Through the direction and funds of NIAID, Southern
Research added a high-throughput screening (HTS)
services to the program in the fall of 2001.
The HTS component allows for screening of large compound libraries against specific validated targets, expanding the primary in vitro screening efforts to a high throughput format when necessary and supports the design and implementation of in silico systems for predicting drug characteristics such as solubility, human intestinal absorption, bioavailability and potential toxicity. These services are available at no cost to the supplier.
Southern Research Institute provides HTS services in Birmingham, AL.
The HTS screening contract, led by
Southern's Ms. Lucile White, receives selected compound libraries from the TAACF and screens these libraries against various target assays that have been modified, validated, and optimized for a high throughput format. The HTS screens are developed through collaborations with investigators who have identified assays for M. tuberculosis targets.
Function
The function of the High Throughput Screening division of the TAACF is threefold:
- To develop and implement biochemical, target-specific Mycobacterium tuberculosis drug screening assays in a high-throughput format;
- To develop and implement M. tuberculosis metabolic stage-specific drug screening assays in a high-throughput format;
- To implement an in silico system for predicting drug-like characteristics such as solubility, human intestinal absorption, bioavailability, and potential toxicities.
Primary HTS drug testing capabilities
- Mycobacterium tuberculosis biochemical target panel (NIAID/SRI-HTS)
- Mycobacterium tuberculosis primary in vitro screen (NIAID/SRI-HTS)
Current M. tuberculosis in vitro targets:
Dihydrofolate Reductase Rv2763c, E.C.1.5.1.3 (DHFR)
DHFR catalyses the reaction, 7,8-dihydrofolate + NADPH _ 5,6,7,8-tetrahydrofolate +NADP. Compounds are assayed against purified, recombinant human and Mtb DHFR. Enzyme activity is determined by the increase in optical density at 490 nm due to the nonenzymatic reduction of MTS (3-[4,5-dimethylthiazol-2-yl]-5-(3-carboxymethoxyphenyl] -2-[4-sulfophenyl-2H-tetrazolium, inner salt) by tetrahydrofolate to a soluble formazan. Reactions are run in 96-well plates with a reaction mix containing phosphate buffer, pH 7, EDTA, 2-mercaptoethanol, NADPH, dihydrofolate, MTS and enzyme. The reaction is initiated upon addition of dihydrofolate and followed for two min to determine velocity.
Initial screening is done with a single concentration of each compound, which is usually 10 µM. If this amount inhibits Mtb DHFR by 90% or more, the assay is repeated, using Mtb and human DHFR, with several concentrations of compound to determine the amount that inhibits the reaction by 50% (IC50). The selectivity of the compound for Mtb DHFR is reported as the ratio of the human DHFR IC50 to the Mtb DHFR IC50. The higher the selectivity ratio, the more selective is the compound for Mtb.
The assay is currently available for screening.
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Mycobacterium tuberculosis
Enoyl-ACP Reductase (InhA, Rv 1484, E.C.
1.3.1.9)
Mycobacteria have two
fatty acid synthases, FAS I (normally
found only in eukaryotes) and FAS II.
FAS I in M. tuberculosis is a
multidomain enzyme that catalyzes
formation of shorter chain saturated
fatty acids (<C26), while FAS II
produces long chain (C26-C56) a-alkyl,
b-hydroxy fatty acids. The FAS II
pathway found in M. tuberculosis
produces mycolic acids, very long chain
(C40-C60) a-branched fatty acids, which
are a major constituent of the
arabinogalactan of the cell wall1.
M. tuberculosis enoyl-ACP
reductase, known as InhA (E.C. 1.3.1.9),
catalyzes the final reduction step of
this pathway which, in an NADH specific
reduction of 2-trans-enoyl ACP,
specifically reduces long chain
substrates (C12-C24) attached to CoA as
a carrier protein. InhA, a functional
homologue of the Escherichia coli
FabI, shows great potential for drug
discovery, as human hosts utilize a
distinctive enzyme system to produce
fatty acids2.
To determine potential
inhibitors of InhA, we have developed a
kinetic assay that measures the InhA-catalyzed
reduction of the carbon-carbon double
bond of 2-trans-dodecenoyl-CoA.
