INHIBITING FtsZ PROTEIN (DE)POLYMERISATION

Abstract: The invention relates to substances with antibiotic activity and methods to treat infections. The invention provides a method to prevent or hamper division or proliferation of a prokaryotic cell or organism comprising inhibiting polymerisation and/or depolymerisation of cell-division protein FtsZ or analogue thereof by contacting said protein with a guanoside triphosphate (GTP) analogue capable of binding to said protein.

Description: Title Inhibiting FtsZ protein (de)polymerlsatιon The invention relates to substances with antibiotic activity and methods to treat infections Antibiotics are organic chemicals w hich, usually at very low concentrations, either kill or inhibit the growth of a micro-organism Originally derived as wholly natural products from secretions of fungi or bacteria (Pemcillium, Streptomyces spp , etc), most antibiotics today are at least in part chemically synthesised To be clinically useful, antibiotics have to exhibit antimicrobial activity, for example by inhibiting division or proliferation of micro-organisms, in concentrations which have no or minimal side effects to the treated host infected with said micro-organism Important antibiotics can be classified into the following groups Beta lactams (penicillin) and beta-lactamase resistant penicillins, Cephalosporins Fusidic acid, Aminoglycosides, Tetracyclines, Chloramphenicol, Rifamycins, Metronidazole, Macrohdes, Glycopeptides, Trimethopπm and sulphonamides, Qumolones, Nitrofurantoin, Mupirocin, Antimycobacterials, and Antifungals Microbes have developed resistance against practically all these different groups of antimicrobial agents The growing incidence of antibiotic resistance in bacterial pathogens represents a major clinical and epidemiological problem [e g increasing multidrug resistance in staphvlococci (MRSA) and Mycobacterium tuberculosis In the past, new families of antibacterial antibiotics were mainly discovered by random screening of collections of microbes isolated from the natural environment and of libraries of chemicals For some time this approach allowed us to stay 'one step ahead' of the evolution of clinical drug lesistance However more recently concentration of efforts on the modification of existing classes of antibiotics in conjunction with a decreasing rate of discoven of new antibiotics has led to a diminution in options for treatment of infections with certain microbes (see http //www socgenmicrobiol org uk/news/sgmhol htm) A solution for the resistance problem would be the identification of novel targets in pathogens Such targets might allow the development of drugs to which microbes are unlikely to have any natural intrinsic resistance, thereby possibly avoiding, or slowing down, the emergence of clinical resistance Among the novel targets being considered are for example bacterial proteins involved in cell division in prokaryotes A particular protein class that was recently defined as potential target is the class of FtsZ proteins and analogues (Desai and Mitchison, BioEssays 20 523-527, 1998) FtsZ is a cell division protein conserved in prokaryotic organisms such as Eubacteria, Archaebacteria, Mycoplasmas, and chloroplasts It is a guanimne-nucleotide binding protein that is thought to be a prokaryotic relative of tubuhn, a protein involved in microtubuli and cytoskeleton formation in eukaryotic cells Although sequence homology between FtsZ and tubuhn is weak, recent studies have shown that FtsZ can polymerise in a GTP-regulated manner into structures similar to those formed after tubu n polymerisation During cell-division prokaryotes, FtsZ localises to a constricting ring structure at the division site FtsZ, thus being a major cell division in protein in prokaryotes could be an excellent target for new antibacterial drugs However, no specific inhibitors of FtsZ polymerisation are known In general, inhibition of polymerisation of FtsZ in vitro maybe considered possible by using various well known broadly acting tubulm inhibitors and analogues, as can be expected due to the structural and functional similarities between the two proteins However, using such inhibitors for the clinical treatment of infections m vivo would at least have detrimental side effects, and potentially be dangerous or lethal to the treated host, said host relying on undisturbed tubu n function The invention provides a method to prevent or hamper division or proliferation of a prokaryotic cell or organism comprising inhibiting polymerisation and/or depolymerisation of cell-division protein FtsZ or analogue thereof by contacting said protein with, preferably hydrolysable a guanoside tπphosphate (GTP) analogue, such as derivable from guanosine or another nucleotide known in the art, capable of binding to said protein In particular, the invention provides a method wherein said analogue is modified at position C2', C5' and or C8, such as for example indicated in figure 2 Modification at C2', C3' C2 and/or C8 especially provides steric bulk or electrostatic hindrance to prevent polymerisation (see