Discovery of a Small Molecule That Inhibits Cell Division by Blocking FtsZ, a Novel Therapeutic Target of Antibiotics

The emergence of bacterial resistance to antibiotics is a major health problem and, therefore, it is critical to develop new antibiotics with novel modes of action. FtsZ, a tubulin-like GTPase, plays an essential role in bacterial cell division, and its homologs are present in almost all eubacteria and archaea. During cell division, FtsZ forms polymers in the presence of GTP that recruit other division proteins to make the cell division apparatus. Therefore, inhibition of FtsZ polymerization will prevent cells from dividing, leading to cell death. Using a fluorescent FtsZ polymerization assay, the screening of >100,000 extracts of microbial fermentation broths and plants followed by fractionation led to the identification of viriditoxin, which blocked FtsZ polymerization with an IC50 of 8.2 μg/ml and concomitant GTPase inhibition with an IC50 of 7.0 μg/ml. That the mode of antibacterial action of viriditoxin is via inhibition of FtsZ was confirmed by the observation of its effects on cell morphology, macromolecular synthesis, DNA-damage response, and increased minimum inhibitory concentration as a result of an increase in the expression of the FtsZ protein. Viriditoxin exhibited broad-spectrum antibacterial activity against clinically relevant Gram-positive pathogens, including methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococci, without affecting the viability of eukaryotic cells. The emergence of bacterial resistance to antibiotics is a major health problem and, therefore, it is critical to develop new antibiotics with novel modes of action. FtsZ, a tubulin-like GTPase, plays an essential role in bacterial cell division, and its homologs are present in almost all eubacteria and archaea. During cell division, FtsZ forms polymers in the presence of GTP that recruit other division proteins to make the cell division apparatus. Therefore, inhibition of FtsZ polymerization will prevent cells from dividing, leading to cell death. Using a fluorescent FtsZ polymerization assay, the screening of >100,000 extracts of microbial fermentation broths and plants followed by fractionation led to the identification of viriditoxin, which blocked FtsZ polymerization with an IC50 of 8.2 μg/ml and concomitant GTPase inhibition with an IC50 of 7.0 μg/ml. That the mode of antibacterial action of viriditoxin is via inhibition of FtsZ was confirmed by the observation of its effects on cell morphology, macromolecular synthesis, DNA-damage response, and increased minimum inhibitory concentration as a result of an increase in the expression of the FtsZ protein. Viriditoxin exhibited broad-spectrum antibacterial activity against clinically relevant Gram-positive pathogens, including methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococci, without affecting the viability of eukaryotic cells. Bacterial infection is a global health hazard. There are a number of very good clinically efficacious antibiotics in use today; however, because bacteria render almost all of these antibiotics less effective because of the development of resistance, new antibiotics with novel mechanisms of action are needed to overcome the emerging resistance problem. To date, there are ∼150–670 essential proteins that have been identified in bacteria as potential drug targets (1Ji Y. Zhang B. Van S.F. Horn Warren P. Woodnutt G. Burnham M.K.R. Rosenberg M. Science. 