uHTS identification of inhibitors of NadD in a Colorimetric assay
The increasing occurrence of multidrug resistant strains of bacterial pathogens poses a significant threat to public health calling for urgent actions. More than half of all S. aureus infections in U.S. hospitals are resistant to common antibiotics such as penicillins and cephalosporins. Some of the strains of MRSA (methicillin-resistant S. aureus), are resistant to "drugs of last resort" such as vancomycin. Therefore, new antibacterial agents acting on previously unexploited novel targets must be urgently found. ..more
BioActive Compounds: 2812
Data Source: Sanford-Burnham Center for Chemical Genomics (SBCCG)
Source Affiliation: Sanford Burnham Medical Research Institute (SBMRI, La Jolla, CA)
Network: NIH Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Number: 1 R03 MH095597-01A1
Assay Provider: Dr. Andrei Osterman, Sanford Burnham Medical Research Institute, La Jolla, CA
The increasing occurrence of multidrug resistant strains of bacterial pathogens poses a significant threat to public health calling for urgent actions. More than half of all S. aureus infections in U.S. hospitals are resistant to common antibiotics such as penicillins and cephalosporins. Some of the strains of MRSA (methicillin-resistant S. aureus), are resistant to "drugs of last resort" such as vancomycin. Therefore, new antibacterial agents acting on previously unexploited novel targets must be urgently found.
Comparative and functional genomics studies identified a therapeutically unexplored target pathway, biosynthesis of an indispensable redox cofactor, nicotinamide adenine dinucleotide (NAD) (2, 6). Targeting of the key essential genes involved in this pathway presents a promising strategy for the development of novel antibiotics(1, 4, 5, 8).
Blocking NAD biosynthesis by inhibition of an essential enzyme, a nicotinic acid mononucleotide adenylyltransferase (NaMNAT) of the NadD family conserved in most bacterial pathogens, leads to growth suppression of Gram-negative and Gram-positive bacteria(3, 7), thus validating it as a druggable target amenable to inhibition by small molecules.
The primary goal of this high-throughput screening (HTS) campaign is to identify potent inhibitors of S. aureus NadD (saNadD). Chemical probes inhibiting saNadD function would provide invaluable tools to help elucidate the efficacy of targeting NAD biosynthesis in bacterial pathogens, and would ultimately lead to the development of new antibacterial therapies.
In this biochemical assay, saNadD adenylyltransferase converts nicotinate mononucleotide (NaMN) in the presence of ATP, into two products, nicotinic acid adenine dinucleotide (NaAD) and inorganic pyrophosphate (PPi). Formation of PPi product is monitored through malachite green-based colorimetric detection of inorganic phosphate (Pi) generated by a coupled pyrophosphatase.
1. Boshoff, H. I., X. Xu, K. Tahlan, C. S. Dowd, K. Pethe, L. R. Camacho, T. H. Park, C. S. Yun, D. Schnappinger, S. Ehrt, K. J. Williams, and C. E. Barry, 3rd. 2008. Biosynthesis and recycling of nicotinamide cofactors in mycobacterium tuberculosis. An essential role for NAD in nonreplicating bacilli. J Biol Chem 283:19329-41.
2. Gerdes, S. Y., M. D. Scholle, M. D'Souza, A. Bernal, M. V. Baev, M. Farrell, O. V. Kurnasov, M. D. Daugherty, F. Mseeh, B. M. Polanuyer, J. W. Campbell, S. Anantha, K. Y. Shatalin, S. A. Chowdhury, M. Y. Fonstein, and A. L. Osterman. 2002. From genetic footprinting to antimicrobial drug targets: examples in cofactor biosynthetic pathways. J Bacteriol 184:4555-72.
3. Huang, N., R. Kolhatkar, Y. Eyobo, L. Sorci, I. Rodionova, A. L. Osterman, A. D. MacKerell, and H. Zhang. 2010. Complexes of Bacterial Nicotinate Mononucleotide Adenylyltransferase with Inhibitors: Implication for Structure-Based Drug Design and Improvement. Journal of Medicinal Chemistry 53:5229-5239.
4. Moro, W. B., Z. Yang, T. A. Kane, C. G. Brouillette, and W. J. Brouillette. 2009. Virtual screening to identify lead inhibitors for bacterial NAD synthetase (NADs). Bioorg Med Chem Lett 19:2001-5.
5. Moro, W. B., Z. Yang, T. A. Kane, Q. Zhou, S. Harville, C. G. Brouillette, and W. J. Brouillette. 2009. SAR studies for a new class of antibacterial NAD biosynthesis inhibitors. J Comb Chem 11:617-25.
6. Osterman, A. L., and T. P. Begley. 2007. A subsystems-based approach to the identification of drug targets in bacterial pathogens. Prog Drug Res 64:131, 133-70.
