Bookmark and Share
BioAssay: AID 1533

Rml C and D inhibition 384-well mixture HTS from 1536-well compound plates

This screen is for compounds that have the potential to be developed into new drugs against tuberculosis (TB) because they inhibit the enzymes required for the formation of the cell wall of the tuberculosis bacterium. New drugs are needed because the rate of cure with the present drugs is very slow, and prevalence of Mycobacterium tuberculosis resistance to present drugs is increasing. Recently, an increase in co-infection of HIV and M. tuberculosis has occurred, and treatment with present drugs results in harmful HIV/TB drug interactions. ..more
_
   
 Tested Compounds
 Tested Compounds
All(158925)
 
 
Active(46)
 
 
Inactive(157823)
 
 
Inconclusive(1058)
 
 
 Tested Substances
 Tested Substances
All(158970)
 
 
Active(46)
 
 
Inactive(157866)
 
 
Inconclusive(1058)
 
 
AID: 1533
Data Source: PCMD (RML 1536-MIXTURE HTS)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
Deposit Date: 2009-03-03

Data Table ( Complete ):           View Active Data    View All Data
Targets
BioActive Compounds: 46
Related Experiments
AIDNameTypeComment
1695Rml C and D dose-response confirmationConfirmatorydepositor-specified cross reference
1696Rml C and D fluorescent artifact dose-response confirmationConfirmatorydepositor-specified cross reference
Description:
Molecular Library Screening Center Network (MLSCN)
Penn Center for Molecular Discovery (PCMD)
Assay Provider: Michael McNeil, Colorado State University, Fort Collins, CO
MLSCN Grant: DA024889-01

This screen is for compounds that have the potential to be developed into new drugs against tuberculosis (TB) because they inhibit the enzymes required for the formation of the cell wall of the tuberculosis bacterium. New drugs are needed because the rate of cure with the present drugs is very slow, and prevalence of Mycobacterium tuberculosis resistance to present drugs is increasing. Recently, an increase in co-infection of HIV and M. tuberculosis has occurred, and treatment with present drugs results in harmful HIV/TB drug interactions.

To identify potential anti-TB agents, we focused on two enzymes that act sequentially in the formation of dTDP-rhamnose (dTDP-Rha), a biosynthetic precursor required for TB cell wall formation and found to be essential for the growth of M. smegmatis and M. tuberculosis. Active compounds inhibit the activity of the two enzymes, dTDP-6-deoxy-D-xylo-4-hexulose 3,5-epimerase (RmlC) and dTDP-6-deoxy-L-lyxo-4-hexulose reductase (RmlD). Enzyme activity is measured by the decrease in fluorescence upon the oxidation of NADPH to NADP. The enzymes are balanced to allow detection of an inhibitor of either enzyme. Here we report the results of the primary screening of 200,000 compounds as mixtures of 4 compounds per well in 384-well plates and retest of the hits as individual compounds.

Mixtures were tested at 14.6 uM, corresponding to concentrations of 3.64 uM from each individual compound, for the 1536-well library primary screen. The four compounds contained in each mixture that showed >30.0% inhibition were reordered from BioFocus DPI and retested individually at a concentration of 5.5 uM. Compounds that gave >30.0% inhibition on retest were tested in dose-response and selected for further study to determine which of the enzymes, RmlC or RmlD, was the target of inhibition.
Protocol
COMPOUND POOLING
The MLSCN 1536-well library were screened as mixtures of 4 compounds per well in 384-well plates. Compounds were supplied by BioFocus DPI as 2.5 mM solutions in DMSO, stored in 1536 V-bottom plates. Mixtures of the 1536-well library of compounds were generated by pintool (384-pin V&P Scientific) transfer of 91 nL of DMSO solution from each four quadrants of a 1536-well compound plate into a single well of a 384-well compound dilution plate containing 25 ul of water and 4 uL of this compound mixture was transferred into a single well of a 384-well assay plate.
Thus the 2.5 mM solutions in each 1536-well plate are mixed 4-per-well in the 384-well compound dilution plate as follows: wells A5, A6, B5, B6, etc (1536-well) transferred into A3, etc (384-well). Followed by dilution compound plate mixture solution transfer into 384-well assay plate as follows: wells A3, A4, A5, A6, etc (384-well) transferred into A3, A4, A5, A6, etc (384-well)
Final mixture composition:
1536-well compound library compound dilution plate: 9.1 uM per compound, 36.8 uM in total mixture. 1536-well assay plate: 3.64 uM per compound, 14.6 uM total mixture.
MATERIALS
The purified rhamnosyl biosynthetic enzymes RmlB, RmlC and RmlD, cloned and expressed in E. coli, were provided by Michael McNeil of Colorado State University. MOPS, TritonX 100 and TDP-Glc were purchased from Sigma. MgCl2 and glycerol were purchased from Fisher. NADPH was purchased from Roche.
The RmlC enzyme substrate, dTDP-4-keto-6-deoxy-D-xylo-hexulose (dTDP-KDX), was synthesized enzymatically by converting d-TDP-glucose (2.5 mg) to dTDP-KDX using RmlB (2.4 ug), in 50 mM MOPS buffer, pH 7.4, at 37 deg C for 1 hr. Aliquots of dTDP-KDX were stored at -80 deg C. Each aliquot was subjected to freeze thaw no more than three times.
Assay plate---Black 384-well plate (Corning 3676)
Compound dilution plate---Polypropylene 384-well V-bottom plate (Greiner 781280)
Compound storage plate---Polypropylene 1536-well V-bottom plate (Greiner 782270)
ASSAY
The high-throughput screen for inhibitors of the Mycobacterium tuberculosis cell wall enzymes, RmlC and RmlD, reported here is based on the decrease in fluorescence observed upon the oxidation of NADPH to NADP. The change in fluorescence is calculated from the difference between the fluorescence read at the beginning of the assay and one after 90 min.

