Drug Targe�ng of Novel An�microbials in the MEP Pathway:U�lizing an in silico and in vitro based screening approach

L. Jeffrey Johnson‡, Paul O. Neilsen‡, Mark Grier‡, Charles Herrmann†, Ashley Wentzel†, 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 Robo�cs, Springfield, New Jersey 07081, United States

An�bio�c resistance is a global problem, and the search for new an�bio�csor bacterial targets con�nues to be a frui�ul area of research, making themethylerythritol phosphate (MEP) pathway an a�rac�ve underexploredtarget.

U�lizing computa�onal methods we virtually screened a set of > 150,000compounds and iden�fied 200 poten�al deoxyxylulose reductoisomerase(DXR) inhibitors.

A medium throughput enzyme inhibitor screen, which kine�cally monitorsinhibi�on of the DXR enzyme, was developed to evaluate these poten�alinhibitors.

OVERVIEW

INTRODUCTION

Methods

CONCLUSIONS & 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 effec�ve assay for measuring DXR inhibi�on.

In Silico screening coordinated with op�mized 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 Inhibitor Screen is available from Echelon Biosciences, Inc.For more informa�on, please visit our website (www.echelon-inc.com)

Presenter Contact Informa�on:L. Jeffrey Johnsonjjohnson@echelon-inc.com(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 analyze bacterial permeability, cell uptake, and efflux pump interac�ons.

Screening Strategies for the Iden�fica�on of Poten�al An�microbials

Fosmidomycin

• Two unrelated essen�al pathways exist in nature for the biosynthesis ofisoprenoid metabolites, which include the methylerythritol phosphate(MEP) pathway, unique to bacteria, and the mevalonate (MVA) pathwayused by humans.

• The individual enzyma�c steps of the MEP pathway are a�rac�ve for thedevelopment of new an�bio�cs targeted against them, as few inhibitors ofthis pathway have been described.

• The third commi�ed step in the MEP Pathway, involving the conversion ofdeoxyxylulose phosphate (DXP) to MEP, was chosen as the target based onthe known inhibitor fosmidomycin.

RESULTSIn Silico Screening: Computa�onal Background

• The published crystal structure (PDB 2v2z) was used to model thebinding 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 ofthe binding site.

• A database of 63,808 molecules was obtained in SDF format andconverted into a 3D database with 5 conforma�ons selected for eachstructure 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. (Performedby Hudson Robotics, Springfield, NJ)

• Many of the top sugges�ons contained similar pharmacophore traits asfosmidomycin. For example, many of the hits suggested a sulfate group(yellow) in subs�tu�on for the phosphate group (yellow) and a subs�tutedaryl 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 ofhydrogen accep�ng groups were presented.

In Vitro Screening: MEP Synthase (DXR) Inhibitor Screen

• Following assay op�miza�on; the fosmidomycin posi�ve control was furtherevaluated and demonstrated an IC50 value of 130 nM, similar to inhibitoryvalues 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 100µM with a third compound inhibi�ng 32% of DXR ac�vity at this sameconcentra�on (C).

NextFollow up hit to lead iden�fica�on in a cell based screening assay.We developed an innova�ve gene�cally-engineered whole-cell (Salmonellatyphimurium) phenotypic screen to iden�fy compounds that selec�velyinhibit the MEP pathway. Hits from this screen produce three significantresults:

• 189 compounds were selected, weighed, and “blindly” plated in a 96 well plate format for screening. (Performed by Frontier Scientific, 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 analyzed blindly at Echelon Biosciences before compounds were decoded by Fron�er Scien�fic

DXR Inhibitor 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)

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Results

1. The compound is an�bacterial.

2. The compound hits the MEP pathway.

3. The compound does not affect the

MVA pathway- presumably with low

poten�al for human toxicity.

M E P C o n d it io n s

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