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BioAssay: AID 1527

Primary biochemical high throughput screening assay to identify inhibitors of VIM-2 metallo-beta-lactamase

Name: Primary biochemical high throughput screening assay to identify inhibitors of VIM-2 metallo-beta-lactamase ..more
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 Tested Compounds
 Tested Compounds
All(290731)
 
 
Active(2575)
 
 
Inactive(288157)
 
 
 Tested Substances
 Tested Substances
All(290893)
 
 
Active(2575)
 
 
Inactive(288318)
 
 
AID: 1527
Data Source: The Scripps Research Institute Molecular Screening Center (VIM-2_INH_EPIABS_1536_%INH)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2009-02-25
Modify Date: 2012-12-26

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 2575
Related Experiments
Show more
AIDNameTypeProbeComment
1854Summary of probe development efforts to identify selective inhibitors of VIM-2 metallo-beta-lactamaseSummary1 depositor-specified cross reference
1856Epi-absorbance-based counterscreen for selective VIM-2 inhibitors: biochemical high throughput screening assay to identify inhibitors of IMP-1 metallo-beta-lactamase.Screening depositor-specified cross reference
1857FRET-based counterscreen assay for selective VIM-2 inhibitors: biochemical high throughput screening assay to identify epi-absorbance assay artifactsScreening depositor-specified cross reference
1860Epi-absorbance-based confirmation biochemical high throughput screening assay to identify selective inhibitors of VIM-2 metallo-beta-lactamase.Screening depositor-specified cross reference
1866Epi-absorbance-based counterscreen assay for selective VIM-2 inhibitors: biochemical high throughput screening assay to identify inhibitors of TEM-1 serine-beta-lactamase.Screening depositor-specified cross reference
1919Epi-absorbance-based dose response biochemical high throughput screening assay for selective inhibitors of VIM-2 metallo-beta-lactamaseConfirmatory depositor-specified cross reference
1920Epi-absorbance-based counterscreen for selective VIM-2 inhibitors: dose response biochemical high throughput screening assay to identify inhibitors of IMP-1 metallo-beta-lactamase.Confirmatory depositor-specified cross reference
1925Epi-absorbance-based counterscreen for selective VIM-2 inhibitors: dose response biochemical high throughput screening assay to identify inhibitors of TEM-1 serine-beta-lactamase.Confirmatory depositor-specified cross reference
1926FRET-based counterscreen for selective VIM-2 inhibitors: dose response biochemical high throughput screening assay to identify epi-absorbance assay artifacts.Confirmatory depositor-specified cross reference
1927FRET-based counterscreen for selective VIM-2 inhibitors: dose response biochemical high throughput screening assay to identify inhibitors of IMP-1 metallo-beta-lactamase.Confirmatory depositor-specified cross reference
2128Late stage results from the probe development efforts to identify selective inhibitors of VIM-2 metallo-beta-lactamase: probe resultsOther depositor-specified cross reference
2184Epi-absorbance-based counterscreen assay for common VIM-2 and IMP-1 inhibitors: biochemical high throughput screening assay to identify inhibitors of TEM-1 serine-beta-lactamase.Screening depositor-specified cross reference
2187Epi-absorbance-based confirmation assay for common VIM-2 and IMP-1 inhibitors: biochemical high throughput screening assay to identify inhibitors of VIM-2 metallo-beta-lactamase.Screening depositor-specified cross reference
2189Epi-absorbance-based confirmation assay for common IMP-1 and VIM-2 inhibitors: biochemical high throughput screening assay to identify inhibitors of IMP-1 metallo-beta-lactamase.Screening depositor-specified cross reference
2317Late stage results from the probe development efforts to identify selective inhibitors of VIM-2 metallo-beta-lactamase: Prior art resultsScreening depositor-specified cross reference
2754Epi-absorbance-based dose response assay for common IMP-1 and VIM-2 inhibitors: biochemical high throughput screening assay to identify inhibitors of VIM-2 metallo-beta-lactamaseConfirmatory depositor-specified cross reference
2755Epi-absorbance-based dose response assay for common IMP-1 and VIM-2 inhibitors: biochemical high throughput counterscreen to identify inhibitors of TEM-1 metallo-beta-lactamaseConfirmatory depositor-specified cross reference
2756Epi-absorbance-based dose response assay for common IMP-1 and VIM-2 inhibitors: biochemical high throughput screening assay to identify inhibitors of IMP-1metallo-beta-lactamaseConfirmatory depositor-specified cross reference
2767Late stage counterscreen results from the probe development effort to identify common IMP-1 and VIM-2 inhibitors: Epi-absorbance-based biochemical dose response assay for inhibitors of TEM-1 metallo-beta-lactamaseConfirmatory depositor-specified cross reference
2768Late stage results from the probe development effort to identify common IMP-1 and VIM-2 inhibitors: Epi-absorbance-based biochemical dose response assay for inhibitors of IMP-1metallo-beta-lactamaseConfirmatory depositor-specified cross reference
2769Late stage results from the probe development effort to identify common IMP-1 and VIM-2 inhibitors: Epi-absorbance-based biochemical dose response assay for inhibitors of VIM-2 metallo-beta-lactamaseConfirmatory depositor-specified cross reference
