DNA-directed RNA polymerase (EC 220.127.116.11) 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: 98
Molecular Library Screening Center Network (MLSCN)
Penn Center for Molecular Discovery (PCMD)
Assay Provider: Dr. Arkady Mustaev, Public Health Research Institute, Newark, NJ
MLSCN Grant: RO3 MH076325-01
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 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.
The high-throughput screen reported here 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.)
This assay is a part of the Molecular Library Screening Center Network (MLSCN).
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. HTS was performed using 10 uM compound.
1. Fill low-volume plate with 4 uL water using Multidrop-micro
2. Add 5 uL transcription assay buffer to columns 1 and 23 using Multidrop-384
3. Add 200 nL of compound (0.5 mM in DMSO) using Evolution pintool
4. Add 1 uL of NTP mix (120 uM) and Um-pppp-G (88 uM) in 5x transcription assay buffer using Multidrop-micro
5. Add 5 uL RNA polymerase (40 ug/mL) and PUC19 plasmid DNA (16 ug/mL) in transcription assay buffer using Multidrop-384
6. Incubate for 5 hr at room temperature
7. Add 10 uL alkaline phosphatase (1/2000 dilution, 5 U/mL) in 250 mM AMPSO.
8. Incubate for 30 min at room temperature
9. Read fluorescence (excitation 355, emission 460) on Envision reader
Data were analyzed in IDBS ActivityBase. Each HTS plate contained compounds (10 uM in 2% DMSO) in columns 3-22, controls (enzyme, no compound) in columns 2 and 24, and blanks (no enzyme) in columns 1 and 23. HTS percent inhibition was calculated for 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:
% Inhibition = 100*(1-((signal-blank mean)/(control mean-blank mean)))
Activity scores were calculated as follows:
For positive percent inhibition, score = percent inhibition
For negative percent inhibition, score = 0
(Note that compounds that fluoresce at excitation 355/emission 460 nm give a signal higher than the plate control that corresponds to a substantial negative percent inhibition)
Activity outcome is reported as follows:
(1) percent inhibition > 20 = active
(2) perecent inhibition < 20 = inactive
Analysis of screening results
Results of testing compounds the MLSCN library of compounds against RNA polymerase are as follows:
Hits (>20% inhibition): 98 compounds
Inactives (<20% inhibition): 62139 compounds
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 Nuzhat Motlekar and 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).
Data Table (Concise)