Strathclyde Minor Groove Binders (S MGBs) as Antimicrobial Agents

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1 Strathclyde Minor Groove Binders (S MGBs) as Antimicrobial Agents Dr. Fraser J. Scott University of Lincoln or

2 utline The origin of the S MGB programme Recent Success of MGB BP 3 against Grampositive bacteria Efforts towards Gram negative active S MGBs Efforts towards TB active S MGBs Programme overview

3 Cozzi et al. Polyamide MGBs Dervan et al. Strathclyde

4 riginal Design Concept at Strathclyde ydrophobic patches ydrogen bonding on floor alkyl group on pyrrole

5 Very Early it Compounds ead et 1 et 2 et 3 Tail

6 Target Identification DA Footprinting TyrT a standard, naturally occurring oligonucleotide commonly used in footprinting. Me R Me Me 2 MS1 and MS2 synthetic oligonucleotides designed to contain all possible 4mers ideal for studying binding of MGBs of this size. Prof Keith Fox, University of Southampton Me Me S 3 R = Me 4 R = ipr Me 2 5

7 Target Identification -alkyl pyrroles read AT Thiazotropsin A can read GC

8 Target Identification 3 G C T G A T C A G C 5 5 C G A C T A G T C G 3 Canonical DA with AT rich sequence showing minor groove shaded to show electrostatic potential (red = negative) Structure of DA 2:1 thiazotropsin A complex calculated from MR data. ote wider minor groove.

9 Anti-Gram-Positive S-MGBs

10 Recent Success of MGB BP 3 against Grampositive bacteria Amide: planar, bond donor and acceptor, hydrolysable. Alkene: planar, non polar, stable to hydrolysis. ne hydrogen bond lost. T m measurements show that loss of a hydrogen bond does not weaken binding to DA oligos in this group of compounds. MGB-BP-3

11 SAR Summary for Antibacterial Activity

12 Current status MGB Biopharma has taken MGB BP 3 to successful completion of Phase I clinical trials for oral treatment of C. diff infections. Differentiation of MGB-BP-3 in Clostridium difficile Infection 1.Broad in vitro activity confirmed against Clostridium difficile 2. Rapid killing effect seen for MGB-BP-3 against C. diff strains, including hypervirulent AP1/027; activity superior to vancomycin 3.Superior activity to vancomycin confirmed in vivo 4.MGB-BP-3 is superior to vancomycin in suppressing sporulation in all C. difficile strains tested 5.MGB-BP-3 is active against vancomycin resistant enterococci (VRE)

13 Current status MGB Biopharma is nearly ready to enter phase I with IV treatments against other Gram positives. Final formulation optimisation and up-scaling has been successfully completed, and pivotal proof of concept and ADME studies assessing IV MGB-BP-3 against Staphylococcus aureus (including MRSA), Streptococcus pyogenes and pneumoniae are complete.

14 Selectivite Toxicity? S27 Cells Effect of AIK-20/25/1 on S27 Cells The Effect of AIK-20/25/1 in the Antimicrobial Assay Against S.aureus 07/02/07 S. Aureus Cytotoxicity (% Control) IC 50 > 30 M n=4 % Control n=4 0.7 M Log Concentration (M) Log conc (M)

15 Selective Toxicity S. aureus without MGB BP3 Brightfield under UV S. aureus with MGB BP3 Brightfield under UV B16Fluc cells with MGB BP3 Brightfield under UV B16Fluc cells with oechst dye Brightfield under UV

16 Selective Toxicity Primary hepatocytes after 24 hwith a highly fluorescent MGB that has antibacterial activity

17 Anti-Gram-egative S-MGBs

18 Entry to Cells Brightfield Fluorescence Bacillus cereus Staphylococcus aureus Listeria monocytogenes

19 Entry to Cells Brightfield Fluorescence Pseudomonas aerugenosa Salmonella enteriditis Escherichia coli

20 Entry to Cells Brightfield Fluorescence E. coli Spheroplast E. coli intact cells

21 The Role of Efflux Efflux pump inhibitor (EPI) PAβ. rganism MIC 80 (µm) MIC 80 with EPI (µm) P. Aeruginosa > E. coli >

22 Synergistic Effects Amoxicillin ( M) MIC S. aureus AIK18/85/4 ( M) Me S Me

23 Synergistic Effects Clinical Isolate Klebsiella pneumoniae 28 MIC 80 for Ceftazidime 64 μg/ml Klebsiella pneumoniae 37 Klebsiella pneumoniae 68 Klebsiella pneumoniae μg/ml 64 μg/ml 1 μg/ml

24 Anti-Mycobacterium Tuberculosis S- MGBs

25 Reto Guler, University of Cape Town Journey to high activity MIC 99 Mtb 37Rv 25 M 25 M 25 M 25 M 25 M 25 M 25 M

26 Reto Guler, University of Cape Town Journey to high activity 25 M 12.5 M 25 M F 25 M 3.1 M S Different S-MGB Series 0.2 M M. Tuberculosis >100 M EK293T (IC 50 )

27 ther Targets for S-MGBs

28 Antiparasitics 1: Sleeping Sickness Michael Barrett University of Glasgow Swiss Tropical and Public ealth Institute Trypanosoma brucei (human) Trypanosoma congolense (cattle) IC 50 = 14 nm IC 50 = 390 nm IC 50 = 110 nm IC 50 = 7 nm

29 Antiparasitics 2: Malaria Victoria Avery, Griffith University, Australia Plasmodium falciparum IC 50 = 39, 38 nm, selectivity > 500 IC 50 = 103, 241 nm, selectivity >200 Two strains, chloroquine sensitive and chloroquine insensitive studied. Selectivity and activity against insensitive strain important. cf. chloroquine IC 50 = 11.3, 177 nm, selectivity > 10 5 for sensitive cells. Pyrimethamine IC 50 = 8.45, na, selectivity = 955 for sensitive cells.

30 Anticancer: human melanoma cell line Chris Carter, University of Strathclyde IC 50 = 140 nm cf. gemcitabine = 1089 nm

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32 Acknowledgements Chemistry Colin Suckling Abedawn Khalaf Roger Waigh Simon Mackay Colin Gibson David Breen ahoum Anthony John Parkinson Judith uggan Gavin Donoghue Claire Bourdin Gail Wilson Angus Wilson Andrew MacDonald Ross Carleton Ryan ichol Biology Elizabeth Ellis Iain unter Gordon Ford Curtis Gemmell ick Tucker Leena ieminen Chris Carter elen Grant Katherine enderson Keith Fox (Southampton) Michael Barrett (Glasgow) Federica Giordani (Glasgow) Reto Guler (Cape Town) Funding Synergy Fund Scottish Enterprise Proof of Concept Leucovorin Royalties, Strathclyde BBSRC MSD SULSA Rosetrees Trust Carnegie Foundation