Fluorescence-based biochemical primary high throughput screening assay to identify inhibitors of the fructose-bisphosphate aldolase (FBA) of M. tuberculosis
Name: Fluorescence-based biochemical primary high throughput screening assay to identify inhibitors of the fructose-bisphosphate aldolase (FBA) of M. tuberculosis. ..more
BioActive Compounds: 8215
Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center (SRIMSC)
Affiliation: The Scripps Research Institute, TSRI
Assay Provider: Mary Jackson, Colorado State
Network: Molecular Library Probe Production Centers Network (MLPCN)
Grant Proposal Number: 1 R21 NS066438-01
Grant Proposal PI: Mary Jackson, Colorado State
External Assay ID: ALD_INH_FLINT_1536_1X%INH PRUN
Name: Fluorescence-based biochemical primary high throughput screening assay to identify inhibitors of the fructose-bisphosphate aldolase (FBA) of M. tuberculosis.
The rise in antibiotic-resistant Mycobacterium tuberculosis and the lack of drugs capable of efficiently eradicating persistent bacilli responsible for life-long infections in humans emphasize the need for novel anti-TB agents with mechanisms of action different from those of existing drugs(1, 2). In fact, the latent form of Mycobacterium tuberculosis infects approximately a third of the global population (3). Class II fructose-1, 6-bisphosphate aldolase (FBA) is a key enzyme of glycolysis/gluconeogenesis induced in M. tuberculosis grown under oxygen-limiting conditions thought to mimic the physical microenvironment encountered by persistent bacilli in pulmonary lesions. Fructose bisphosphate aldolase (FBA) catalyzes the conversion of fructose bisphosphate into glyceraldehyde phosphate and dihydroxyacetone phosphate in a reversible fashion. As a result, this enzyme is a likely target for molecular tools to kill multi-drug-resistant as well as persistent M. tuberculosis (2).
Selective inhibition of FBA is expected to prevent M. tuberculosis from growing on host-derived fatty acids during persistent infection. Although ubiquitous in living organisms, FBAs can be divided into two classes which differ in their structure and reaction mechanism. While class I FBAs are the only type found in mammals, prokaryotes produce class II FBAs. The absence of class II FBAs from mammalian cells and the specificity of their structure and catalytic mechanism should make it possible to design specific inhibitors of class II enzymes that target pathogenic bacteria without affecting the host's gluconeogenetic and glycolytic pathways.
1. Siegel, R.E., Emerging gram-negative antibiotic resistance: daunting challenges, declining sensitivities, and dire consequences. Respir Care, 2008. 53(4): p. 471-9.
2. Fonvielle, M., M. Coincon, R. Daher, N. Desbenoit, K. Kosieradzka, N. Barilone, B. Gicquel, J. Sygusch, M. Jackson, and M. Therisod, Synthesis and biochemical evaluation of selective inhibitors of class II fructose bisphosphate aldolases: towards new synthetic antibiotics. Chemistry, 2008. 14(28): p. 8521-9.
3. Pegan, S.D., K. Rukseree, S.G. Franzblau, and A.D. Mesecar, Structural basis for catalysis of a tetrameric class IIa fructose 1,6-bisphosphate aldolase from Mycobacterium tuberculosis. J Mol Biol, 2009. 386(4): p. 1038-53.
Primary, PRUN, bacteria, tuberculosis, M. Tb. TB, infection, aldolase, fructose-bisphosphate aldolase, FBA, ALD, NADH, oxidation, NAD, fluorescence, fluor, FLINT, inhibition, enzyme, GDH, inhibitor, inhibit, decrease, screen, HTS, high throughput screen, 1536, Scripps Florida, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.
The purpose of this assay is to identify compounds that act as inhibitors of fructose bisphosphate aldolase (FBA) of M. tuberculosis, as monitored by loss (oxidation) of NADH in an enzymatic reaction. Fructose bisphosphate aldolase (FBA) catalyzes the conversion of fructose bisphosphate (FB) into the triose product glyceraldehyde 3 phosphate (G3P) in a reversible fashion. The G3P is converted to dihydroxyacetone phosphate (DHAP) by the helper enzyme triose phosphate isomerase (TPI). A second helper enzyme, glycerol phosphate dehydrogenase (GDH), converts the dihydroxyacetone phosphate to glycerol-3-phosphate with the concomitant oxidation of NADH to NAD, and thus the FBA activity is monitored by the reduction of well fluorescence as measured at 450 nm upon excitation at 340 nm. Compounds are tested in singlicate at a final nominal concentration of 1 uM.
Prior to the start of the assay, 5 uL /well of Buffer A (50 mM HEPES, 0.01% Triton X-100, 10% Glycerol, pH8.0) supplemented with 140 ng/mL FBA, 400 nM ZnCl2, 160 uM NADH and the helper enzymes GDH-TPI (4 U/mL) was dispensed into all wells of a 1536-well plate. Next, 10 nL of test compounds were delivered to each well using a PinTool. DMSO only was dispensed to negative control wells whereas a final concentration of 75 nM of reference compound TD3 was dispensed as a positive control. The assay was then initiated by dispensing 5 uL of Buffer A supplemented with 40 uM of FBP substrate. Plates were incubated at room temperature for 20 minutes before fluorescence was measured (Ex. 340 nm; Em. 450 nm) using the ViewLux plate reader (Perkin Elmer).
The percent inhibition for each compound was calculated as follows:
%_Inhibition = 100 * ( ( Test_Compound - Median_Low_Control ) / ( Median_High_Control - Median_Low_Control ) )
Test_Compound is defined as wells containing test compound.
Low_Control is defined as wells containing DMSO.
High_Control is defined as wells containing Compound TD3 (75 nM final)
PubChem Activity Outcome and Score:
Two values were calculated: (1) the average percent inhibition of all test compounds and (2) three times their standard deviation. The sum of these two values was used as a cutoff parameter. Any compound that exhibited a greater % inhibition than this cutoff parameter was declared active.
Potential fluorescent compounds that showed a percent inhibition value significantly higher than the one calculated for the High_Control (i.e. greater than the average % inhibition +/- 3 S.D. of the High_Control wells) were removed from the hit-cutoff calculation.
The reported PubChem Activity Score has been normalized to 100% observed inhibition. Negative % inhibition values are reported as activity score zero.
The PubChem Activity Score range for active compounds is 100-1, and for inactive compounds 1-0.
List of Reagents:
FBA enzyme (Assay Provider)
ZnCl2 (Fisher Scientific, part Z33-500)
NADH (EMD Biosciences, part 481913)
GDH-TPI (Sigma, part G1881)
HEPES (EMD Biosciences, part EM-5310)
Triton X-100 (Sigma, part T8787)
Glycerol (Fisher, part AC327255000)
Glyceraldehyde-3-phosphate (Sigma, part D7137)
TD3 reference control (Assay Provider)
1536-well plates (Corning, part 7298)
Due to the increasing size of the MLPCN compound library, this assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. In this case the results of each separate campaign were assigned 'Active/Inactive" status based upon that campaign's specific compound activity cutoff value. All data reported were normalized on a per-plate basis. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, and compounds that quench or emit fluorescence within the well. All test compound concentrations reported are nominal; the specific concentration for a particular test compound may vary based upon the actual sample provided by the MLSMR.
BAO: version: 1.4b1090
BAO: bioassay specification: assay stage: primary
BAO: bioassay specification: assay biosafety level: bsl1
BAO: assay format: biochemical format: protein format: single protein format
BAO: bioassay specification: assay measurement type: endpoint assay
BAO: bioassay specification: assay readout content: assay readout method: regular screening
BAO: bioassay specification: assay readout content: content readout type: single readout
BAO: meta target: molecular target: protein target: enzyme: lyase
BAO: meta target detail: binding reporter specification: interaction: protein-small molecule
BAO: detection technology: fluorescence: fluorescence intensity
** Test Concentration.
Data Table (Concise)