fig 1) whereas modification at C5' is particularly useful to increase membrane passage of the analogue In a particular embodiment, C8 is modified with a group providing electrostatic repulsion, such as a bromine or a group of comparable size and or electrostatic properties. Hereby, the invention provides a method to hamper division of a prokaryotic cell comprising inhibiting polymerization and/or depolymerization of cell division protein FtsZ or analogue thereof by contacting said protein with a guanoside trophosphate (GTP) analogue capable of binding said protein, wherin said GTP analogue is modified at position C2', C3', and/or C2 and in particular wherein said analogue is essentially not binding to or at least essentially not hampering the function of a GTP-binding protein, such as tubuhn, from a eucaryotic cell. Though the GTP binding pocket and the mechanism of polymerization of FtsZ and for example tubuhn seem to very similar, the differences are sufficient to make an analogue that discriminates between the two molecules. For instance Br-GTP does not inhibit the polymerization of Tubuhn (Muraoka, M., Fukuzawa, H., Nishida, A., Okano, K, Tsuchihara, T , Shimoda, A , Suzuki, Y., Sato, M , Osumi, M , and Sakai, H (1999) The effects of various GTP analogues on microtubule assembly Cell Struc. Funct. 24: 101-109), whereas it inhibits the polymerization of FtsZ. For example, for modification of the C8 position a 8-Br-guanosιde or a 8-Br- Guanoside monophosphate can be used as starting points. Other likely substitutes are 8-(substιtuted)alkyl, 8-(substιtuted)aryl, 8-(substιtuted)amιno, 8- (substιtuted)hydroxy, 8-halogen, 8-carboxyhc acid and acid derivatives, 8- sulphonic acid and acid derivatives, and 8-substιtuted heterocychc systems The invention also provides a new antibiotic or pharmaceutical composition comprising such a guanoside tπphosphate (GTP) analogue capable of binding to a prokaryotic cell-division protein FtsZ or analogue thereof, said analogue essentially not capable of binding to a GTP-binding site of a GTP- binding protein from a eukaryotic cell. Serious side-effects are not to be expected when treating a eukaryotic host with said pharmaceutical. It allows treatment of a eukaryotic cell against a prokaryotic infection, with minimal or no side effects to said eukaryotic cell In one embodiment, the invention provides use of said pharmaceutical or antibiotic substance for the (preventive) treatment of a eukaryotic cell culture possibly infected with a prokaryotic organism. Such cell cultures often are infected with bacteria, Infections that now can be treated by a pharmaceutical according to the invention In another preferred embodiment, the invention provides use of said pharmaceutical or antibiotic substance for the (preventive) treatment of a eukaryotic organism or cell therein possibly infected with a prokaryotic organism In particular animals, varying from humans receiving or taking medical care, to animals under veterinary care, such as domestic animals (e g food animals or small animals) are preferably treated with a pharmaceutical according to the invention Bacterial infections can now be treated with a new antibiotic substance to which no resistance has been developed, and likely will not or only at a minor scale will develop since the necessary mutations in the FtsZ protein would render it inactive as well, thereby limiting the chances that bacteria rapidly become resistent The invention is further explained in the detailed description without limiting the invention thereto Detailed description Function and structure of FtsZ FtsZ is a highly conserved protein that appears to be present in all prokaryotes, where it has an essential role in cell division (Lutkenhaus, J 1993 FtsZ ring in bacterial cytokinesis Mol Microbiol 9 403-409 ) It assembles at midcell into a cytoskeletal structure designated the FtsZ ring (Z ring) that directs the process of septation (Addinal, S G , and J Lutkenhaus 1996 FtsZ- spirals and -arcs determine the shape of the invaginatmg septa in some mutants of Eschenchia coh (Mol Microbiol 22 231-237 Bi, E and J Lutkenhaus 1991 FtsZ ring structure associated ) It was suggested that the Z ring formed through the polymerization of FtsZ, and all subsequent work supports this hypothesis (Lutkenhaus, J 1993 FtsZ ring in bacterial cytokinesis Mol Microbiol 9 403-409 ) One activity of this ring is to recruit other division proteins to the division site where they participate m formation of the septum (Lutkenhaus J 1993 FtsZ ring in bacterial cytokinesis Mol Microbiol 9 403 409 ) FtsZ has limited sequence identity to eukaryotic tubulins, suggesting that it is an ancestral homologue (Mukherjee, A , and J Lutkenhaus 1994 Guanine nucleotide-dependent assembly of FtsZ into filaments J Bacteriol 176 2754- 2758 ) The recent solution of the structures of FtsZ and tubuhn support this conclusion, as the structures are remarkably similar (Lowe, J , and L A Amos 1998 crystal structure of the bacterial cell-division protein FtsZ Nature 391:203-206; Nogales, E., S. G. Wolf, and K. H. Downing. 