2001; 293: 2266-2269Crossref PubMed Scopus (357) Google Scholar, 2Kobayashi K. Ehrlich S.D. Albertini A. Amati G. Andersen K.K. Arnaud M. Asai K. Ashikaga S. Aymerich S. Bessieres P. Boland F. Brignell S.C. Bron S. Bunai K. Chapuis J. Christiansen L.C. Danchin A. Debarbouille M. Dervyn E. Deuerling E. Devine K. Devine S.K. Dreesen O. Errington J. Fillinger S. Foster S.J. Fujita Y. Galizzi A. Gardan R. Eschevins C. Fukushima T. Haga K. Harwood C.R. Hecker M. Hosoya D. Hullo M.F. Kakeshita H. Karamata D. Kasahara Y. Kawamura F. Koga K. Koski P. Kuwana R. Imamura D. Ishimaru M. Ishikawa S. Ishio I. Le Coq D. Masson A. Mauel C. Meima R. Mellado R.P. Moir A. Moriya S. Nagakawa E. Nanamiya H. Nakai S. Nygaard P. Ogura M. Ohanan T. O'Reilly M. O'Rourke M. Pragai Z. Pooley H.M. Rapoport G. Rawlins J.P. Rivas L.A. Rivolta C. Sadaie A. Sadaie Y. Sarvas M. Sato T. Saxild H.H. Scanlan E. Schumann W. Seegers J.F.M.L. Sekiguchi J. Sekowska A. Seror S.J. Simon M. Stragier P. Studer R. Takamatsu H. Tanaka T. Takeuchi M. Thomaides H.B. Vagner V. van Dijl J.M. Watabe K. Wipat A. Yamamoto H. Yamamoto M. Yamamoto Y. Yamane K. Yata K. Yoshida K. Yoshikawa H. Zuber U. Ogasawara N. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 4678-4683Crossref PubMed Scopus (1144) Google Scholar, 3Hutchison III, C.A. Peterson S.N. Gill S.R. Cline R.T. White O. Fraser C.M. Smith H.O. Venter J.C. Science. 1999; 286: 2165-2169Crossref PubMed Scopus (735) Google Scholar, 4Akerley B.J. Rubin E.J. Novick V.L. Amaya K. Judson N. Mekalanos J.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 966-971Crossref PubMed Scopus (326) Google Scholar, 5Forsyth R.A. Haselbeck R.J. Ohlsen K.L. Yamamoto R.T. Xu H. Trawick J.D. Wall D. Wang L. Brown-Driver V. Froelich J.M. Kedar G.C. King P. McCarthy M. Malone C. Misiner B. Robbins D. Tan Z. Zhu Z.-Y. Carr G. Mosca D.A. Zamudio C. Foulkes J.G. Zyskind J.W. Molecular Microbiology. 2002; 43: 1387-1400Crossref PubMed Scopus (409) Google Scholar). Of those, only 15 are proven therapeutic targets. To overcome resistance, existing antibiotics have been modified extensively in an attempt to maintain activity against their targets. Because efficacious modification is becoming increasingly difficult, targeting new, unexplored essential proteins to identify new classes of small molecular weight compounds is critical for the discovery of new, non-cross resistant antibacterial agents. FtsZ is a GTPase and an essential protein for cell division with homologs in almost all species of eubacteria and archaea (6Bramhill D. Annu. Rev. Cell Dev. Biol. 1997; 13: 395-424Crossref PubMed Scopus (204) Google Scholar, 7Rothfield L. Justice S. Garcia-Lara J. Annu. Rev. Genet. 1999; 33: 423-448Crossref PubMed Scopus (238) Google Scholar, 8Erickson H.P. Trends Cell Biol. 1997; 7: 362-367Abstract Full Text PDF PubMed Scopus (196) Google Scholar). FtsZ shows limited sequence similarity to tubulin; however, the three-dimensional structure of FtsZ shows significant similarities with α and β tubulin (9Nogales E. Wolf S.G. Downing K.H. Nature. 1998; 391: 199-203Crossref PubMed Scopus (1799) Google Scholar). FtsZ is the most abundant of all bacterial cell division proteins, totalling ∼10,000–20,000 copies per single bacterium (10Bi E. Dai K. Subbarao S. Beall B. Lutkenhaus J. Res. Microbiol. 1991; 142: 249-252Crossref PubMed Scopus (24) Google Scholar). During vegetative growth, bacterial cells divide into two identical copies. The first stage in the cell replication process is localization of the FtsZ protein at the site of cell division where self-polymerization occurs. The polymerized FtsZ recruits other cell division proteins, including FtsA (11Addinall S.G. Lutkenhaus J. J. Bacteriol. 