This reduction occurs in the presence of
NADH to yield acyl-ACP and NAD+.
Overview of
InhA Reaction.
Initial screening is
done with a single concentration of each
compound, which is usually 10
mM.
If this amount inhibits Mtb InhA
by 20% or more, the assay is repeated in
dose response format to determine the
compound concentration which inhibits
the reaction by 50% (IC50).
For additional
information on this assay, please see
http://www.srdiscovery.com/posters.html
1Kuo
MR, et. al. Targeting
tuberculosis and malaria through
inhibition of Enoyl reductase: compound
activity and structural data. J Biol
Chem. 2003 Jun 6; 278(23):20851-9.
2Heath
RJ, White SW, Rock CO. Lipid
biosynthesis as a target for
antibacterial agents. Prog Lipid Res.
2001 Nov;40(6):467-97. Review.
3Gill
RB, et. al.
A High Throughput
Screening Assay for Inhibitors of
Mycobacterium tuberculosis Enoyl ACP
Reductase (InhA).
Society of Biomolecular
Screening Annual Meeting. 2004 Sept.
10-15,
Orlando, FL.
4Fletcher
TM III, et. al. A High
Throughput Screen for the
Mycobacterium Tuberculosis Enoyl
Acyl Carrier Protein Reductase, InhA.
Society of Biomolecular
Screening Annual Meeting. 2003 Sept.
20-25, Portland, OR.
The assay is
currently available for screening.
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Isocitrate Lyase (ICL,
Rv0467, E.C.4.1.3.1) - Malate Synthase
(MS, Rv1837c, E.C. 2.3.3.9); Combined
Isocitrate lyase (ICL)
and malate synthase (MS) are the two
enzymes of the glyoxylate shunt of the
tricarboxylic acid cycle. These enzymes
allow the utilization of C2
substrates such as acetate or fatty
acids, during periods of carbon
starvation. Evidence suggests that this
pathway is required for growth and
persistence of Mycobacterium
tuberculosis (Mtb) in vivo,
specifically during the pathogenic
infection of host macrophages.
Inhibitors of this pathway are expected
to be active against persistent
organisms which are recalcitrant to
present therapeutic strategies.
Using isocitrate as
the starting substrate for ICL, we can
detect the corresponding production of
free CoA by the second enzyme MS. Free
CoA reacts with
5,5¢-dithiobis(2-nitrobenzoic acid) (DTNB)
to produce the colored thionitrobenzoate
dianion which can be measured spectrophotometrically
at 450 nm1.
Initial screening is done with a single
concentration of each compound, which is
usually 10µM. Compounds that inhibit
the coupled ICL-MS assay by more than
20% are then assayed specifically
against ICL and MS in uncoupled
reactions.
1Smith CV, et.al.
Biochemical and structural studies of
malate synthase from Mycobacterium
tuberculosis. J Biol Chem.
278:1735-43, 2003.
The assay is currently
available for screening.
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Isocitrate
Lyase Rv0465, E.C.4.1.3.1 (ICL)
Isocitrate lyase (ICL) is an important
enzyme in the glyoxylate cycle during
carbohydrate starvation in
Mycobacterium tuberculosis (Mtb);
it catalyzes the cleavage of isocitrate
to glyoxylate and succinate, allowing
the organisms to survive on acetate or
fatty acids. The glyoxylate cycle is
not present in higher animals, and due
to its necessity for survival for the
persistent phase of the infection, ICL
is considered an ideal drug target for
Mtb. The reaction mixture
contains MOPS buffer (pH 6.8), MgCl2,
NADH, isocitrate, lactic dehydrogenase (LDH),
and ICL. The kinetic reaction is
initiated by the addition of ICL.
Enzyme activity is determined by
coupling the ICL reaction to LDH and
measuring the oxidation of NADH to NAD
at 340 nm1.
TCA Cycle and Glyoxylate Cycle
Initial screening is done with a single
concentration of each compound, which is
usually 10µM. Compounds that inhibit
the ICL assay by more than 20% are then
screened against the coupled enzyme LDH
to eliminate compounds that are
inhibiting LDH. ICL specific actives are
then screened in a dose response format
to determine the concentration that
inhibits ICL by 50% (IC50).
