Rml C and D dose-response confirmation
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
BioActive Compounds: 14
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.
We have earlier reported the results of screening 200,000 compounds of the MLSCN library (AID 1532 and AID 1533), and identified compounds with >30.0% inhibition as hits. Here we report the dose-response results of the hit compounds. The identified hits were reordered from Biofocus DPI and tested in dose-response in triplicate from a top concentration of 55 #M. Compounds giving IC50 <55 uM were selected for further study to rule out fluorescent artifacts and to determine which of the enzymes, RmlC or RmlD, was the target of inhibition.
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)
Serial dilution compound plate---Polypropylene 384-well V-bottom plate (Greiner 781280)
The 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.
1. Serial dilute each compound in DMSO, 16 two-fold dilutions from 2.5mM to 76nM,
using tips on Evolution.
2. Dispense 4 ul of water into 384-well assay plate using Microdrop.
3. Pintool transfer compound twice (2x110 nL) from a 384-well dilution dose-response
plate into a 384-well assay plate.
4. 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
5. 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
6. Add 1 ul 83.5 mM MOPS buffer, into columns 1 and 23(blank), using microdrop.
7. Read NADPH fluorescence (Excitation 340/Emission 460 nm) at time 0 and after 90
minutes, at RT.
Data were analyzed in IDBS ActivityBase. Each dose-response assay 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. Each compound column (3-22) contained 16 two-fold dilutions of a single compound, ranging in concentration from 55 #M to 1.7 nM. Percent activity was calculated for each concentration of 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:
% Activity = 100*((∆signal-∆blank mean)/(∆control mean-∆blank mean))
IC50 scores were calculated as follows:
(1) Score = 5.75 x (pIC50-3), where pIC50 = -log(10) of Mean IC50 in mol/L
(2) For IC50 >55 uM (highest concentration tested), Score = 0
Activity Score Range:
For active compound, Score = 24-7
For Inactive compound, Score = 0
(1) IC50 <55 uM = active
(2) IC50 >55 uM (highest concentration tested)= inactive
ANALYSIS OF DOSE-RESPONSE RESULTS
Number of compounds tested = 388
Number of actives = 372
[Many of the actives identified were fluorescent at the excitation/emission wavelengths monitored in the assay (360/460 nm) and the corresponding dose-response curves may result from dose-dependent changes in fluorescence rather than enzyme inhibition. Studies to rule out fluorescent artifacts will be reported in a follow-up AID].
This assay was submitted to the PCMD (Scott L. 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.
Please direct correspondence to Sharmila Sivendran (firstname.lastname@example.org).
Data Table (Concise)