1996; 178: 7167-7172Crossref PubMed Scopus (158) Google Scholar, 12Dewar S.J. Donachie W.D. J. Bacteriol. 1993; 175: 7097-7101Crossref PubMed Google Scholar, 13Dai K. Lutkenhaus J. J. Bacteriol. 1991; 173: 3500-3506Crossref PubMed Scopus (225) Google Scholar), ZipA (14Hale C.A. de Boer P.A. Cell. 1997; 88: 175-185Abstract Full Text Full Text PDF PubMed Scopus (396) Google Scholar, 15Liu Z. Mukherjee A. Lutkenhaus J. Mol. Microbiol. 1999; 31: 1853-1861Crossref PubMed Scopus (117) Google Scholar), FtsK (16Begg K.J. Dewar S.J. Donachie W.D. J. Bacteriol. 1995; 177: 6211-6222Crossref PubMed Google Scholar), FtsQ (17Chen J.C. Weiss D.S. Ghigo J.M. Beckwith J. J. Bacteriol. 1999; 181: 521-530Crossref PubMed Google Scholar, 18Buddelmeijer N. Aarsman M.E.G. Kolk A.H.J. Vicente M. Nanninga N. J. Bacteriol. 1998; 180: 6107-6116Crossref PubMed Google Scholar), FtsL (19Barondess J.J. Carson M. Guzman Verduzco L.M. Beckwith J. Res. Microbiol. 1991; 142: 295-299Crossref PubMed Scopus (6) Google Scholar, 20Ghigo J.M. Weiss D.S. Chen J.C. Yarrow J.C. Beckwith J. Mol. Microbiol. 1999; 31: 725-737Crossref PubMed Scopus (72) Google Scholar), FtsW (21Khattar M.M. Begg K.J. Donachie W.D. J. Bacteriol. 1994; 176: 7140-7147Crossref PubMed Google Scholar, 22Wang L. Khattar M.K. Donachie W.D. Lutkenhaus J. J. Bacteriol. 1998; 180: 2810-2816Crossref PubMed Google Scholar), FtsI (22Wang L. Khattar M.K. Donachie W.D. Lutkenhaus J. J. Bacteriol. 1998; 180: 2810-2816Crossref PubMed Google Scholar, 23Weiss D.S. Chen J.C. Ghigo J.M. Boyd D. Beckwith J. J. Bacteriol. 1999; 181: 508-520Crossref PubMed Google Scholar), and FtsN (24Addinall S. Cao C. Lutkenhaus J. Mol. Microbiol. 1997; 25: 303-309Crossref PubMed Scopus (138) Google Scholar), either by direct physical interaction or by secondary protein-protein interactions, leading to the formation of a Z-ring and the initiation of the complex process of septation. All of these cell division proteins are localized at mid-cell, working in concert to constrict the cell and resulting in cell division. Because FtsZ is essential for bacterial cell division and shows a high degree of similarity among bacterial species, it presents an excellent novel target for antibacterial drug discovery. In this study, we identified viriditoxin as an inhibitor of bacterial cell division and studied its mechanism of action and broad-spectrum antibacterial properties. Isolation, Purification, and Identification of Viriditoxin—Aspergillus sp. (MF6890) was isolated from herbivore dung collected in Arizona. It was grown on a seed medium consisting of 4 g of yeast extract, 8 g of malt extract, 4 g of glucose, and 1.5 g of junlon (Nihon Junyaku Co. Ltd, Tokyo, Japan) in 1 liter of distilled water at pH 7.0. After 4 days, it was transferred to a vermiculite-based production medium consisting of (per liter) 150 g of glucose, 20 g of glycerol, 4 g of yeast extract, 1 g of sodium nitrate, 3 g of monosodium glutamate, 0.5 g of Na2HPO4, 1 g of MgSO4.7H2O, and 8 g of CaCO3. The fermentation broth was extracted with methyl ethyl ketone and successively chromatographed on Sephadex LH 20 in methanol followed by reversed phase high pressure liquid chromatography affording 2 mg/liter of viriditoxin. The structure of viriditoxin was elucidated by 1H, 13C, and two-dimensional NMR methods and mass spectral studies. Protein Purification—FtsZ and FtsZT65C were purified as described (25Trusca D. Bramhill D. Anal. Biochem. 