1998 Structure of the alphabeta tubulin dimer by electron crystallography. Nature 391:199-203.). In addition to these sequence and structural similarities there is functional similarity, as both tubulin and FtsZ are GTPases that can polymerize into higher-order structures. Like tubulin's GTPase, FtsZ's GTPase activity increases dramatically with the protein concentration (Davies, D. R., E. A. Padlan, and S. Sheriff. 1990. Antibody-antigencomplexes. Annu. Rev. Biochem. 59:439-473; 54. Lu, C, J. Strieker, and H. P. Erickson. 1998. FtsZ from escherichia coli, Azotobacter vinelandii, and Thermotoga maritima-quantitation, GTP; (Wang, X., and J. Lutkenhaus. 1993.) The FtsZ protein of Bacillus subtilis is localized at the division site and has GTPase activity that is dependent upon FtsZ concentration. (Mol. Microbiol. 9:435-442., Wang, X., and J. Lutkenhaus. 1996. FtsZ ring: the eubacterial division apparatus conserved in archaebacteria. Mol Microbiol 21:313-319.). Both tubuline as well as FtsZ have a low basal rate of polymerisation at protein concentrations too low to support polymerization and a significantly increased rate at concentrations above the critical concentration needed for assembly. Because many inhibitors of tubuhn assembly exist, investigating the effects of tubulin inhibitors on FtsZ assembly should provide important information for FtsZ structure and function. Tubuhn inhibitors have been classified into four categories: (i) colchicine and its structural analogues, such as colcemid and podophyllotoxin; (ii) vinblastine and its analogues vincristine and maytansine; (iii) the metal ions Ca 2+ , Cu 2+ , and Hg 2+ ; and (iv) aminonaphthalenes, such as bis-ANS. Different inhibitors bind to tubulin at different sites and presumably arrest tubulin assembly by different mechanisms. It was shown that colchicine, colcemid, benomyl and vinblastine have no effect on Ca 2+ -induced FtsZ polymerization (Yu, X.-C, and W. Margolin. 1998. Inhibition of assembly of bacterial cell division protein FtsZ by the hydrophobic dye 5.5'-Bis-(8-anilino-l-naphthalenesulfonate). J. Biol. Chem. 273: 10216-10222), suggesting that FtsZ interacts with these drugs in a manner distinct from that of tubulin. However, it was also shown that another tubulin inhibitor, bis-ANS, effectively inhibits FtsZ polymerization. Because bis-ANS is a fluorescent probe that measures protein hydrophobic surface properties, bis-ANS was used and a related compound, ANS, to probe directly for FtsZ conformational changes induced by Ca 2+ binding. Titration of FtsZ with bis-ANS indicated that FtsZ has one high affinity binding site (presumably the GTP-bindmg pocket) and multiple low affinity binding sites. Although ANS binds to FtsZ, it has no effect on the polymerization reaction Tubuhn assembles into microtubules that are composed of protofilaments, head-to-tail polymers of the ab dimer (Desai, A , and T. J. Mitchison. 1997. Microtubule polymerization dynamics Annu Rev Cell Biol. 13:83-117 ). FtsZ also assembles into protofilaments (Erickson, H. P , and D. Stoffer. 1996 protofilaments and rings, two conformations of the tubuhn family conserved from bacterial FtsZ to alpha/beta and gamma tubuhn. J Cell Biol. 135 5-8., 0 Mukher ee, A., and J Lutkenhaus. 1994. Guamne nucleotide-dependent assembly of FtsZ into filaments. J. Bacteriol. 176:2754-2758), although it is not known how these protofilaments are arranged in the Z ring. FtsZ readily polymerizes in the presence of DEAE-dextran, a polycation that also promotes tubu n polymerization (Erickson, H. P , and D. Stoffer. 1996 Protofilaments and 5 rings, two conformations of the tubulin family conserved from bacterial FtsZ to alpha/beta and gamma tubuhn. J. Cell Biol. 135:5-8., Mukherjee, A., and J Lutkenhaus. 1994 Guamne nucleotide-dependent assembly of FtsZ into filaments. J Bacteriol. 176.2754-2758). In the presence of DEAE-dextran, the FtsZ protofilaments tend to bundle or form sheets. Assembly of FtsZ into bundles 0 of protofilaments at neutral pH was also observed by Bramhill and Thompson (Bramhill, D , and C M Thompson. 1994 GTP-dependent polymerization of Escherichia co FtsZ protein to form tubules Proc ) but was later reported to be due to an inadvertent drop in the pH below 6 (Erikson, H P , D W Taylor, K.A Taylor, and D Bramhill 1996 Bacterial cell division protein Ftsz assembles into 5 protohlament sheets and mimrings, structural homologs of tubulin polymers Proc Natl Acad Sci USA 93 519-523). More recently, assembly of FtsZ into bundles of protofilaments was observed in the presence of 7 to 25 mM Ca 2+ , a concentration 1, 000-fold higher than the physiological concentration (Yu, X -C , and W Margolin 1997 Ca 2+ -medιated GTP-dependent dynamic assembly of 0 bacterial cell division protein FtsZ into asters and polymer networks in vitro EMBO J 16 5455-5463 ) Although these various conditions have been used to promote FtsZ assembly, FtsZ can readily assemble in their absence (Mukherjee, A , and J Lutkenhaus 1998 Dynamic assembly of FtsZ regulated by GTP hydrolysis EMBO J 17 462-469 ) Under such conditions, FtsZ assembles into a " > mixture of single protofilaments and polymers that contain two to three protofilaments. Importantly, under these more physiological conditions the FtsZ polymers, like microtubules, are dynamic, depolymerizmg as the GTP is exhausted (Mukherjee, A., and J. Lutkenhaus. 