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Novel Allosteric Inhibitors of the Bacterial Protein RmlA Targeting L-rhamnose biosynthesis in bacteria Chemical library in place Robust HT screen Hit Identification Chemical and biological validation Prof James Naismith Prof David Gray Used to study biological processes Potential therapeutic M. Alphey, L. Pirrie, N.J. Westwood*, J.H. Naismith* et al., ACS Chemical Biology, 2012, ASAP The Biosynthesis of L-Rhamnose: a key role for RmlA Novel Inhibitors of the bacterial protein RmlA Allosteric sites Active sites Chemical library in place Active sites Targeting L-rhamnose biosynthesis in bacteria 15,667 member Screening library Dundee Drug Discovery Unit Robust HT screen Purified protein assay using RmlA from P. aeruginosa Hit Identification Allosteric sites Chemical and biological validation Used to study biological processes Potential therapeutic J.H. Naismith et al., EMBO J, 2000, 19, 6652; D.B. Nikolov et al., Nat. Struct. Biol, 2001, 8, 545. Hits from HTS bind in the Allosteric Pocket Targeting L-rhamnose biosynthesis in bacteria 15,667 member Screening library Dundee Drug Discovery Unit Chemical library in place Robust HT screen Purified protein assay using RmlA from P. aeruginosa 2 HITS both with a pyrimidinedione core structure IC50s in low mM range Hit Identification Chemical and biological validation Hits bound in the allosteric pocket Used to study biological processes M. Alphey, Naismith Lab. Potential therapeutic Structure-based Optimisation of Hits Reagents and Conditions: (a) Ac2O, 80oC, 2 h then 10% aq. NaOH, 85oC, 1 hr, 51%; (b) Br2, NaHCO3, MeOH, 0 oC to rt, 30 min then 30% aq. amine, 70 oC, 4 h, 69% for 12a; 30% (12b); 28% (12c); 11% (12d); 8% (12e); (c) for 8a-e phenylsulfonyl chloride, pyridine, DCM, rt, 16 h, 65% for 8a 54% (8b); 54% (8c); 60% (8d); 51% (8e); 44-85% for 8g-w; for 8f benzoyl chloride, pyridine, DCM, rt, 16 h, 73%; (d) NaNO2, AcOH-H2O, 70 oC to rt, 1 h, 72%; (e) Na2S2O4, NH4OH, 6 h, 75%; (d) phenylsulfonyl chloride or benzoyl chloride, pyridine, DCM, rt, 26 h, 71% for 15a; 75% for 15b. Structure-based Optimisation of Hits IC50 = 1.14 mM IC50 = 73 nM L. Pirrie, Westwood lab and M. Alphey, Naismith Lab.