449774Late stage counterscreen results from the probe development efforts to identify common IMP-1 and VIM-2 inhibitors: wildtype E. coli growth inhibition dose response assay (MIC: minimum inhibitory concentration)Other depositor-specified cross reference
463099Late stage assay provider counterscreen results from the probe development efforts to identify common IMP-1 and VIM-2 inhibitors: IMP1-transformed E. coli growth inhibition dose response assay in the presence of imipenemOther depositor-specified cross reference
463100Late stage assay provider counterscreen results from the probe development efforts to identify common IMP-1 and VIM-2 inhibitors: VIM-2-transformed E. coli growth inhibition dose response assay in the presence of imipenemOther depositor-specified cross reference
504620Late stage assay provider results from the probe development efforts to identify selective inhibitors of VIM-2 metallo-beta-lactamase: VIM-2-transformed E. coli growth inhibition in the presence of imipenem (synergy)Confirmatory depositor-specified cross reference
624079Late stage assay provider results from the probe development efforts to identify nonselective inhibitors of VIM-2 metallo-beta-lactamase: Absorbance-based biochemical assays to determine the ability of probe candidates and selected analogs to inhibit VIM-2Confirmatory1 depositor-specified cross reference
624080Late stage assay provider results from the probe development efforts to identify inhibitors of VIM-2 metallo-beta-lactamase (nonselective): Growth inhibition of clinically relevant VIM-2 transformed P. aeruginosa (PA641) in the presence of imipenem (synergy)Other depositor-specified cross reference
624081Late stage assay provider results from the probe development efforts to identify inhibitors of VIM-2 metallo-beta-lactamase (nonselective): VIM-2-transformed E. coli growth inhibition in the presence of imipenem (synergy)Other1 depositor-specified cross reference
624082Late stage assay provider results from the probe development efforts to identify inhibitors of VIM-2 metallo-beta-lactamase (nonselective): Growth inhibition of clinically relevant New Delhi metallo-beta-lactamase-1 (NDM-1)-transformed K. pneumoniae (BAA-2146) in the presence of imipenem (synergy)Other depositor-specified cross reference
624083Late stage assay provider results from the probe development efforts to identify nonselective inhibitors of VIM-2 metallo-beta-lactamase: Absorbance-based biochemical assays to determine the ability of probe candidates and selected analogs to inhibit VIM-2Confirmatory1 depositor-specified cross reference
624084Late stage assay provider results from the probe development efforts to identify nonselective inhibitors of VIM-2 metallo-beta-lactamase: Absorbance-based biochemical assays to determine the ability of probe candidates and selected analogs to inhibit IMP-1Confirmatory1 depositor-specified cross reference
624085Late stage assay provider results from the probe development efforts to identify nonselective inhibitors of VIM-2 metallo-beta-lactamase: Absorbance-based biochemical assays to determine the ability of probe candidates and selected analogs to inhibit IMP-1Confirmatory1 depositor-specified cross reference
624090Late stage assay provider results from the probe development efforts to identify nonselective inhibitors of VIM-2 metallo-beta-lactamase: Absorbance-based biochemical assays to determine the ability of probe candidates and selected analogs to inhibit AmpCConfirmatory depositor-specified cross reference
624092Late stage assay provider results from the probe development efforts to identify nonselective inhibitors of VIM-2 metallo-beta-lactamase: Absorbance-based biochemical assays to determine the ability of probe candidates and selected analogs to inhibit TEM-1Confirmatory depositor-specified cross reference
624095Late stage assay provider results from the probe development efforts to identify inhibitors of VIM-2 metallo-beta-lactamase (nonselective): Growth inhibition of clinically relevant IMP-1 transformed P. aeruginosa (KN20) in the presence of imipenem (synergy)Other depositor-specified cross reference
624096Late stage assay provider results from the probe development efforts to identify inhibitors of VIM-2 metallo-beta-lactamase (nonselective): Growth inhibition of clinically relevant VIM-2-transformed Acinetobacter species (YMC07/8/B3323) in the presence of imipenem (synergy)Other2 depositor-specified cross reference
624097Late stage assay provider results from the probe development efforts to identify inhibitors of VIM-2 metallo-beta-lactamase (nonselective):IMP-1-transformed E. coli growth inhibition in the presence of imipenem (synergy)Other1 depositor-specified cross reference
2319Late stage results from the probe development efforts to identify selective inhibitors of VIM-2 metallo-beta-lactamase: probe resultsOther same project related to Summary assay
2715Summary of probe development efforts to identify common inhibitors of VIM-2 and IMP-1 metallo-beta-lactamases (IMP-1 inhibitors)Summary same project related to Summary assay
Description:
Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center (SRIMSC)
Center Affiliation: The Scripps Research Institute (TSRI)
Assay Provider: Peter Hodder, TSRI
Network: Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Proposal Number: 1 R21 NS059451-01 Fast Track
Grant Proposal PI: Peter Hodder, TSRI

