RNA polymerase dose-response confirmation
DNA-directed RNA polymerase (EC 18.104.22.168) 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 ..
BioActive Compounds: 17
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 22.214.171.124) 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 have been reported previously (AID 559). Actives from the HTS were tested in dose-response using the umbelliferone release assay described above. Results of the dose-response confirmation are reported here.
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.
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.
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 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).
Activity scoring is based on the linear-log formula developed by Eduard Sergienko at the San Diego Center for Chemical Genomics. The activity score reported here is calculated from the results of follow-up IC50 testing on compounds that showed >20% inhibition in the primary HTS:
Activity score = IC50 score #1 + IC50 score #2 + IC50 score #3.
IC50 scores were calculated as follows:
(1) Score = 5.75 x (pIC50-3), where pIC50 = -log(10) of IC50 in mol/L
(2) For IC50 >50 uM (zero in IC50 column), score was calculated from percent activity at maximum concentration tested in assay (50 uM):
Score = [5.75 x (0-3)] + [(100-percent activity at max concentration)/1.75]
Compounds that gave percent inhibition >20 in the primary HTS were judged to be hits and these compounds were selected for follow-up IC50 testing. 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 in all three IC50 determinations = active
(2) IC50 >50 uM in all IC50 determinations = inactive
(3) IC50 <50 uM in one or more determinations & >50 uM in one or more = inconclusive
Analysis of screening results
Results of retesting compounds that gave percent inhibition >20 in the primary HTS were as follows:
Hits (>20% inhibition in both locations) = 105
Hits active in IC50 = 17 (16% retest rate)
This assay was submitted to the PCMD (Scott Diamond, Director; University of Pennsylvania) by Arkady Mustaev, Public Health Research Institute, Newark, NJ. Assay development was carried out by Edinson Lucumi and Andrew Napper, HTS was conducted by Edinson Lucumi, and data were submitted by Andrew Napper.
Our thanks go to Parag Shah and Bill Denney for enormous help in setting up the HTS lab and troubleshooting its operation.
Please direct correspondence to Andrew Napper (firstname.lastname@example.org).
* Activity Concentration. ** Test Concentration.
Data Table (Concise)