1998. Dynamic assembly of FtsZ regulated by GTP hydrolysis. EMBO J. 17:462-469.). Thus, GTP hydrolysis plays 5 a role in regulating FtsZ polymer dynamics similar to its role in dictating microtubule dynamics. MATERIALS AND METHODS 0 See for references:(den Blaauwen, T., N Buddelmeijer, M. E. G. Aaarsman, C. M. Hameete, and N. Nanninga. 1999. Timing of the FtsZ assembly in Escherichia coh. J. Bacteriol. 181 (17): 5167-5175; Lu, C, and H. P. Erickson 1998 Purification and assembly of FtsZ. Methods Enzymol. 298-305-313., Mukherjee, A., K. Dai, and J Lutkenhaus. 1993. Escherichia coh cell division 5 protein FtsZ is a guamne nucleotide binding protein. Proc. Natl. Acad. Sci. USA 90: 1053-1057., Mukherjee, A., and J. Lutkenhaus. 1998. Dynamic assembly of FtsZ regulated by GTP hydrolysis. EMBO J. 17:462-469; Mukherjee, A , and J Lutkenhaus. 1998 Purification, assembly, and localization of FtsZ. methods Enzymol. 296-305., Mukherjee, A., and J. Lutkenhaus. 1999 Analysis of FtsZ 0 assembly by light scattering and detemmation of the role of divalent metal cations. J. Bacteriol 181.823-832.) All assays are excecuted with Escherichia coh or Bacilus subtilhs FtsZ, and can easily be adapted for FtsZ of other organisms Purification of FtsZ T Escherichia coh transformed with pRRE6 was grown overnight at 37°C in Luna broth with ampicil n (100 μg/mL) The overnight culture was diluted 100-fold into 3 L of fresh medium and grown with shaking at 37°C At OD600 of 0 3, 0 5 mM isopropyl-β-D-thiogalactopyranoside (IPTG) was added and the 30 culture grown for another 3 h. The culture was then chilled, harvested at 4 °C washed once with 20 mM T is-HCI (pH 7.9) and the pellet stored frozen at -70 °C From this step onwards, all procedures were carried out at 4°C The frozen pellet was thawed in 30 ml of 50 mM Tris-HCI (pH 7 9), 50 mM KC1, 1 mM EDTA, 10% glycerol (buffer A) and lysed by passage through a French 35 press The lysate was spun at 10 000 rpm for 10 mm to remove debris and unbroken cells. High-speed centrifugation of the 10,000 rpm supernatant was done at 38,000 rpm for 1 h in a Beckmann 50.2 Ti rotor to remove the membrane fraction and the supernatant fractionated with 30% ammonium sulfate (16.6 g/lOOml of extract). The ammonium sulfate precipitate was recovered by centrifugation at 12 000 rpm for 20 mm, resuspended in buffer A and dialyzed extensively against the same buffer. The dialyzed sample was passed through a DEAE column (bed volume, 25 ml) equilibrated with buffer, washed with 5 volumes of buffer A and eluted with 200 ml of a 50 mM up to 1 M KC1 gradient The pooled peak fractions (elutmg at 200-250 mM KC1) of the DEAE column were then loaded (a maximum of 50 mg of protein) onto a Sephacryl S-300HR (Pharmacia) column (1.6 cm x 70 cm) and eluted with buffer A. The pooled fractions were concentrated using a Centπprep 10 to 8-10 mg/mL, dialyzed against 50 mM HEPES-NaOH (pH 7.2). 0.1 mM EDTA and 10% glycerol, ahquoted and stored frozen at -70 °C The total yield of FtsZ was between 80 and 100 mg per 3 L of culture Polymerization assay in general For the standard polymerization assay, FtsZ was incubated at 30°C in 50 mM morphohne ethanesulfomc acid (MES)-NaOH (pH 6.5)- 10 mM MgCk- 50 mM KC1 (polymerization buffer) for various time periods. Polymerization was initiated by adding 1 mM GTP to the prewarmed reaction mixture. In some experiments the pH, the concentration of GTP, or some other component of the polymerization reaction was altered, such changes are indicated where relevant Pιperazme-N,N'-bιs(2-ethanesulfonιc acid) (PIPES)-NaOH (50 mM) was used for polymerization at pH 6 9, and HEPES-NaOH (50 mM) was used for polymerization at pH 7 5 and 7.9 Polymerization assay for electron microscopy For electron microscopic analysis, 1 mM GTP was added to FtsZ at 200 μg ml (5 μM) in the appropriate buffer and incubated for 10 min at 30°C A 10 μL aliquot was withdrawn from reaction mixture and placed on a carbon coated copper grid (410 mesh size) for 2 min It was then blotted dry, negative-stamed with 1% uranyl acetate and immediately blotted dry again Polymerization assay by centrifugation For assay by centrifugation, polymerization of FtsZ (200 μg ml was initiated in the appropriate buffer by adding 1 mM GTP, and the reaction was immediately centrifuged at 25°C in a Beckman TLA 100.2 rotor at 80,000 rpm for 15 min. The amount of FtsZ present in the pellet was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Coomassie Brilliant Blue stained protein bands were quantitated using a Dexstra DF-2400T scanner (Dextra Corp., Taipei, Taiwan) and Sigmascan sofware (Jandel Corp., San Rafael, CA, USA). Light scattering of FtsZ polymerization. FtsZ polymerization was measured by 90 ° angle light scattering in a Hitachi fluorescence spectrophotometer with both the excitation