2002; 307: 322-329Crossref PubMed Scopus (15) Google Scholar) with some modification. Briefly, BL21(DE3)pLysS (Novagen) was transformed with plasmid pET11a-ftsZwt or pET11a-ftsZT65C and induced by adding 1 mm IPTG 1The abbreviations used are: IPTG, isopropyl-1-thio-β-d-galactopyranoside; MIC, minimum inhibitory concentration; MTS, 3-(4,5-dimethylthiazol-2yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; wt, wild type. to mid-log culture (A 600 = 0.5 ∼ 0.6). Cultures were harvested after 1 h at 37 °C. To purify FtsZ and FtsZT65C, the cells were allowed to lyse for 30 min at 0 °C in Buffer A (40 mm Tris-Cl, pH 8.0, 5 mm EDTA, 140 mm NaCl, and 10% sucrose) containing 1 mg/ml lysozyme and Complete protease inhibitor cocktail (Roche Applied Science). The lysed cells were then sonicated with four 30-s bursts using a microprobe. After centrifugation at 40,000 rpm in a 45 Ti rotor for 1 h at 4 °C, the supernatant was adjusted to 10 mm MgCl2, solid ammonium sulfate was added (0.17 g/ml), and the cells were centrifuged for 1 h again. Ammonium sulfate pellets of FtsZwt (wild type) were resuspended in Buffer B (100 mm Tris-Cl, pH 7.4, 5 mm MgCl2, 80 mm KCl, and 10% glycerol) while ammonium sulfate pellets of FtsZT65C were suspended in buffer B with 1 mm reducing agent Tris-(2-carboxyethyl)phosphine hydrochloride (TCEP-HCl). FtsZwt or FtsZT65C was loaded separately onto a DEAE-Sepharose Fast Flow column (Amersham Biosciences) and eluted with a KCl gradient from 80 to 600 mm KCl. The fractions were analyzed by SDS-gel electrophoresis. The FtsZ- or FtsZT65C-containing fractions were pooled and concentrated using Amicon. The fluorescein-labeled FtsZT65C was prepared by incubating the purified FtsZT65C with 2 mm fluorescein iodoacetamide for 25 min in the same buffer on ice, and the free dye was removed by using a Sephadex G25 column. All proteins were aliquoted and stored at –80 °C. FtsZ Polymerization Assay—The assay was done as described previously (25Trusca D. Bramhill D. Anal. Biochem. 2002; 307: 322-329Crossref PubMed Scopus (15) Google Scholar) with a minor modification. Briefly, the NUNC 96-well Silent Screen filter plates were pre-washed with wash Buffer C (100 mm Tris-Cl, pH 7.4, 67.5 mm KCl, 1.5 mm magnesium acetate, and 0.1% Tween.) followed by centrifugation at 3,000 rpm at room temperature for 15 min in a Beckman tabletop centrifuge. The fluorescence-labeled FtsZT65C (FtsZT65C-fluorescein) was pre-centrifuged at 100,000 × g for 20 min at 4 °C to removes any pre-existing polymers. The FtsZ polymerization assay was done by mixing 1 μm fluorescence-labeled FtsZT65C with a serial dilution of inhibitors in 200 μl of Buffer D (50 mm Tris pH 7.4, 34 mm KCl, 0.75 mm magnesium acetate, 2.5 mm CaCl2, 0.5 mm GTP, and 2% Me2SO) and initiated with 50 μg/ml DEAE-dextran. After incubation at 37 °C for 15 the filter plates were centrifuged at 3,000 rpm for 15 and the were on the with The of was 3,000 and 40,000 IC50 was using GTPase activity was and analyzed as described (25Trusca D. Bramhill D. Anal. Biochem. 2002; 307: 322-329Crossref PubMed Scopus (15) Google Scholar, J. Y. S. P. of Scholar) with a small modification. Briefly, this assay was at 37 °C in a buffer containing 50 mm pH 7.4, 34 mm KCl, 0.75 mm magnesium acetate, 2.5 mm CaCl2, 50 μg/ml 2% GTP, and pre-centrifuged FtsZ or The of GTP and the proteins as as the of the are in the to were by the of and the supernatant was of GTPase activity with or without of viriditoxin. The GTPase assay was described in the of 3 and In this study, the GTP of wild FtsZ and with or without 200 μg/ml of viriditoxin was at the on the The of FtsZ and were at 1 was at μm The concentration of GTP was μm with the of a of The other were identical to were in of viriditoxin on GTPase activity of GTP was described in the of 3 and In this study, GTPase activity was at 37 °C for 15 min with the of a serial dilution of viriditoxin. The concentration of GTP was μm with the of a of were in IC50 of and was with an at 20 × against of the was to the for is as the concentration of Assay—The of viriditoxin on macromolecular was as described Chen Science. 