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ESCMID Online Lecture Library © by Author Mikael Elofsson Department of Chemistry Umeå Centre for Microbial Research Laboratories for Molecular Infection Medicine Sweden Laboratories for Chemical Biology Umeå Chemical Biology Consortium Sweden [email protected] Type III Secretion Inhibitors as Antivirulence© by author Agents ESCMID Online Lecture Library Microbial Infections Virulence - The ability to cause disease © by author ESCMID Online Lecture Library Umeå Centre for Microbial Research www.ucmr.umu.se Type III Secretion A common virulence system in Gram negative bacteria Animal pathogens Plant pathogens Yersinia spp. Erwinia spp. Salmonella spp. Xanthomonas spp. Chlamydia spp. Pseudomonas syringae Bordetella spp. Ralstonia solanacearum Shigella spp. © by author Pseudomonas aeruginosa enteropathogenic and enterohaemorrhagic ESCMIDEscherichia Online coli Lecture Library Inhibitors Research tools and starting points for drug development Human Pathogenic Yersinia spp. © by author ESCMID Online Lecture Library Type III Secretion - Required for Full Virulence © by author ESCMID Online Lecture Library Needle: length 50-60 nm; inner diam. 2-3 nm; outer diam. 7 nm Type III Secretion in Yersinia Adhere Inject Subvert Proliferate © by author InjectionESCMID of toxins Online - Yersinia Lecture outer proteins Library (Yops) Inhibitors of Type III Secretion in Yersinia T3S Inhibitor Adhere © byPhagocytosis author Clearance ESCMIDThe inhibitor Online disarms Lecture the bacterium Library Novel mode of action Active on resistant strains Less selective pressure Screening Luciferase reporter gene assay in Y. pseudotuberculosis 9,400 commercial compounds 30 confirmed hits Chem. & Biol. 10(2003)241 © by author Post-Screening Assays ESCMID OnlineProtein secretionLecture Library Bacterial growth Gene expression Infection models (ex vivo, in vivo) Statistical Molecular Design Minimize number of compounds and maximize information Computational methods using chemical descriptors to select representative - Building blocks - Target compounds © by author Design ESCMID Online Lecture Library Evaluation Synthesis Curr. Med. Chem. 17(2010)2001 Multivariate QSAR Mathematical model to quantitatively link chemistry and biology © by author ESCMID Online Lecture Library 29 compounds External validation set J. Med. Chem. 50(2007)6177 Salicylidene Acylhydrazides Active against multiple species Yersinia spp. Pseudomonas aeruginosa Salmonella spp. ©Chlamydia by author spp. Shigella spp. ESCMID OnlineEHEC and Lecture EPEC Library Erwinia spp. Inf. Immun. 73(2005)3104, 75(2007)3478, 77(2009)4209, FEBS Lett. 581(2007)587, PNAS 103(2006)14556, Antimicrob. Agents Chemother. 51(2007)2631 and 2867, J. Bact. 191(2009)563 Ex Vivo Yersinia Infection Model Macrophages (J774A) are infected with Y. pseudotuberculosis (96-well plates) Macrophage viability Calcein AM green fluorescence © by author ESCMID Online Lecture Library Chlamydia spp. © by author ESCMID Online Lecture Library Chlamydia spp. - Challenges Clinically relevant (eye and lung infections, STD) Antibiotic resistance (5-20% recurrence) Persistence (long-term infection without symptoms) Many aspects of the infectious cycle are poorly understood Obligate intracellular© pathogenby author No general tools for genetic manipulation ESCMID The functions Onlineof type III secretion Lecture not fully Library clarified Salicylide acylhydrazides inhibit Chlamydia C. trachomatis infection of HeLa cells DMSO Control 6.125 μM 12.5 μM © by author ESCMID Online Lecture Library The inhibitors do not prevent uptake The inhibitors target EB to RB differentiation The inhibitors target RB to EB re-differentiation FEBS Lett. 581(2007)587, PNAS 103(2006)14556, Inf. Immun. 