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 [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 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)
0 5 1 0 1 5 2 0 2 5 3 0 3 5 4 0-2 0
0
2 0
4 0
6 0
8 0
1 0 0
F u ll R e a c tio n
F u ll R e a c tio n (+ fo s m id o m y c in )
T im e , m in u te s
�-N
AD
PH
Co
nc
en
tra
tio
n(�
M)
0 1 2 3 4 5 6 7 8 9 1 0-1 0
0
1 0
2 0
3 0
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F u ll R e a c tio n
F u ll R e a c tio n (+ fo s m id o m y c in )P o te n t ia l In h ib ito r
T im e , m in u te s
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Co
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(�M
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D X RIn h ib it io n
F o sm id o m y c in
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3
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C o n c e n tra t io n , (� M )
DX
RA
cti
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IC 50 = 1 3 0 n M
0 1 2 3 4 5 60 .0
0 .1
0 .2
0 .3
0 .4
0 .5
0 .6
P la te N u m b e r
OD
(34
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m)
N e g a tiv e c o n tro l (-D X R )P o s itiv e c o n tro l (F u ll R x n )
Z ' = 0 .7 1 4
(A) (B) (C)
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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2 0
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6 0
7 0
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1 1 0
1 2 0
C o m p o u n d C o n c . (µ g /m L )
%In
hib
itio
n
M V A C o n d it io n s
0 .1 1 1 0 1 0 0 1 0 0 0
-2 0
-1 0
0
1 0
2 0
3 0
4 0
5 0
6 0
7 0
8 0
9 0
1 0 0
1 1 0
1 2 0
C o m p o u n d C o n c . (µ g /m L )
%In
hib
itio
n
W h o le -C e ll M E P P a th w a y -S e le c tiv e A n tib a c te r ia l S c re e n
C o m p o u n d 2
F o s m id o m y c in C o m p o u n d 1
C h lo ra m p h e n ic o l
0 1 0 2 0 3 0 4 00 .0
0 .1
0 .2
0 .3
0 .4
0 .5
0 .6
� -N A D P H O n lyF u ll E n z y m a tic R e a c tio n
T im e , m in u te s
�-N
AD
PH
Ab
s.(3
40
nm
) B u ffe r O n ly
(A)
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