and emission wavelengths set at 350 nm and a slit width of 1.5 nm. FtsZ was added to a final concentration of 500 μg ml (12.5 μM) or as specified in the appropriate buffer to a fluorometer cuvette with a 1-cm path length. The cuvette was then placed in a cuvette chamber that was maintained at 30°C by a circulating water bath, and data were collected for 8 min to establish a baseline. Subsequently, the cuvette was removed and 1 mM GTP or any other nucleotide was added to achieve a final reaction volume of 300 μl. The reaction mixture was gently stirred with a pipette tip, and the cuvette was returned to the cuvette chamber for data collection for a specified period of time. The reading at time zero is the first reading taken after the cuvette was returned to the chamber following nucleotide addition. The elapsed time for the nucleotide addition step was 20 to 30 s. The net change in light scattering following nucleotide addition was plotted as a function of time. FtsZ GTPase activity (1) The GTPase activity of FtsZ is measured in either 50 mM HEPES-NaOH (pH 7.2) or 50mM MES-NaOH (pH 6.5) with 10 mM MgC , 200 mM KC1. A standard reaction contains FtsZ at 200 μg/ml (5 μM) and the reaction is initiated by adding 1 mM [g- 2 P]GTP (100-200 cpm/pmol) (Amersham, Arlington Heights. IL) and incubating at 30 °C.The GTPase activity requires Mg 2+ and is stimulated by K ~ . For optimal activity. 200 mM KC1 is used as the rate decreases at lower KC1 concentration. To measure the amounts of released 32 P, a 5 μL aliquot of the reaction mixture is withdrawn at 5 min intervals and added to 95 μL of 0.55 M HC10 4 containing 0.55 mM KH 2 P0 4 followed by the addition of 150 μL of 20 mM Na2Mo0 . The mixture is vortexed and after 2 min at room temperature, 300 μL of isopropyl acetale (Acros Organics, Geel, Belgium) is added, vortexed for 15 s, and spun for 2 min in an Eppendorf centrifuge to separate the two phases The organic (upper phase) (100 μL) is withdrawn and added to 5 mL of Econo-safe (Research products Intl Corp., Mount Prospect, IL) and counted in a hquid scintilation counter. Alternatively the formation of [α- 32 P]GDP from [α- 32 P]GTP can be determined by separating them on polyethyleneamme cellulose plates. FtsZ GTPase activity (2) GTPase activity was monitored by measuring the release of phosphate using a colorimetric assay to determine the amount of free phosphate in the samples A phosphorous stock solution (Sigma Diagnostics, St. Louis, MO, USA) was used for calibration. FtsZ at various concentrations was incubated at 30 °C in 50 mM Mes-NaOH (pH6.5), 50 mM KC1, 5 mM MgCl 2+ and CaCl 2+ and EGTA at various concentrations At t=0 the reaction was started by the addition of GTP to 1 mM At different time points 30 μL samples were withdrawn and transferred to 200 μl of a malachite green solution on ice. After 5 mm 30 μl of 34 % w/v citric acid was added and the absorption at 660 nm was read immediately with a Spectramax 340 plate reader (Molecular Devices Corp Sunnyvale, Ca, USA) GTP binding assay GTP binding GTP binding was monitored by the covalent cross-linking of [α- J2 P]GTP to FtsZ essentially as described by De Boer et al., (de Boer, P A J , R E Crossley and L. I Rothfield 1992 The essential bacterial cell division protein FtsZ is a GTPase Nature 359.254-256 ) Purified FtsZ (3 μg) was incubated in 20 mL 50 mM Mes-NaOH (pH 6 5), 100 mM KC1, 4 mM MgCl 2+ , 1 mM EDTA, 0 1 mM EGTA, 0 5 mM DTT containing 0 1 μM [α]GTP and non- radiolabeled competitors Samples were incubated 15 min at 0 °C, then subjected to photoaffinity cross-linking for 5 min at 0°C using a 254-nm UV lamp at a distance of 2 cm. Samples were analyzed by SDS-PAGE on 12% gels. The gels were dried and exposed to Kodak Biomax MR film. Autoradiograms were densitometrically scanned using a Dextra DF-2400T scanner (Dextra Corp., Taipei, Taiwan) and Sigmascan software (Jandel Corp., San Rafael, CA) Imunofluorescence microscopy visualization of FtsZ Fixation and permeabilzation. Cells were fixed in 2.8% formaldehyde (FA) and 0.04% glutaraldehyde (GA) for 15 min at room temperature. For the permeabihsation, the cells were collected at 7,000 rpm for 5 mm, washed twice in PBS pH 7.2 and subsequently incubated in 0.1% Triton X-100 in PBS for 45 mm at room temperature The cells were washed three times in PBS and incubated in PBS containing 100 μg/mL lysozyme and 5 mM EDTA for 45 min at room temperature. Finally the cells were washed three times in PBS. In situ lmmuno-fluoresence labeling. Non-specific binding sites were blocked by incubating the cells in 0.5% (w/v) blocking reagents (Boehringer, Mannheim, Germany) in PBS for 30 min at 37 °C. Incubation with primary antibodies, i.e. a monoclonal antibody against FtsZ (Voskuil, J. L. A., C. A. M. Westerbeek, C. Wu, A. H. J. Kolk, and N Nanninga. 1994. Epitope mapping of Escherichia coli cell division protein FtsZ with monoclonal antibodies. J. Bacteriol. 