HTS PROTOCOL: 1536-well compound library mixture screen
1.Dispense 25 ul of water into 384-well compound dilution plate using Multidrop.
2.Pintool transfer compound (92 nL) from each four quadrants of a 1536-well compound plate (2.5 mM) into a single well of a 384-well compound dilution plate containing 25 uL of water. Transfer 4 uL of this mixture solution into a 384-well assay plate.
3.Add 5 ul 83.5 mM MOPS buffer containing (2x) RmlC, (2x) RmlD, and (2x) NADPH using multidrop. Final buffer composition: 50mM MOPS, pH 7.4 with 1 mM MgCl2, 10% glycerol and 0.01% tritonX-100 - Rml C final concentration: 2.63x10-4 ug/ul - Rml D final concentration: 6.53x10-4 ug/ul - NADPH final concentration: 25 uM
4.Add 1 ul (10x) substrate (TDP-KDX) in 83.5 mM MOPS buffer, into columns 2-22 and 24, using microdrop. - TDP-KDX final concentration: 200 uM
5.Add 1 ul 83.5 mM MOPS buffer, into columns 1 and 23(blanks), using microdrop.
6.Read NADPH fluorescence (Excitation 340/Emission 460 nm) at time 0 and after 90 minutes, at RT.
RETEST PROTOCOL
Same as HTS protocol, except for steps 1 and 2:
1.Dispense 4 uL of water into 384-well assay plate using microdrop.
2.Pintool transfer compound (110 nL) from 384-well compound (0.5 mM) plate into 384-well assay plate.
DATA ANALYSIS
Data were analyzed in IDBS ActivityBase. Each HTS plate contained compounds in columns 3-22, controls (100% activity) in columns 2 and 24, and blanks (0% activity, no TDP-KDX) in columns 1 and 23. HTS percent inhibition was calculated for each compound from the change in fluorescence over 90 min (∆Signal), calculated from t=0 and t=90 min reads, and the mean of the change in plate controls and blanks over the 90 min using the following equation:
% Inhibition = 100*[1-((∆Signal-∆Blankmean)/(∆Controlmean-∆Blankmean))]
Comment
ACTIVITY SCORING
Activity scores of 40-13 represent active compounds; 12-0 represent inactives. Scores were calculated as follows:
Percent inhibition >30.0 in primary HTS, >30.0 in both retests, and mean retest percent inhibition >100:
Score = 40
Percent inhibition >30.0 in primary HTS, >30.0 in both retests, and mean retest percent inhibition between 100 & 0:
Score = 0.4 x mean percent inhibition on retest
Percent inhibition >30.0 in primary HTS, >30.0 in only one retest, and lower retest percent inhibition >0:
Score = 0.4 x lower percent inhibition on retest
Percent inhibition >30.0 in primary HTS, between 30 & 0 in prior 384-well mixture HTS:
Score = 0.4 x lower (384-well mixture) percent inhibition
Percent inhibition >30.0 in primary HTS, <0 in prior 384-well mixture HTS:
Score = 0
Percent inhibition >30.0 in primary HTS, >30.0 in only one retest, and lower retest percent inhibition <0:
Score = 0
Percent inhibition >30.0 in primary HTS, <30.0 in both retests, and mean percent inhibition on retest >0:
Score = 0.4 x mean percent inhibition on retest
Percent inhibition >30.0 in primary HTS, <30.0 in both retests, and mean percent inhibition on retest <0:
Score = 0
Percent inhibition >100 in primary HTS, compounds not retested:
Score = 30
Percent inhibition between 100 & 30 in primary HTS and compound unavailable for retest:
Score = 0.3 x percent inhibition in primary HTS
Percent inhibition between 30.0 & 0 in primary HTS:
Score = 0.3 x percent inhibition in primary HTS
Percent inhibition <0 in primary HTS:
Score = 0
ACTIVITY OUTCOME
Activity outcome is reported as follows:
(1) Percent inhibition between 30 and 110 in primary HTS, >30 in both retests = active
(2) Percent inhibition between 30 and 110 in primary HTS, <30 in one or both retests = inactive
