Mycobacterium tuberculosis BioA enzyme inhibitor Measured in Biochemical System Using Plate Reader - 2163-03_Inhibitor_Dose_DryPowder_Activity
Mycobacterium tuberculosis (Mtb), Tuberculosis (TB), bioA, BioA enzyme, biotin biosynthesis, KAPA (7-keto-8-aminopelargonic acid), DAPA (7,8-diaminopelargonic acid) ..more
Mycobacterium tuberculosis (Mtb), Tuberculosis (TB), bioA, BioA enzyme, biotin biosynthesis, KAPA (7-keto-8-aminopelargonic acid), DAPA (7,8-diaminopelargonic acid)
BioA catalyzes the reversible transamination between KAPA and DAPA in the biotin biosynthetic pathway. BioD catalyzes the irreversible ATP-dependent carbonylation of DAPA to provide dethiobiotin (DTB), and this step drives the BioA reaction forward. Dethiobiotin is detected by diplacement of a fluorescent dethiobiotin probe (Fl-DTB) from streptavidin resulting in an increase in the fluorescent signal. The essence of the assay lies in the fluorescence quenching of Fl-DTB by streptavidin and restoration of fluorescence upon release from streptavidin. Another critical feature of the assay that enables it to be performed in a continuous format is the rapid reversible binding of dethiobiotin and Fl-DTB as a result of the substantially weaker binding affinities of these molecules for streptavidin, which contrasts with the functionally irreversible binding of biotin.
Uninhibited BioA enzyme catalyzes the biotin biosynthetic pathway to produce dethiobiotin which displaces coupled Fl-DTB, resulting in increased fluorescent signal.
Inhibited BioA enzyme does not produce dethiobiotin and no increase in fluorescent signal is observed.
followup assays on hits will eliminate autofluorescent compounds and compounds the inhibit the enzyme BioD, the enzyme the catalyzes the BioA product to dethiobiotin.
Inactive compounds show a high fluorescenct signal as dethiobiotin is produced.
Active compounds will show a decreased fluorescent signal and can be expressed as a %inhibition relative to the control inhibitor.
Black hi-base Aurora 1536-square well assay ready plates are used. These plates contain 7.5 nL pre-dispensed compounds at 8 point dose(10mM-78uM stock)in DMSO in compound wells, 7.5nL of DMSO in 128 negative control wells, and 1.5uL of positive enzyme inhibitor (EDTA) is in 128 positive control wells.. The assay components are added directly to the plates. The final compound screening concentration is 10uM-78nM.
Prepare reaction buffer and DAPA initiation solution fresh each day. DAPA intitation solution should be replenished with freshly made stock every 8 hours.
1. Dispense 1.5uL of positive inhibitor control EDTA into positive control wells of the assay plate.
2. Dispense 3.75 uL of reaction buffer into all wells of the assay plate
3. Dispense 3.75 uL of DAPA initiation solution into all wells of the assay plate
4. Incubate 45 min at 25 degrees C
5. Dispense 1.5 uL 500 mM EDTA into negative control and compound wells of the assay plate
6. Allow to equilibrate 5 min
7. Read on Viewlux microplate reader: Ex 485, em 530, cutoff 530 (FITC filters)
PRESENCE OF CONTROLS: Neutral control wells (NC; n=48) and positive control wells (PC; n=48) were included on every plate.
EXPECTED OUTCOME: Active compounds result in decreasing readout signal.
ACTIVE CONCENTRATION LIMIT:
For each sample, the highest valid tested concentration (Max_Concentration) was determined and the active concentration limit (AC_limit) was set to equal (2)(Max_Concentration).
The raw signals of the plate wells were normalized using the 'Neutral Controls Minus Inhibitors' method in Genedata Assay Analyzer (v10.0.2):
The median raw signal of the intraplate neutral control wells was set to a normalized activity value of 0.
The median raw signal of the intraplate positive control wells was set to a normalized activity value of -100.
Experimental wells values were scaled to this range.
PATTERN CORRECTION: The plate pattern correction algorithm 'Runwise Pattern (Multiplicative)' in Genedata (v10.0.2) was applied to the normalized plate data.
MEASUREMENT USED TO DETERMINE ACTIVE CONCENTRATION (AC): AC50
AC values were calculated using the curve fitting strategies in Genedata Screener Condoseo (7.0.3).
AC values were calculated up to the active concentration limit described for each sample.
pAC was set to equal -1*log10(AC)
Activity_Outcome = 1 (inactive) when:
a) compound shows activity but in a direction opposite to the expected outcome
in these cases, values describing curve fitting parameters (Sinf, S0, Hill Slope, log_AC50, log_AC50_SE) are set to null
b) curve fit is constant where activity is > -30% and < 30% at all tested concentrations, or
c) AC > AC_limit
Activity_Outcome = 2 (active) when:
AC <= AC_limit
Activity_Outcome = 3 (inconclusive) when:
a) Curve fitting strategy resulted in a constant fit with activity >= -70% but <= -30%, or
b) The fit was deemed not valid due to poor fit quality.
If PUBCHEM_ACTIVITY_OUTCOME = 1 (inactive) or 3 (inconclusive),
then PUBCHEM_ACTIVITY_SCORE = 0
If PUBCHEM_ACTIVITY_OUTCOME = 2 (active)
then PUBCHEM_ACTIVITY_SCORE = (10)(pAC)
Scores relate to AC in this manner:
120 = 1 pM
90 = 1 nM
60 = 1 uM
30 = 1 mM
0 = 1 M
When the active concentration (AC) is calculated to be greater than the highest valid tested concentration (Max_Concentration), the PUBCHEM_ACTIVITY_SCORE is calculated using Max_Concentration as the basis.
When the active concentration (AC) is calculated to be less than the lowest tested concentration, the PUBCHEM_ACTIVITY_SCORE is calculated using the lowest tested concentration as the basis.
The individual dose data point columns ('Activity_at_xxuM') reported here represent the median of valid (unmasked) replicate observations at each concentration. These values are the inputs to a curve fitting algorithm.
All other data columns represent values which are derived during the curve fitting algorithm; this may sometimes include automatic further masking of some replicate data points.
Occasionally this results in perceived inconsistencies: for example, between the derived 'Maximal_Activity' and the apparent most active data point.
* Activity Concentration. ** Test Concentration.
Data Table (Concise)