176: 1886-1893.) diluted in blocking buffer was carried out for 60 mm at 37 °C The cells were washed three times with PBS containing 0.05% (v/v) Tween-20. Incubation with secondary antibodies, donkey-anti-mouse conjugated with Cy3 (Jackson ImmunoResearch laboratories, INC West Grove, Pennsylvania) or donkey-anti- mouse conjugated with Alexa(TM) 546 (Molecular Probes, Eugene, OR), diluted in blocking buffer was carried out for 30 min at 37 °C The cells were washed three times in PBS, 0 05% Tween-20 The nucleoids were stained with DAPI (4,6-dιammo-2-phenyhndole) at a final concentration of 0.5 μg/ml in H2O The cells were washed once in H2O and resuspended in PBS Microscopy and image analysis Cells were immobilized on agarose slides as described by Van Helvoort and photographed with a cooled Princeton CCD camera (Princeton Instruments, SARL, Utrecht, The Netherlands) mounted on an Olympus BX-60 fluorescence microscope. Images were taken using the program IPlab 3 1a (Signal Analytics, Vienna, Virginia). In all experiments the cells were first photographed m the phase contrast mode, then with a DAPI fluorescence filter (U-MWU, excitation at 330-385 nm) and finally with an Alexa filter (U-MNG, ex. 530-550 nm) or with a GFP filter (U-MNB, ex.470-490 nm). The three photographs were stacked and the length of each cell was measured in the phase contrast image, the nucleoid separation was determined in the DAPI image and the presence of rings or foci in the fluorescence image. Interactive measurements were performed as structured point collection on a Macintosh 7200 computer using the public domain program Object-Image 1.62' by Norbert Vischer (University of Amsterdam, http://simon.bio.uva nl/object-image.html), which is based on NIH Image by Wayne Rasband. Synthesis of 2'- and 8- derivatives of GTP A simple, convenient and rapid solid phase synthesis of 2'-0- methylribonucleoside 5'-tπphosphate and their a-thio analogues have been published (Gaur, R. K., B. S. Sproat, and G. krupp. 1992. Novel solid phase synthesis of 2'-0-methylπbonucleosιde 5'-trιphosphates and their α-thio analogues, Tetz. Letff. 33- 3301-3304). The 3'-hydroxyl group of the 5'-0- dιmethyloxytrιtyl-N-Acetyl-2'-0-methylrιbonucleosιde is attached to an amino functionahsed solid support (e.g CPG) via a succmate linkage, which serves the dual purpose of anchor and protecting group All the chemical modifications can be performed whilst the nucleotide is attached to the support, enabling excess reagents to be removed by simple washing under argon. In addition the commercial availability of the fully protected 2'-0-methylrιbonucleosιde functionahsed supports renders this approach very attractive. For modifications of the C8 postion the commercially available 8-Br-guanosιde and the 8-Br- guanoside monophosphate (Sigma B1893 and B2131) can be used as starting points Substitution at the C8 position of GTP causes an inhibition of the polymerization or depolymerization of FtsZ depending on the concentration of the nucleotide GTP hydrolysis is essential for the polymerization and depolymerization reaction of FtsZ. FtsZ was shown not be able to hydrolyze 8-Br-GTP and hence its inhibitory function. The interaction between the HCl loop of adjacent FtsZ molecules is a requirement for GTP hydrolysis and determines the accessibility of, the for the hydrolysis required divalent cations The presence of the relative bulky bromide on the C8 position interferes with the interaction of the HC1 loop and the GTP binding site In this respect it is foreseeable that a C8 chloro or fluoro substituent provides still sufficient bulk (steric hindrance) to inhibit (de)polymerιzatιon of FtsZ Although, the GTP binding pocket and the mechanism of polymerization of FtsZ and tubulin seem to be very similar, the differences are sufficient to make an analogue that discriminates between the two molecules For instance, the Br-GTP does not inhibit the polymerization of tubuhn (Muraoka, M , Fukuzawa, H , Nishida, A , Okano, K , Tsuchihara, T , Shimoda, A , Suzuki, Y , Sato, M , Osumi, M , and Sakai, H (1999), The effects of various GTP analogues on microtubule assembly Cell Struc Funct 24 101-109), whereas it inhibits the polymerization of Fts Although not related, in Lu et al , (1999) Cell Struct And Function 285-290 the use of a GTP analogue that is not substituted on the C2', C3', C2, C5, or C8 position is mentioned Z Tubuhn is clearly capable of hydrolyzmg the Br-GTP, suggesting a somewhat different coordination of the magnesium ion comparedto FtsZ and a difference in mterfacial spacing The fine tuning of the specificity and the affinity of the C8-substιtuted GTPwill therefore strongly depend on the characteristics of the introduced group Small changes in size or electrostatic properties could have an important impact on the selectivity and affinity of the molecule Several approaches can be envisaged to facilitate the membrane passage of the GTP analogue Several of the mentioned positions on the GTP analogue can be substituted with a labile group such as a thio-ether or disulfide, which is coupled to a hydrophobic group like a fatty acid chain of variable length Once the hydrophobic GTP analogue has passed the membrane barrier the labile nature of the linking group will assure