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BioAssay: AID 1392

RNA polymerase SAR

DNA-directed RNA polymerase (EC 2.7.7.6) is responsible for bacterial RNA synthesis and as such is essential for bacterial gene expression. Owing to its central role in DNA transcription, the enzyme RNA polymerase is the target of various natural antibiotics. The best known is rifampicin, a potent and broad-spectrum anti-infective agent that is particularly effective against intracellular more ..
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 Tested Compounds
 Tested Compounds
All(52)
 
 
Active(15)
 
 
Inactive(31)
 
 
Inconclusive(6)
 
 
 Tested Substances
 Tested Substances
All(52)
 
 
Active(15)
 
 
Inactive(31)
 
 
Inconclusive(6)
 
 
AID: 1392
Data Source: PCMD (RNA POL SAR)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
Deposit Date: 2008-09-11

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 15
Depositor Specified Assays
AIDNameTypeComment
559RNA polymerasescreening
826RNA polymerase dose-response confirmationconfirmatory
Description:
Screening Center: Penn Center for Molecular Discovery
Center Affiliation: University of Pennsylvania
Network: Molecular Library Screening Center Network (MLSCN)
Assay Provider: Arkady Mustaev, Public Health Research Institute, Newark, NJ
Grant number: MH076325-01

DNA-directed RNA polymerase (EC 2.7.7.6) is responsible for bacterial RNA synthesis and as such is essential for bacterial gene expression. Owing to its central role in DNA transcription, the enzyme RNA polymerase is the target of various natural antibiotics. The best known is rifampicin, a potent and broad-spectrum anti-infective agent that is particularly effective against intracellular pathogens, such as Mycobacterium tuberculosis, for which it is one of the most widely used chemotherapeutic agents. However, the emergence of drug-resistant bacteria has become a major public health problem, so the discovery of novel RNA polymerase inhibitors is an important goal.

A high-throughput screen was designed to discover novel inhibitors of E. coli RNA polymerase. The screen consisted of an end-point assay monitoring the release of the fluorophore umbelliferone (Um). RNA polymerase catalyzes the polymerization of RNA on a DNA template by incorporation of adenine, cytosine, guanosine, and uracil from the corresponding nucleotide triphosphates, ATP, CTP, GTP, and UTP, with concomitant release of pyrophosphate (pp). In this assay GTP was replaced with Um-pppp-G, which releases Um-ppp upon incorporation of guanosine into RNA. Addition of alkaline phosphatase in a subsequent detection step cleaves Um-ppp to release the Um fluorophore. (See Koslov M., Bergendahl V., Burgess R., Goldfarb A., Mustaev A. Anal. Biochem. 342 (2005), 206-213 for a description of the development of this assay.)

Primary HTS results and confirmatory dose-response testing have been reported previously (AIDs 559 and 826). An active series of sulfonamides were synthesized and tested to determine SAR. These compounds were tested in dose-response using the umbelliferone release assay described above, and the results are reported here.
Protocol
Materials

The synthesis of Um-pppp-G and the purification of E. coli RNA polymerase and PUC19 plasmid DNA were carried out by Mustaev and co-workers as previously described (Koslov M., et al. Anal. Biochem. 342 (2005) 206-213). ATP, CTP, and UTP were purchased from Roche Applied Science; alkaline phosphatase was from New England Biolabs (Cat #M0290S); buffers and other reagents were from Sigma. Low-volume 384-well black plates were from Corning (Item #3676).

Stock solutions were made up as follows and stored at -80 C:

(1)Um-pppp-G: 12 mM in water
(2)NTP mix: 25 mM each of ATP, CTP, and UTP in water
(3)DNA template: 2 mg/mL of PUC19
(4)RNA polymerase: 3 mg/mL
(5)Transcription assay buffer (10x):
a.HEPES, pH 8.0 (200 mM)
b.NaCl (1 M)
c.Magnesium chloride (100 mM)
d.Manganese chloride (15 mM)
e.EDTA (1 mM)

Alkaline phosphatase was stored as supplied by the vendor at -20 C.

AMPSO buffer, pH 9.2 (250 mM) was stored at room temperature.

Assay

RNA polymerase (20 ug/mL) was incubated with DNA template (8 ug/mL), NTP mixture (12 uM each of ATP, CTP, and UTP), and Um-pppp-G (8.8 uM) in 10 uL of transcription assay buffer (see above) for 5 hr at room temperature. Um fluorophore was released by addition of 10 uL of alkaline phosphatase (1/2000 dilution, 5 U/mL) in 250 mM AMPSO, pH 9.2. Fluorescence (excitation 355, emission 460) was read after an additional 30 min at room temperature. IC50 values were determined as described below.

IC50 protocol

1. Serial dilute compounds at 50x concentration in DMSO (16 two-fold dilutions from 2.5 mM to 75 nM)
2. Fill low-volume plate with 4 uL water using Multidrop-micro
3. Add 5 uL transcription assay buffer to columns 1 and 23 using Multidrop-384
4. Add 200 nL of compound in DMSO (prepared as in (1) above) using Evolution pintool
5. Add 1 uL of NTP mix (120 uM) and Um-pppp-G (88 uM) in 5x transcription assay buffer using Multidrop-micro
6. Add 5 uL RNA polymerase (40 ug/mL) and PUC19 plasmid DNA (16 ug/mL) in transcription assay buffer using Multidrop-384
7. Incubate for 5 hr at room temperature
8. Add 10 uL alkaline phosphatase (1/2000 dilution, 5 U/mL) in 250 mM AMPSO.
9. Incubate for 30 min at room temperature
10. Read fluorescence (excitation 355, emission 460) on Envision reader

Data analysis

Data were analyzed in IDBS ActivityBase. Each IC50 plate contained compounds in columns 3-22, controls (enzyme, no compound) in columns 2 and 24, and blanks (no enzyme) in columns 1 and 23. Each column 3-22 contained 16 two-fold dilutions of a single compound, ranging in concentration from 50 uM to 1.5 nM. Percent activity was calculated for each dilution of each compound from the signal in fluorescence units (FU) and the mean of the plate controls and the mean of the plate blanks using the following equation:

% Activity = 100*((signal-blank mean)/(control mean-blank mean))

Dose response curves of percent activity were fit using XLfit equation 205 (four parameter logistic fit with maximum percent activity and minimum percent activity fixed at 100 and 0, respectively).
Comment
Activity scoring

Activity scoring is based on the linear-log formula developed by Eduard Sergienko at the San Diego Center for Chemical Genomics:

Activity scores were calculated as follows:

(1) Score = 20 x (pIC50-3.2), where pIC50 = -log(10) of IC50 in mol/L
(2) For IC50 >50 uM, score was calculated from percent activity at maximum concentration tested in assay (50 uM):
Score = (80-percent activity at max concentration)/2

Activity Outcome

IC50 values were determined as described in protocol above. The percent activity at the maximum concentration is reported and can be used to estimate the potency of compounds for which the IC50 values were >50 uM.

Activity outcome is reported as follows:

(1) IC50 <50 uM = active
(2) IC50 >50 uM, percent activity at 50 uM < 70 = inconclusive
(3) IC50 >50 uM, percent activity at 50 uM > 70 = inactive

Contributors

This assay was submitted to the PCMD (Scott Diamond, Director; University of Pennsylvania) by Arkady Mustaev, Public Health Research Institute, Newark, NJ. Compounds were synthesized by Michael Myers, dose-response testing was conducted by Edinson Lucumi and Chun-Hao Chiu, and data were submitted by Andrew Napper.

Correspondence

Please direct correspondence to Andrew Napper (napper@seas.upenn.edu).
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1QualifierString
2IC50 #1*FloatμM
3IC50 #1 Hill slopeFloat
4IC50 #1 R-squaredFloat
5IC50 #1 min concentrationFloatμM
6IC50 #1 percent activity at min concentrationFloat%
7IC50 #1 max concentrationFloatμM
8IC50 #1 percent activity at max concentrationFloat%
9IC50 #1 signal at 0.00152 microM (0.00152μM**)Float
10IC50 #1 signal at 0.00305 microM (0.00305μM**)Float
11IC50 #1 signal at 0.00610 microM (0.0061μM**)Float
12IC50 #1 signal at 0.01221 microM (0.01221μM**)Float
13IC50 #1 signal at 0.02441 microM (0.02441μM**)Float
14IC50 #1 signal at 0.04883 microM (0.04883μM**)Float
15IC50 #1 signal at 0.09766 microM (0.09766μM**)Float
16IC50 #1 signal at 0.19531 microM (0.19531μM**)Float
17IC50 #1 signal at 0.39063 microM (0.39063μM**)Float
18IC50 #1 signal at 0.78125 microM (0.78125μM**)Float
19IC50 #1 signal at 1.5625 microM (1.5625μM**)Float
20IC50 #1 signal at 3.125 microM (3.125μM**)Float
21IC50 #1 signal at 6.25 microM (6.25μM**)Float
22IC50 #1 signal at 12.5 microM (12.5μM**)Float
23IC50 #1 signal at 25 microM (25μM**)Float
24IC50 #1 signal at 50 microM (50μM**)Float
25IC50 #1 control meanFloat
26IC50 #1 control standard deviationFloat
27IC50 #1 number of control wellsInteger
28IC50 #1 control percent CVFloat%
29IC50 #1 blank meanFloat
30IC50 #1 blank standard deviationFloat
31IC50 #1 number of blank wellsInteger
32IC50 #1 blank percent CVFloat%
33IC50 #1 signal-background ratioFloat
34IC50 #1 plate Z-factorFloat

* Activity Concentration. ** Test Concentration.

Data Table (Concise)
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