(3) Percent inhibition between 30 and 110 in primary HTS, <30 in prior 384-well mixture HTS = inactive
(4) Percent inhibition between 30 and 110 in primary HTS, compound unavailable for retest = inconclusive
(4) Percent inhibition >110 in primary HTS = inconclusive
(5) Percent inhibition <30 in primary HTS = inactive
ANALYSIS OF SCREENING RESULTS
Primary HTS plate statistics were as follows:
Screening concentration = 3.64 uM (each compound in 4-compound mixture)
14.6 uM (total concentration of mixture)
Number of plates = 128
Median Z-factor = 0.6
Mean control percent CV = 7.91
Maximum control percent CV = 12.6
A hit cut-off of 30.0% inhibition was selected. Based on this cutoff, a hit rate of 0.97% was observed (assuming observed activity originated from only one compound in each 4-compound mixture):
Total compounds screened: 159,098
Hits (>30% inhibition): 1,536 wells equivalent to 6,059 compounds
Hits (30-110% inhibition): 5,118 compounds
Hits (30-110% inhibition) after excluding 2,214 compounds inactive in prior 384-well mixture HTS: 5,118-2,214 = 2,904 compounds
Inactives (<30% inhibition): 39,255 wells equivalent to 153,038 compounds
Hits reclassified as inactives based on prior 384-well HTS results: 2,214 compounds
Hit rate (all hits >30% inhibition): (1,536/159,098)*100 = 0.97%
[Of the 1536 wells that showed >30% inhibition, 234 gave between 110 and 2090% inhibition. These large values represent an INCREASE in fluorescence over the 90 min assay. Enzymatic conversion of NADPH to NADP results in a decrease in fluorescence, so the fluorescence increases are most likely artifacts due to intrinsic compound fluorescence. Previous dose-response confirmation following 384-well mixture HTS confirmed that all the compounds that showed >100% inhibition were artifacts due to an increase in fluorescence. Therefore, in this study only those mixtures giving between 30 and 110% inhibition were retested. All compounds contained in the mixtures giving >110% inhibition have been flagged as inconclusive.]
Retest plate statistics were as follows:
Screening concentration = 5.5 uM
Number of plates = 9
Median Z-factor = 0.47
Mean control percent CV = 9.74
Maximum control percent CV = 12.6
A hit cut-off of 30% inhibition was selected. Based on this cutoff, 53 out of 2,723 compounds were active, giving a retest hit rate of 1.94%. However, given the likelihood that activity observed in the primary HTS originated from only one compound in each 4-compound mixture retested, the retest rate becomes 3.45% based on the 1536 mixtures active in the primary HTS:
Total compounds retested: 2,723
Hits (>30% inhibition): 53
Inactives (<30% inhibition): 2,670
Retest hit rate: (53/2723)*100 = 1.94%
CONTRIBUTORS
This assay was submitted to the PCMD (Scott Diamond, Director; University of Pennsylvania) by Michael McNeil (PI), Colorado State University, Fort Collins, CO. Assay development and HTS was carried out by Sharmila Sivendran and data were submitted by Sharmila Sivendran and Andrew Napper.
CORRESPONDENCE
Please direct correspondence to Sharmila Sivendran (ssharm@seas.upenn.edu)
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1HTS percent inhibition (3.6μM**)Float%
2HTS percent inhibition (prior 384-well compound plates) (5.5μM**)Float%
3Lower retest percent inhibition (5.5μM**)Float%
4Mean retest percent inhibition (5.5μM**)Float%
5Retest percent inhibition #2 (5.5μM**)Float%
6Retest percent inhibition #2 (5.5μM**)Float%

** Test Concentration.
Additional Information
Grant Number: DA024889-01

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
PageFrom: