AID	Name	Type	Comment
588337	Summary of the probe development efforts to identify inhibitors of the fructose-bisphosphate aldolase (FBA) of M. tuberculosis	summary	Summary (FBA inhibitors)
588335	Counterscreen for inhibitors of the fructose-bisphosphate aldolase (FBA) of M. tuberculosis: Absorbance-based biochemical high throughput Glycerophosphate Dehydrogenase-Triosephosphate Isomerase (GDH-TPI) full deck assay to identify assay artifacts	screening	Counterscreen (GDH inhibitors in singlicate)
588726	Fluorescence-based biochemical primary high throughput screening assay to identify inhibitors of the fructose-bisphosphate aldolase (FBA) of M. tuberculosis	screening	Primary screen (FBA inhibitors in singlicate)
651616	Fluorescence-based biochemical high throughput confirmation assay for inhibitors of the fructose-bisphosphate aldolase (FBA) of M. tuberculosis	screening	Confirmation screen (FBA inhibitors in triplicate)

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_3XIC50 DRUN

Name: Fluorescence-based biochemical high throughput dose response assay for inhibitors of the fructose-bisphosphate aldolase (FBA) of M. tuberculosis.

Description:

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.

References:

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.

Keywords:

DRUN, dose response, titration, CRC, triplicate, 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.

Assay Overview:

The purpose of this assay is to determine dose response curves for available samples of compounds identified as active in a set of previous experiments entitled, "Fluorescence-based biochemical primary high throughput screening assay to identify inhibitors of the fructose-bisphosphate aldolase (FBA) of M. tuberculosis" (AID 588726).

Compounds 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 triplicate using a 10-point, 1:3 dilution series starting at a maximum nominal concentration of 10 uM.

Protocol Summary:

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 ) )

Where:

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)

For each test compound, percent activation was plotted against compound concentration. A four parameter equation describing a sigmoidal dose-response curve was then fitted with adjustable baseline using Assay Explorer software (Accelrys Inc). The reported IC50 values were generated from fitted curves by solving for the X-intercept value at the 50% inhibition level of the Y-intercept value. In cases where the highest concentration tested (i.e. 10 uM) did not result in greater than 50% inhibition, the IC50 was determined manually as greater than 10 uM.

PubChem Activity Outcome and Score:

Compounds with an IC50 greater than 10 uM were considered inactive. Compounds with an IC50 equal to or less than 10 uM were considered active.

Activity score was then ranked by the potency of the compounds with fitted curves, with the most potent compounds assigned the highest activity scores.

The PubChem Activity Score range for active compounds is 100-1, and for inactive compounds 0-0.

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. The MLSMR was not able to provide all compounds selected for testing.

Assay: Dictionary: Version: 0.1

Assay: CurveFit [1]: Equation: =( ( [Maximal Response] * [Concentration]^[Hill Slope] ) / ( [Inflection Point Concentration]^[Hill Slope] + [Concentration]^[Hill Slope] ) ) + [Baseline Response]

Assay: CurveFit [1]: Mask: Excluded Points

Grant Number: 1 R01 CA136699-01A1