7. Sorci, L., Y. Pan, Y. Eyobo, I. Rodionova, N. Huang, O. Kurnasov, S. Zhong, A. D. MacKerell, Jr., H. Zhang, and A. L. Osterman. 2009. Targeting NAD biosynthesis in bacterial pathogens: Structure-based development of inhibitors of nicotinate mononucleotide adenylyltransferase NadD. Chem Biol 16:849-61.
8. Vilcheze, C., B. Weinrick, K.-W. Wong, B. Chen, and W. R. Jacobs. 2010. NAD+ auxotrophy is bacteriocidal for the tubercle bacilli. Molecular microbiology 76:365-77.
1) NadD enzyme was provided by Dr. Osterman's laboratory
2) NaMN substrate, ATP, pyrophosphatase (Ppase), & BSA were purchased from Sigma-Aldrich (Cat # N7764, A7699, I5907, & A7888 respectively)
3) Pi Colorlock Gold (c) Kit was purchased from Innova Biosciences (Cat# 303-0030 or 303-0125)
4) HEPES Buffer: 25 mM HEPES pH 7.5
5) Control Buffer: 25 mM HEPES pH 7.5, 10 mM MgCl2, 0.1 mg/mL BSA
6) NadD Buffer: 25 mM HEPES pH 7.5, 0.1 mg/mL BSA
7) Substrate Buffer: 25 mM HEPES pH 7.5, 0.1 mM ATP, 20 mM MgCl2, 0.1 mg/mL BSA.
8) Colorlock Gold (c)/Accelerator Mix: 0.99X Colorlock Gold (c), 0.01X Accelerator (from Innova kit)
9) Stabilizer Mix: 0.6X Stabilizer (from Innova kit) in H2O
10) 1536-well black clear bottom plates were obtained from Corning (Cat # 3891)
1) Using LabCyte Echo, transfer 50 nL of test compounds from a 2 mM compound source plate into assay plate Cols. 5-44 (Final concentration of test compounds is 20 uM, 1 % DMSO). Transfer 50 nL of 100% DMSO into assay plate Col. 4 & 45-47.
3) Using Thermo Combi nL, dispense 7.5 uL of HEPES Buffer to columns 1-3 & 48; dispense 2.5 uL of HEPES Buffer to columns 46-47.
4) Using Thermo Combi nL, dispense 2.5 uL of 3 nM NadD enzyme in NadD Buffer to columns 4-45.
5) Using Thermo Combi nL, dispense 2.5 uL of 0.025 mM NaMN and 0.8 U/mL Ppase in Substrate Buffer to columns 4-47.
6) Spin plates at 1500 rpm for 1 minute with Eppendorf centrifuge 5810.
7) Incubate for 15 minutes at room temperature.
8) Using Thermo Combi nL, dispense 1.5 uL of Colorlock Gold (c)/Accelerator Mix to columns 1-48
9) Spin plates at 1500 rpm for 1 minute with Eppendorf centrifuge 5810
10) Incubate for 15 minutes at room temperature
11) Using Thermo Combi nL, dispense 1 uL of Stabilizer Mix solution to columns 1-48
12) Spin plates at 1500 rpm for 1 minute with Eppendorf centrifuge 5810.
13) Incubate for 15 minutes at room temperature
14) Read plates on Perkin Elmer Envision at 620 nm in absorbance mode
Compounds that demonstrated a normalized or corrected activity of >= 30% at 20 uM concentration are defined as inhibitors of the reaction.
The experimental values were normalized by the difference between values from neutral and stimulator control wells in each plate. Then normalized data was corrected to remove systematic plate patterns due to artifacts such as dispensing tip issues etc. Further information about data correction is available at http://www.genedata.com/products/screener.html.
To simplify the distinction between the inactives of the primary screen and of the confirmatory screening stage, the Tiered Activity Scoring System was developed and implemented.
Activity scoring rules were devised to take into consideration compound efficacy, its potential interference with the assay and the screening stage that the data was obtained. Details of the Scoring System will be published elsewhere. Briefly, the outline of the scoring system utilized for the assay is as follows:
1) First tier (0-40 range) is reserved for primary screening data. The score is correlated with % activity in the assay:
a. If outcome of the primary screen is inactive, then the assigned score is 0
b. If outcome of the primary screen is inconclusive, then the assigned score is 10
c. If outcome of the primary screen is active, then the assigned score is 20
Scoring for Single concentration confirmation screening is not applicable to this assay.
d. If outcome of the single-concentration confirmation screen is inactive, then the assigned score is 21
e. If outcome of the single-concentration confirmation screen is inconclusive, then the assigned score is 25
f. If outcome of the single-concentration confirmation screen is active, then the assigned score is 30
This scoring system helps track the stage of the testing of a particular SID. For the primary hits which are available for confirmation, their scores will be greater than 20. For those which are not further confirmed, their score will stay under 21.
2) Second tier (41-80 range) is reserved for dose-response confirmation data and is not applicable in this assay
3) Third tier (81-100 range) is reserved for resynthesized true positives and their analogues and is not applicable in this assay
** Test Concentration.
Data Table (Concise)