Drug Targe �ng of Novel An�micr obials in the MEP Pathway: U�lizing an in silico and in vitro based scr eening appr oach L. Jeffrey Johnson ‡ , Paul O . Neilsen ‡ , Mark Grier ‡ , Charles Herrmann † , Ashley Wentz el † , Alan H. Katz* , Andrew Witschi*, Mark L. Nelson ‡† ‡ Echelon Biosciences, Inc. Salt Lake City, Utah 84103, United States † Fron�er Scien�fic Services, Inc. Newark, Delaware 19711, United States *Hudson R obo�cs, Springfield, New Jersey 07081, United States An�bio�c resistance is a global problem, and the search for new an�bio�cs or bacterial targets con�nues to be a frui�ul area of research, making the methylerythritol phosphate (MEP) pathway an a�rac�ve underexplored target. U�lizing computa�onal methods we virtually screened a set of > 150,000 compounds and iden�fied 200 poten�al deoxyxylulose reductoisomerase (DXR) inhibitors. A medium throughput enzyme inhibitor screen, which kine�cally monitors inhibi�on of the DXR enzyme, was developed to evaluate these poten�al inhibitors. OVERVIEW INTRODUCTION Methods CONCL USIONS & ACKNOWLEDGEMENTS Booth #1449 Our work highlights the poten�al of mul�ple discovery pla� orms to target the bacterial MEP pathway for iden�fying future novel an�bio�cs. The DXR Inhibitor Screen is quick, convenient, and an eff ec�ve assay for measuring DXR inhibi�on. In Silico screening coordinated with op�miz ed compound libraries remains a promising method for early iden�fica�on of poten�al inhibitors. We thank Dr. Colin Ferguson, Dr. Charles Testa, and Dr. Mark Brown for the early development of MEP associated reagents. DXR Inhibit or Scr een is available from Echelon Biosciences, Inc. For more informa�on, please visit our website ( www.echelon-inc.com) Presenter Contact Informa�on: L. Jeffrey Johnson [email protected] (801) 588-0455 ext. 375 In Silico Virtual Screen of the DXR enzyme and compound library In Vitro Inhibitor Screen developed to validate enzyma�c target Whole-Cell Based Screening approach to confirm hits and further analyz e bacterial permeability, cell uptake, and efflux pump interac�ons. Scr eening Strategies for the Iden�fica�on of Poten�al An�microbials Fosmidomycin • Two unrelated essen�al pathways exist in nature for the biosynthesis of isoprenoid metabolites, which include the methylerythritol phosphate (MEP) pathway, unique to bacteria, and the mevalonate (MVA) pathway used by humans. • The individual enzyma�c steps of the MEP pathway are a�rac�ve for the development of new an�bio�cs targeted against them, as few inhibitors of this pathway have been described. • The third commi�ed step in the MEP Pathway, involving the conversion of deoxyxylulose phosphate (DXP) to MEP, was chosen as the target based on the known inhibitor fosmidomycin. RESULTS In Silico Screening: Computa�onal Background • The published crystal structure (PDB 2v2z) was used to model the binding site. • The an�bio�c fosmidomycin and a series of known inhibitors (2-6) were used as a training set to determine plausible conforma�ons of the binding site. • A database of 63,808 molecules was obtained in SDF format and converted into a 3D database with 5 conforma�ons selected for each structure for further evalua�on in docking experiments. Results • The library was docked into the binding site using ROCS (OpenEye Scien�fic) that iden�fied 200 suggested compounds for in vitro screening. (Performed by Hudson Robocs, Springfield, NJ) • Many of the top sugges�ons contained similar pharmacophore traits as fosmidomycin. For example, many of the hits suggested a sulfate group (yellow) in subs�tu�on for the phosphate group (yellow) and a subs�tuted aryl or alkyl chain for the middle linker group (green) in fosmidomycin. • A wide range of varia�ons was suggested for combina�ons of the carbonyl (red) and hydroxyl amine moiety (blue), but in general a combina�on of hydrogen accep�ng groups were presented. In Vitro Screening: MEP Synthase (DXR) Inhibitor Screen • Following assay op�miz a�on; the fosmidomycin posi�ve control was further evaluated and demonstrated an IC 50 value of 130 nM, similar to inhibitory values described previously (A). • The inhibitor screen when averaged demonstrated a Z’ factor of 0.714 (B). • Two of the 200 test compounds showed modest inhibi�on of 5 - 10% at 100 μM with a third compound inhibi�ng 32% of DXR ac�vity at this same concentra�on (C). Next Follow up hit to lead iden�fica�on in a cell based screening assay. We developed an innova�ve gene�cally-engineered whole-cell (Salmonella typhimurium) phenotypic screen to iden�fy compounds that selec�vely inhibit the MEP pathway. Hits from this screen produce three significant results: • 189 compounds were selected, weighed, and “blindly” plated in a 96 well plate format for screening. (Performed by Froner Scienfic, Newark, DE) • Compound plates were transferred to Echelon Biosciences for tes�ng • Plated compounds were pre-incubated with the DXR enzyme with shaking for 10 minutes. • Deoxyxylulose Phosphate (DXP) substrate was then added to start the reac�ons. • The absorbance was recorded in kine�c mode at 340 nm. • Data was analyz ed blindly at Echelon Biosciences before compounds were decoded by Fron�er Scien�fic DXR Inhibit or Screen The DXR Inhibitor Screen monitors a decrease in β-NADPH levels which directly corresponds with the conversion of the DXP substrate to MEP product (A). The screen will monitor compounds for inhibi�on of DXR ac�vity, as demonstrated by the fosmidomycin control inhibitor, shown at 100 μM (B). (B) 0 5 10 15 20 25 30 35 40 -20 0 20 40 60 80 100 Full Reaction Full Reaction (+ fosmidomycin) Time, minutes -NADPH Concentration ( M) 0 1 2 3 4 5 6 7 8 9 10 -10 0 10 20 30 40 50 60 Full Reaction Full Reaction (+ fosmidomycin) Potential Inhibitor Time, minutes -NADPH Concentration, ( M) DXR Inhibition Fosmidomycin 0.01 0.1 1 10 100 -1 0 1 2 3 4 Concentration, ( M) DXR Activity IC 50 = 130 nM 0 1 2 3 4 5 6 0.0 0.1 0.2 0.3 0.4 0.5 0.6 Plate Number OD (340 nm) Negative control (-DXR) Positive control (Full Rxn) Z' = 0.714 (A) (B) (C) Results 1. The compound is an�bacterial. 2. The compound hits the MEP pathway. 3. The compound does not aff ect the MVA pathway- presumably with low poten�al for human toxicity. MEP Conditions 0.1 1 10 100 1000 -20 -10 0 10 20 30 40 50 60 70 80 90 100 110 120 Compound Conc. (μg/mL) % Inhibition MVA Conditions 0.1 1 10 100 1000 -20 -10 0 10 20 30 40 50 60 70 80 90 100 110 120 Compound Conc. (μg/mL) % Inhibition Whole-Cell MEP Pathway-Selective Antibacterial Screen Compound 2 Fosmidomycin Compound 1 Chloramphenicol 0 10 20 30 40 0.0 0.1 0.2 0.3 0.4 0.5 0.6 -NADPH Only Full Enzymatic Reaction Time, minutes -NADPH Abs. (340 nm) Buffer Only (A)