1996; PubMed Scopus (357) Google Scholar). with FtsZ was using in Molecular Scholar), a to the K. J. Bacteriol. 1991; 173: PubMed Scopus Google Scholar). The cells were transformed with the plasmid The of viriditoxin against the transformed cells were at of IPTG in medium containing 25 μg/ml of and 5 μg/ml at 37 °C for 20 All were from A polymerization assay been (25Trusca D. Bramhill D. Anal. Biochem. 2002; 307: 322-329Crossref PubMed Scopus (15) Google Scholar) for identification of novel Using this assay, we >100,000 extracts of microbial fermentation broths and a of All the extracts were for and in an assay O. R. J. Bacteriol. PubMed Google Scholar) to the of the only of these are of of the most extracts from an sp. led to the of viriditoxin which was first in from D. Scopus Google Scholar). the of the structure of viriditoxin was and was to have an of to D. Scopus Google Scholar). The structure was in K. K. S. K. Scopus Google Scholar). The spectral mass and of the isolated was identical to the of viriditoxin K. K. S. K. Scopus Google Scholar). a in that it a of to at to for 10 J.G. PubMed Scopus Google Scholar). it was that it on or J.G. PubMed Scopus Google Scholar). The activity of purified viriditoxin was first confirmed in the polymerization It the polymerization with an IC50 of 8.2 μg/ml = To that viriditoxin wild FtsZ we the GTPase activity of the wild FtsZ with this study, we used purified wild FtsZ and The for GTP of wild FtsZ and are an of μm = and = The of is of of the of wild FtsZ is of of The of wild FtsZ was with in P. 1999; PubMed Scopus Google Scholar), and the of wild FtsZ was with L. Simon M. H.P. J. Biol. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar), the assay were a concentration of μm GTP with 1 μm wild FtsZ or 1 μm at 37 °C, the of GTP were and Because L. Simon M. H.P. J. Biol. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar) is the of GTP of wild FtsZ and are and that proteins have GTPase properties. Because the polymerization of FtsZ is and the GTPase activity of FtsZ is by self-polymerization P. 1999; PubMed Scopus Google Scholar), we first GTP in which the polymerization of was GTP in min was only 2 per of protein. we the of viriditoxin on GTPase activity with In the presence of 200 μg/ml viriditoxin, GTP was 2 per of protein in with the inhibition of polymerization by this of viriditoxin. In the GTP of wild FtsZ and identical without the inhibitor was and per of and of viriditoxin into the GTP assay an IC50 of 7.0 μg/ml = To the of viriditoxin on cell division, a assay was D. S. C. Bramhill D. J. Bacteriol. 1998; 180: PubMed Google Scholar). E. cells the the inhibitor of FtsZ polymerization and GTPase that is induced by the leading to D. S. C. Bramhill D. J. Bacteriol. 