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BioAssay: AID 1532

Rml C and D inhibition 384-well mixture HTS

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
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 Tested Compounds
 Tested Compounds
All(106271)
 
 
Active(417)
 
 
Inactive(105278)
 
 
Inconclusive(576)
 
 
 Tested Substances
 Tested Substances
All(106289)
 
 
Active(417)
 
 
Inactive(105296)
 
 
Inconclusive(576)
 
 
 Related BioAssays
 Related BioAssays
AID: 1532
Data Source: PCMD (RML 384-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 ):           Active    All
BioActive Compounds: 417
Depositor Specified Assays
AIDNameTypeComment
1696Rml C and D fluorescent artifact dose-response confirmationconfirmatory
1695Rml C and D dose-response confirmationconfirmatory
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 22.0 uM, corresponding to concentrations of 5.5 uM from each individual compound, for the 384-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 384-well library was screened as mixtures of 4 compounds per well in 384-well plates. Compounds were supplied by BioFocus DPI as 10 mM solutions in DMSO, stored in 384-well V-bottom plates. Dilution compound plates of the 384-well compound library were made at 0.5 mM from the 10 mM plates. Mixtures of the 384-well library of compounds were generated using a pintool (384-pin, V&P Scientific) transfer of 110 nL of DMSO solution from a single well of four 384-well dilution compound plates, at 0.5 mM in DMSO, into a single well of a 384-well assay plate containing 4 uL of water.
Thus the 0.5 mM solutions in each 384-well plate are mixed 4-per-well in the 384-well assay plate as follows: wells A3(plate 1), A3(plate 2), A3(plate 3), A3(plate 4), etc (384-well) transferred into A3, etc (384-well)

Final mixture composition:
384-well compound library assay plate: 5.5 uM per compound, 22.0 uM in 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)

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. 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: 384-well compound library mixture screen
1.Dispense 4 ul of water into 384-well assay plate using Microdrop.
2.Pintool transfer compound (110 nL) from four 384-well dilution compound plates (0.5 mM) into one 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 multidrop. - TDP-KDX final concentration: 200 uM
5.Add 1 ul 83.5 mM MOPS buffer, into columns 1 and 23(blank), using microdrop.
6.Read NADPH fluorescence (Excitation 340/Emission 460 nm) at time 0 and after 90 minutes, at RT.

RETEST PROTOCOL

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 a single 384-well compound plate (0.5 mM) 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 90 min, using the following equation:

% Inhibition = 100*[1-((∆Signal-∆Blankmean)/(∆Control mean-∆Blank mean))]
Comment
ACTIVITY SCORING

Activity 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, >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 and compound unavailable for retest:
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 >30.0 in primary HTS, >30 in both retests = active
(2) Percent inhibition >30.0 in primary HTS, <30 in one or both retests = inactive
(3) Percent inhibition >30.0 in primary HTS, compound unavailable for retest = inconclusive
(4) Percent inhibition <30.0 in primary HTS = inactive

ANALYSIS OF SCREENING RESULTS

Primary HTS plate statistics were as follows:

Screening concentration = 5.5 uM (each compound in 4-compound mixture)
22.0 uM (total concentration of mixture)
Number of plates = 85
Median Z-factor = 0.65
Mean control percent CV = 8.00
Maximum control percent CV = 11.5

A hit cut-off of 30.0% inhibition was selected. Based on this cutoff, a hit rate of 0.75% was observed (assuming observed activity originated from only one compound in each 4-compound mixture):

Total compounds screened: 106,290
Hits (>30.0% inhibition): 792 wells equivalent to 3119 compounds
Inactives (<30.0% inhibition): 26408 wells equivalent to 103,171 compounds
Hit rate: (792/106,290)*100 = 0.75%

[Of the 792 wells that showed >30% inhibition, 178 gave between 100 and 1717% 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. Nevertheless, the 710 compounds in these wells were selected for retesting together with those giving <100% inhibition.]

Retest plate statistics were as follows:

Screening concentration = 5.5 uM
Number of plates = 10 (tested in duplicate)
Median Z-factor = 0.59
Mean control percent CV = 7.00
Maximum control percent CV = 9.91

A hit cut-off of 30% inhibition in both tests was selected. Based on this cutoff, 417 out of 2543 compounds were active, giving a retest hit rate of 16.4%. 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 65.6% based on the 792 mixtures active in the primary HTS:

Total compounds retested: 2543
Confirmed active (>30% inhibition in both tests): 417
Inactives (<30% inhibition in one or both tests): 2543-417 =2126
Retest hit rate: (417/792)*100 = 65.6%
Inconclusive (>30% inhibition in HTS, unavailable for retest): 3119-2543 = 576

[Of the 417 compounds that gave >30% inhibition, 171 gave between 100 and 3682% 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. Nevertheless, these 171 compounds were flagged as active and selected for dose-response testing together with those giving <100% inhibition.]

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 (5.5μM**)Float%
2Lower retest percent inhibition (5.5μM**)Float%
3Mean retest percent inhibition (5.5μM**)Float%
4Retest percent inhibition #1 (5.5μM**)Float%
5Retest percent inhibition #2 (5.5μM**)Float%

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

Data Table (Concise)
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