75(2007)3478 Fortunate Polypharmacology The salicylidene acylhydrazides can chelate metal ions including FeII Many pathogenic bacteria and viruses depend on iron The inhibitors also target Neisseria gonorrhoeae: MIC 3-12 μM No effect on the endogenous microflora at 100 μM Lactobacillus crispatus/jensenii Int. J. Antimicr. Agents 36(2010)145 In addition the inhibitors target Herpes simplex type© 2by (HSV author-2): MIC 1 -3 μM Human Immunodeficiency Virus 1 (HIV-1): EC50 1-7 μM ESCMIDInt. J.Online Antimicr. Agents Lecture 40(2012)354 Library Low toxicity (cell, cervicovaginal histology) Stability in semen and vaginal fluid simulants The inhibitors have potential as general microbiocides Activity In Vivo C. trachomatis vaginal infection of C3H/HeJ mice Vaginal administration Compound in sucrose/phosphate buffer (dose: 0.05 mL, 1 mM) Treatment: -48 h, -1 h, 0 h 4 h, 8 h, and then once daily for 5 days Vaginal swabs collected twice a week for one month © by author ESCMID Online Lecture Library J. Inf. Dis. 204(2011)1313 Isolation of Putative Targets Lysate from enterohaemorrhagic E. coli was treated with affinty reagent Eluted proteins were identified by MS/MS anlysis 19 putative targets (16 bands) © by author ESCMID Online Lecture Library J. Biol. Chem. 286(2011)29922 Targets Transcriptomics link three proteins Wrba, Tpx, Folx to T3S regulation Direct bindning: Far western analysis, AUC, and NMR spectroscopy Example: Tpx is a thiol peroxidase that protects against oxidative stress Blue: reduced SH SH Compound docked to the site identified Grey: oxidized S-S by NMR spectroscopy © by author ESCMID Online Lecture Library PLoS ONE 7(2012)e32217 The inhibitors appear to stabilize and/or activate the target proteins Acetaldehyde dehydrogenase - AdhE A bifunctional enzyme important in anaerobic metabolism Far western with biotinylated compounds show binding to the C-terminal domain © by author ESCMID Online Lecture Library Acetaldehyde dehydrogenase - AdhE Phenotype of E. coli O157 △adhe Decreased levels of T3SS gene and protein expression Increased levels of flagella gene and protein expression © by author E. coli ESCMIDO157 △adhe has Online flagella but Lecture do not attach Library to host cells via the T3SS T3S and virulence in Y. pseudotuberculosis △adhe is unaffected compared to wt Acetaldehyde dehydrogenase - AdhE X X © by author ESCMID Online Lecture Library Strangely the E. coli O157 △adhe is non-motile Acetaldehyde dehydrogenase - AdhE Increased acetate concentration might lead to increased acetylation of the chemotaxis protein CheY Acetylation of CheY leads to tumbling and a non-motile phenotype © by author ESCMID Online Lecture Library WT AdhE mutant Acetaldehyde dehydrogenase - AdhE Disulfiram/Antabuse is used to treat alcoholism The compound inhibits acetaldehydedehydrogenase Effect on AdhE and T3S? Disulfiram inhibits T3S in© Y. by tuberculosis author with IC50 around 40 μM (reporter gene assay, YopH phosphatase assay, western analysis) DisulfiramESCMID inhibits replication Online of C. Lecturepneumoniae Libraryand C. trachomatis in HeLa cells IC50 12 and 25 μM Acetaldehyde dehydrogenase - AdhE C. rodentium is model for T3SS-mediated E. coli O157 infection in mice Treatment with disulfiram significantly decreased colonization © by author ESCMID Online Lecture Library AdhE might be a target for novel antibacterial drugs! Mechanism of T3SS inhibition? Erwinia amylovora Plant pathogen that cause fire blight in pears and apples In the USA it causes economic losses of 100 million USD annually T3SS system coded by the hrp gene cluster ME0054 inhibits promoter activities of hrpN, dspE, hrpA, and hrpL genes GFP reporter, IC50 <10 μM Microarray analysis: T3SS genes Iron uptake system genes ME0054 reduced growth of E. amylovora on stigmas© andby author disease development in pistils of crabESCMID apple flowers Online. Lecture Library ME0054 Andrew Roe Mikael Rhen Youfu Zhao Ariel Blocker Ed Galyov Birgitta Henriques Normark Charlotta Sundin Ron Quinn Anders Blomberg Ellena Peterson © by author Carl Tryggers S tiftelse ESCMID Online Lecture Library The Team Sara Spjut Markus Dahlgren Caroline Zetterström Maciej Pudelko Anna Kauppi Weixing Qian Mikael Hillgren Tofeeq Ur-Rehman Rémi Caraballo © byChristoffer author Öberg Åsa Gylfe Per-Anders Enquist ESCMID Online Lecture Library Olli Salin Mickael Mogemark Alka Marwaha Susanne Johansson Fredrik Wallner .
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