1998; 180: PubMed Google Scholar), were used to the of any inhibitors of replication an of cell division. (A 600 = cells were with the of a serial dilution of viriditoxin for The cells were a for the formation of The cells at 50 as as at and μg/ml viriditoxin cells in mid-log we a in which production is The containing was grown to phase and then induced with IPTG for resulting in to was with cells by IPTG, the cells To that viriditoxin with FtsZ in we a assay with the E. we the of IPTG without the of viriditoxin and that 8 μm IPTG on the viability of the transformed cells the described The of IPTG 8 μm cell and we the of the expression of FtsZ on of viriditoxin using a serial dilution of IPTG at of and the of IPTG, the of viriditoxin was 25 the of or 1 μm IPTG, the increased to 50 or 8 μm IPTG, the increased to The was with of viriditoxin was for its against clinically relevant bacterial The of viriditoxin are in I. It the of of and methicillin-resistant Staphylococcus aureus with of μg/ml. The of and resistant of Staphylococcus was with of μg/ml against of and vancomycin-resistant and It the of all of and including and with to the of viriditoxin was μg/ml against a wild E. it inhibitory activity against the with a of 25 that plays a role in the of the of bacterial In it activity against and cells IC50 a for eukaryotic against resistant number and aureus were at followed by incubation at °C with a serial dilution of compounds in Biosciences) for 20 aureus were at followed by incubation at °C with a serial dilution of compounds in Biosciences) for 20 sp. were at followed by incubation at °C with a serial dilution of compounds in Biosciences) for 20 were at followed by incubation at °C with a serial dilution of compounds in Biosciences) for 20 E. E. E. E. were at followed by incubation at °C with a serial dilution of compounds in containing lysed Biosciences) for 20 were at followed by incubation at °C with a serial dilution of compounds in or broth for 20 were at followed by incubation at °C with a serial dilution of compounds in for Cell IC50 was using cell 2 × cells were at 37 °C with a serial dilution of compounds for were at followed by incubation at °C with a serial dilution of compounds in Biosciences) for 20 were at followed by incubation at °C with a serial dilution of compounds in containing lysed Biosciences) for 20 were at followed by incubation at °C with a serial dilution of compounds in or broth for 20 were at followed by incubation at °C with a serial dilution of compounds in for IC50 was using cell 2 × cells were at 37 °C with a serial dilution of compounds for in a new in that viriditoxin FtsZ polymerization and GTPase To this is the first small to as a cell division inhibitor by FtsZ Because the three-dimensional structure of FtsZ is to we the tubulin inhibitors and in FtsZ polymerization and GTPase to of these compounds any inhibition of FtsZ polymerization or GTPase activity in the FtsZ from tubulin and the viriditoxin from tubulin The from the cell the with the E. that were because of and confirmed the target of viriditoxin. Viriditoxin was in a modified O. R. J. Bacteriol. PubMed Google Scholar), that it was in The increased by of FtsZ expression solid that viriditoxin with FtsZ in the In cell using wild S. aureus = and the E. = 25 viriditoxin and cell all macromolecular in the IC50 was the activity as a result of a new mode of action. The broad-spectrum antibacterial activity of this including methicillin-resistant S. vancomycin-resistant Enterococci, and other resistant that the activity of this is to of a new viriditoxin was identified by its activity against the purified E. FtsZ the in activity of viriditoxin against Gram-positive bacteria a high of the target in these clinically the structure of the FtsZ is very in the that is by viriditoxin, and this the to develop resistance in the viriditoxin a drug it a for drug discovery as as an for in the of cell division. are to and for the critical of the and for their and the of the BL21(DE3)pLysS and for their to the