentapeptide. An important step in the synthesis of peptidoglycan is the transfer of cytoplasmic precursors to the periplasm. This is commonly accepted to involve the activity of dedicated proteins as FtsW, another division ring protein essential for division. DIVINOCELL has succeeded in obtaining sufficient amounts of purified FtsW from the Gram-negative Chlorobium tepidum that will be used to obtain structural data on this protein to initiate its exploitation as a new potential inhibitable target. The direction of peptidoglycan synthesis has been described to shift from lateral elongation during growth, to septal synthesis at division. DIVINOCELL has obtained evidence showing that MreB, together with specific division ring proteins, is involved in this shift and has developed procedures to identify the interacting partners by colocalization.

SCREENING, HIT VALIDATION AND LEAD SELECTION

Cell and secondary assays. Once a hit compound able to inhibit bacterial proliferation has been identified, its chemical properties need to be optimised to convert it into a lead or, furthermore into a candidate compound that may be tested for its antimicrobial efficiency prior to its consideration for preclinical and clinical trials. Assays to report the cessation of division in intact cells are required to validate hits in whole cells. Valuable help at this stage is also contributed by the design of assays to test the antimicrobial properties using as little as possible amounts of the hit compounds. DIVINOCELL is exploiting observations on the differential gene expression levels found in cells undergoing cell division arrest by deprivation of division ring proteins, namely FtsZ. These results are used to construct plasmids reporting the absence of division as a fluorescent signal and to tailor strains sensitive to lower antimicrobial concentrations. Impairment of the expression of other genes involved in global regulation by means of sRNA overproduction to block translation is also exploited in the partnership.
Positive assays are also of a great help for screening as they can be used advantageously to detect inhibitors. DIVINOCELL has developed strains that upon inhibition of septation can withstand lysis induced by vancomycin, an antibiotic that leads to the synthesis of a weaker peptidoglycan. Furthermore, it has been found that other compounds that modify the cell wall can be used as alternatives to vancomycin. The full set of reaction conditions has been optimized for these assays.
In vivo detection of the interaction between division ring components using the FRET signal generated by two proteins fused to a suitable donor-aceptor fluorescent pair is a valuable procedure to validate the action of interaction inhibitors. A suitable pair of fluorescent proteins, mCherry and mKO, able to detect sufficiently low levels of protein as those found inside the intact wild type bacteria was tested using them to obtain fusions to penicillin-binding proteins involved in the biosynthesis of the sacculus and to evidence their interaction with other morphogenetic proteins. Work is progressing to circumvent the need for fixation required to avoid alterations during the long period needed for the development of the signal generated by mKO. Results already available indicate that one of the fluorescent protein alternatives tested may perform as required for in vivo assays.

Screening. During the first reporting period DIVINOCELL has initiated the set up of screening activities. Virtual filtering of FtsZ inhibitors based on the architecture of the GTP binding site has produced a collection of chemical structures in which the 50 compounds having a higher interaction potential have been determined. By adjusting the stringency of the search, results can be extended to include up to 1000 compounds showing weaker interactions.
Initial progress in wet screening has performed a first test running a collection of extracts obtained from actinomycete bacteria through an assay reporting the activity of inhibitors of the cell wall synthesis. This approach has already yielded nearly 20 compounds active on E. coli. An assay suitable to detect inhibitors of FtsZ activity in a high throughput facility has been optimized and an initial test has yielded a restricted collection of over 150 compounds that are being rescreened under more stringent conditions. Finally, the implementation of interaction assays has proceeded by constructing the elements required to implement reverse yeast two hybrid assays to test protein-protein interactions.

TECHNOLOGY TO DESIGN ASSAYS AND IMPROVE LEADS

An important task of DIVINOCELL is the generation of new technologies to supply advanced tools to perform assays to select cell division inhibitors. Developments already implemented include the integration of the ZipA protein as a single molecule in nanodiscs made with E. coli lipids restrained by a modified ApoA protein. These nanodiscs are freely soluble and their ZipA protein is able to interact with FtsZ polymers formed in the presence of GTP proving that nanodiscs are a convenient system to study the interaction of membrane bound proteins with their partners. Additionally giant vesicles produced from natural E. coli membranes have been obtained and have been used to observe the localization of FtsZ and FtsA, both in isolation or together, in the presence or absence of GTP. Additional experiments conducted on FtsZ and variants in which the lateral interaction between monomers have been abolished are supplying relevant data to refine the description of polymer formation. The  mutations affect the ability of FtsZ filaments to reorganize on a lipid surface and modify the shape of the aggregates. These results will be used to refine the assay conditions for this relevant target.
Molecular Dynamics simulations have been successfully used, as mentioned above, to describe in detail the FtsZ activity and are being extended to the analysis of interactions involving other proto-ring components.
Progress in the visualization of the E. coli division ring inside the cell have been obtained by using cryotomography and 3D reconstruction of electron microscopy images. The technology is being validated for its use in detecting alterations introduced by new inhibitors by using images from cells in which septation has been inhibited genetically or by well known inhibitors of peptidoglycan synthesis.

Expected final results, potential impact and use.

DIVINOCELL aims at finding inhibitors to block a function, cell division, that has the advantage of being essential and present in the vast majority of pathogens. The assembly of the divisome has peculiarities in Gram-negatives, such as the concerted interactions in the assembly of the FtsZ, ZipA and FtsA components of the proto-ring. These properties are being exploited to discover new antimicrobials to combat this class of bacteria which are responsible for many serious community and hospital acquired infections. The need to find new drugs to block their proliferation is more pressing given the alarming rise in the frequency of antibiotic resistant strains, with some of them being resistant to most of the antibiotics currently used.

As beneficiaries in DIVINOCELL have described elsewhere (Álvarez and Vicente, 2007. Expert Opin. Ther. Patents. 17: 667-674, and corrigendum) “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. For example, the recent releases of dalbavancin (a semisynthetic glycopeptide), telavancin (a lipoglycopeptide), ceftobiprole (a new β- lactam derived from cephalosporin), doripenem (a carbapenem β- lactam antibiotic) and tigecycline (similar to tetracycline) are based on derivatives of already known antibiotics. Only daptomycin (which binds to the membrane, causing rapid depolarisation and whose clinical development to circumvent its pharmacologically related toxicity was resumed after 20 years) released to the market with a new dosing schedule, linezolid (an oxazolidinone that prevents the formation of the translation initiation complex) and retapamulin (also acting on the 50S ribosomal subunit) have been described in the last decade as genuine new agents.”

DIVINOCELL will contribute to remedy this situation by using the divisome and the septum, two well-known and, surprisingly, underexploited structures, essential for bacterial survival, as the targets to be inhibited by a totally new class of anti-infectives. Some of these compounds, being derived from artificial scaffolds are less likely to induce an immediate resistance mechanism, as pathogens may have never evolved in their presence.

Quoting from another of DIVINOCELL publications (Vicente et al., 2006. FEMS Microbiol. Rev. 30: 841-852) “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”.

New medicines to attack Gram-negative pathogens will decrease the burden of infectiosus disease and have a highly beneficial social and economic impact in Europe and beyond. The translational steps of the project will be developed by 4 companies in close collaboration with the 7 academic partners having well-proven expertise in molecular microbiology, protein chemistry, structural biology, biophysics, imaging and bioinformatics.