For
additional information on this assay
please see
http://www.srdiscovery.com/posters.html
1 Blossom Sneed,
Sabrina van-Ginkel, Rachel Gill, Larry
Ross, Melinda Ingrum Sosa, Sara Cooley,
Anthony Pate, Anna Manouvakhova, David
Barnett, Lucile White, Thomas M Fletcher
III, James C Sacchettini. A High
Throughput Screening Assay for
Isocitrate Lyase Inhibitors of
Mycobacterium Tuberculosis. Society
of Biomolecular Screening 10th
Annual Conference and Exhibition.
September, 2004, Orlando, FL.
The assay is currently
available for screening.
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Malate
Synthase(MS, Rv1837c, E.C. 2.3.3.9)
Malate synthase is one of
the two enzymes of the glyoxylate shunt
of the tricarboxylic acid cycle.
Briefly, MS is incubated with the two
substrates, glyoxylate and acetyl-CoA,
for 25 min. The amount of CoA-SH formed
is measured by titrating the free thiol
group with 5,5'-dithiobis(2-nitrobenzoic
acid) [DTNB] and monitoring the
absorbance at 450 nm1.
Overview of Tricarboxylic
Acid (TCA)
Cycle in M. tuberculosis.
Initial screening is done
with a single concentration of each
compound, which is usually 10µM.
Compounds that inhibit the MS assay by
more than 20% are defined as actives.
These compounds are then screened in a
dose response format to determine the
concentration that inhibits MS by 50%
(IC50).
1
Smith CV, et.al.
Biochemical and structural studies of
malate synthase from Mycobacterium
tuberculosis. J Biol Chem.
278:1735-43, 2003.
The assay is currently
available for screening.
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Pantothenate
Synthetase Rv3602c, EC 6.3.2.1 (PanC)
Pantothenate
Synthetase (PS) catalyzes the formation
of pantothenate from D-pantoate and β-alanine
in bacteria, yeast and plants. This is
an important precursor for the
biosynthesis of two essential cofactors,
coenzyme A and acyl carrier protein. PS
is therefore considered a potential drug
target for Mycobacterium tuberculosis
(Mtb). The reaction mixture
contains HEPES buffer (pH 7.8), MgCl2,
β-alanine, ATP, potassium
phosphoenolpyruvate, NADH, pantoate,
lactate dehydrogenase (LDH), pyruvate
kinase, myokinase, and PS. The kinetic
reaction is initiated with the addition
of PS. The second product of the PS
reaction, AMP, is measured by a series
of catalysis steps (myokinase, pyruvate
kinase and LDH). The final step is the
oxidation of NADH to NAD by LDH which is
measured at 340 nm1.
Mtb H37Rv Pathway: pantothenate
biosynthesis
Initial
screening is done with a single
concentration of each compound, which is
usually 10µM. Compounds that inhibit
the PS assay by more than 20% are then
screened in an assay that contains
myokinase, pyruvate kinase, and LDH to
eliminate compounds that are inhibiting
steps other than PS. PS specific
actives are then screened in a dose
response format to determine the
concentration that inhibits PS by 50%
(IC50).
For additional information on this assay
please see
http://www.srdiscovery.com/posters.html
1
Blossom Sneed, Larry Ross, Melinda
Ingrum Sosa, Sara Cooley, Anthony Pate,
Anna Manouvakhova, David Barnett, E.
Lucile White, Thomas M Fletcher III,
ShuiShu Wang, David Eisenberg, Celia W
Goulding. A High Throughput Screening
Assay for Pantothenate Synthetase
Inhibitors of Mycobacterium
Tuberculosis. Society of
Biomolecular Screening 10th
Annual Conference and Exhibition.
September, 2004, Orlando, FL.
The assay is currently
available for screening.
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FtsZ Rv2150c, (FtsZ)
FtsZ is the first non-regulatory
element to appear at the septum. FtsZ
and tubulin form a unique family of
GTPases.
The assay is
currently in development.
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