the removal of the hydrophobic part of the molecule and allow the GTP analogue to inhibit the (de)polymerιzatιon of FtsZ Depending on the molecular properties of both hpid and peptide, creation of bilayer defects such as phase separation or membrane thinning, pore formation, promotion of nonlamellar hpid structures or bilayer disruption by the carpet model or detergent-like action, may occur A combination of antimicrobial peptides and the GTP analogue would facilitate the entrance of the analogue in the bacterial cell A major advantage of this approach is the availability of species specific antibacterial peptides, which would prevent eucaryote membrane passage of the GTP analogue. Figure legends Figure 1 Light scattering of FtsZ polymerisation FtsZ polymerisation was measured by 90° angle hght scattering in a Hitachi fluorescence spectrophotometer with both the excitation and the emission wavelengths set at 350 nm and a sht width of 1.5 nm FtsZ added to a final concentration of 12.5 microM in 50 mM MES/NaOH, pH 6.5, 50 mM KC1, 5 mM MgCl 2+ , 10m CaCl 2+ to a quartz cuvette with a 1 cm path length The cuvette was then placed in a cuvette chamber that was maintained at 30° C by a circulating waterbath and data were collected for 8 min to establish a basehne. Subsequently, the cuvette was removed and dGTP was added to a final concentration of 0.5 mM. The reaction mixture was gently stirred with a pipette tip and the cuvette was returned to the chamber The elapsed time for the nucleotide addition step was 2-3 seconds. The net change in light scattering following nucleotide addition was plotted as a function of time Figure 2. GTP backbone R = Phosphate group(s) or substituents, there of X = substituents of different size or polarity Figure 3 Light scattering of FtsZ polymerisation FtsZ polymerisation was measured by 90° angle light scattering in a Hitachi fluorescence spectrophotometer with both the excitation and the emission wavelength set at 350 nm and a slit width of 3 0 nm. FtsZ added to a final concentration of 2 5 micro in 50 mM MES/NaOH, pH 6 5, 50 mM KC1 5 mM MgC12+, 10 mM CaC12+ to a quartz cuvette with a 1 cm path length The cuvette was then placed in a cuvette chamber that was maintained at 30°C by a circulating waterbath and data were collected for 1 mm to estabhsh the baseline. Subsequently, GTP or a mixture of 8-Br-GTP and GTP (1:1, 1:2, or 1.4) was added to a final concentration of 20 microM The reaction mixture was gently mixed and the polymerisation reaction was followed m time. The elapsed time for the nucleotide addition was 2-3 seconds. The net change in light scattering following nucleotide addition was plotted as a function of time. Further references: 1. Epand, R. M., and H. J. Vogel. 1999. Diversity of antimicrobial peptides and their mechanism of action. Biochim. Biophys. Acta. 1462: 11-28. 2. Lowe, J , and L. A. Amos. 1998. Crystal structure of the bacterial cell-division protein FtsZ. Nature. 391:203-206 3. Lowe, J., and L. A. Amos. 1999. Tubuhn-like proteofilaments m Ca2+ -induced FtsZ sheets. EMBO J. 18:2364-2371. 4. Shai, Y. 2000. mechanism of the binding, insertion and destabihzation of phosphohpid bilayer membranes by a-hehcal antimicrobial and cell non-selective membrane-lytic peptides. Biochim. Biophys. Acta. 1462:55-70.

Claims: CLAIMS A method to hamper division of a prokaryotic cell comprising inhibiting polymerisation and/or depolymerisation of cell-division protein FtsZ or analogue thereof by contacting said protein with a guanoside tπphosphate (GTP) analogue capable of binding to said protein, wherein said GTP analogue is modified at position C2', C5' and or C8 A method to hamper division of a prokaryotic cell comprising inhibiting polymerisation and/or depolymerisation of cell-division protein FtsZ or analogue thereof by contacting said protein with a guanoside triphosphate (GTP) analogue capable of binding to said protein, wherein said GTP analogue is modified at position C2', C3' and/or C2 A method to hamper division of a prokaryotic cell comprising inhibiting polymerisation and/or depolymerisation of cell-division protein FtsZ or analogue thereof by contacting said protein with a guanoside triphosphate (GTP) analogue capable of binding to said protein, wherein said GTP analogue is modified at position C2', C2 and/or C8 A method to hamper division of a prokaryotic cell comprising inhibiting polymerisation and/or depolymerisation of cell-division protein FtsZ or analogue thereof by contacting said protein with a guanoside triphosphate (GTP) analogue capable of binding to said protein, wherein said GTP analogue is modified at position C8 . A method to hamper division of a prokaryotic cell comprising inhibiting polymerisation and/or depolymerisation of cell-division protein FtsZ or analogue thereof by contacting said protein with a GTP analogue capable of binding to said protein, wherein said GTP analogue is essentially not hampering division of a eukaryotic cell . A method according to claim 5 wherein said analogue is essentially not inhibiting polymerisation of tubuhn . A method according to claim 5 wherein said analogue is essentially not capable of binding to a GTP-bindmg site of a GTP-binding protein from a eukaryotic cell 8 . A method according to anyone of claim 1 to 4 wherein said GTP analogue is essentially not inhibiting polymerisation of tubulin 9 . A method according to anyone of claim 1 to 4 wherein said GTP analogue is essentially not capable of binding to a GTP-bindmg site of a GTP-binding protein from a eukaryotic cell 10 . A pharmaceutical composition comprising a GTP analogue capable of binding to a prokaryotic cell-division protein FtsZ or analogue thereof, wherein said GTP analogue is modified at position C2', C3', C5\ C2 and/or C8 11 . A pharmaceutical composition comprising a GTP analogue capable of binding to a prokaryotic cell-division protein FtsZ or analogue thereof, said GTP analogue essentially not inhibiting polymerisation of tubuhn 12 . A pharmaceutical composition according to claim 10 or 11 essentially not capable of binding to a GTP-bmdmg site of a GTP-bmdmg protein from an eukaryotic cell 13 . A composition according to claim 11 or 12 wherein said GTP analogue is modified at position C2', C3\ C5', C2 and/or C8 14 . Use of a GTP analogue capable of binding to a prokaryotic cell-division protein FtsZ or analogue thereof, wherein said GTP analogue is modified at position C2', C3, C5', C2 and/or C8, as a pharmaceutical 15 . Use of a GTP analogue capable of binding to a prokaryotic cell-division protein FtsZ or analogue thereof, said GTP analogue essentially not inhibiting polymerisation of tubuhn, as a pharmaceutical 16 . Use of a GTP analogue capable of binding to a prokaryotic cell-division protein FtsZ or analogue thereof, said GTP analogue essentially not capable of binding to a GTP-bindmg site of a GTP-binding protein from an eukaryotic cell, as a pharmaceutical 17 . Use according to claim 15 or 16 wherein said pharmaceutical is for the treatment of a eukaryotic cell against a prokaryotic infection 18 . Use according to claim 17 wherein said eukaryotic cell is part of a cell culture 19 . Use according to claim 17 wherein said eukaryotic cell is part of an organism 20 . Use according to claim 17 wherein said organism is an animal 21 . Use according to anyone of claims 17 to 20 wherein said prokaryotic ^ infection is a bacterial infection . Use of a GTP analogue capable of binding to a prokaryotic cell-division protein FtsZ or analogue thereof, wherein said GTP analogue is modified at position C2', C3' C5', C2 and or C8, for the preparation of a medicament for the treatment of an eukaryotic organism against a prokaryotic infection. . Use of a GTP analogue capable of binding to a prokaryotic cell-division protein FtsZ or analogue thereof, said GTP analogue essentially not inhibiting polymerisation of tubuhn, for the preparation of a medicament for the treatment of an eukaryotic organism against a prokaryotic infection. . Use of a GTP analogue capable of binding to a prokaryotic cell-division protein FtsZ or analogue thereof, said GTP analogue essentiaUy not capable of binding to a GTP-bmdmg site of a GTP-bmding protein from an eukaryotic cell for the preparation of a medicament for the treatment of an eukaryotic organism against a prokaryotic infection . A method to treat an eukaryotic cell against a prokaryotic infection comprising inhibiting polymerisation and/or depolymerisation of cell-division protein FtsZ or analogue thereof by contacting said protein with a guanoside triphosphate (GTP) analogue capable of binding to said protein, wherein said GTP analogue is modified at position C2', C3\ C5\ C2 and or C8. . A method to treat a eukaryotic cell against a prokaryotic infection comprising inhibiting polymerisation and/or depolymerisation of cell-division protein FtsZ or analogue thereof by contacting said protein with a guanoside triphosphate (GTP) analogue capable of binding to said protein, said analogue essentially not inhibiting polymerisation of tubuhn . A method to treat a eukaryotic cell against a prokaryotic infection comprising inhibiting polymerisation and/or depolymerisation of cell-division protein FtsZ or analogue thereof by contacting said protein with a guanoside triphosphate (GTP) analogue capable of binding to said protein, said analogue essentially not capable of binding to a GTP-binding site of a GTP-bindmg protein from an eukaryotic cell 8 . A method according to claim 26 or 27 wherein said eukaryotic cell is part of a cell culture 9 . A method according to claim 26 or 27 wherein said eukaryotic cell is part of an organism 0 . A method according to claim 29 wherein said organism is an animal

Inventors: DEN BLAAUWEN TANNEKE, KOOMEN GERRIT-JAN, NANNINGA NANNE, DEN BLAAUWEN, TANNEKE, KOOMEN, GERRIT-JAN, NANNINGA, NANNE
Applicants: DEN BLAAUWEN TANNEKE, INST MOLECULAR CELL BIOLOGY, INST OF MOLECULAR CHEMISTRY, KOOMEN GERRIT JAN, NANNINGA NANNE, DEN BLAAUWEN, TANNEKE, INSTITUTE FOR MOLECULAR CELL BIOLOGY, INSTITUTE OF MOLECULAR CHEMISTRY, KOOMEN, GERRIT-JAN, NANNINGA, NANNE
Application year: 2000
Main classification: A61K31
IPC codes: A61K31, A61P31