External Assay ID: VIM-2_INH_EPIABS_1536_%INH

Name: Primary biochemical high throughput screening assay to identify inhibitors of VIM-2 metallo-beta-lactamase

Description:

The emergence of gram-negative bacteria that exhibit multi-drug resistance, combined with the paucity of new antibiotics, poses a public health challenge (1). The production of bacterial beta-lactamase enzymes, in particular, is a common mechanism of drug resistance (2-4). The beta-lactamases evolved from bacteria with resistance to naturally-occurring beta-lactams or penams (5), agents which inhibit the transpeptidase involved in cell wall biosynthesis (6). Human medicine adapted these agents into synthetic antibiotics such as penicillins, cephalosporins, carbapenems, and monobactams that contain a 2-azetidone ring (5, 7). The metallo-beta-lactamases (MBL) are zinc-dependent class B beta-lactamases that hydrolyze the beta-lactam ring, rendering the antibiotic ineffective (6, 8). Increasingly, nosocomial beta-lactam antibiotic resistance arises in P. aeruginosa, Enterobacteriaceae, and other pathogenic bacteria via gene transfer of B1 MBLs (4, 9), including IMP (active on IMiPenem) (10) and VIM (Verona IMipenemase) (11, 12). For two of these enzymes, VIM-2 and IMP-1, no inhibitors exist for clinical use (6, 9). Thus, the identification of MBL inhibitors would provide useful tools for reducing nosocomial infections and elucidating their mechanism of action (13).

References:

1. Siegel, R.E., Emerging gram-negative antibiotic resistance: daunting challenges, declining sensitivities, and dire consequences. Respir Care, 2008. 53(4): p. 471-9. PMID: 18364060.
2. Gupta, V., An update on newer beta-lactamases. Indian J Med Res, 2007. 126(5): p. 417-27. PMID: 18160745.
3. Bradford, P.A., Extended-spectrum beta-lactamases in the 21st century: characterization, epidemiology, and detection of this important resistance threat. Clin Microbiol Rev, 2001. 14(4): p. 933-51, table of contents. PMID: 11585791.
4. Sacha, P., Wieczorek, P., Hauschild, T., Zorawski, M., Olszanska, D., and Tryniszewska, E., Metallo-beta-lactamases of Pseudomonas aeruginosa--a novel mechanism resistance to beta-lactam antibiotics. Folia Histochem Cytobiol, 2008. 46(2): p. 137-42. PMID: 18519228.
5. Koch, A.L., Bacterial wall as target for attack: past, present, and future research. Clin Microbiol Rev, 2003. 16(4): p. 673-87. PMID: 14557293.
6. Jin, W., Arakawa, Y., Yasuzawa, H., Taki, T., Hashiguchi, R., Mitsutani, K., Shoga, A., Yamaguchi, Y., Kurosaki, H., Shibata, N., Ohta, M., and Goto, M., Comparative study of the inhibition of metallo-beta-lactamases (IMP-1 and VIM-2) by thiol compounds that contain a hydrophobic group. Biol Pharm Bull, 2004. 27(6): p. 851-6. PMID: 15187432.
7. Abeylath, S.C. and Turos, E., Drug delivery approaches to overcome bacterial resistance to beta-lactam antibiotics. Expert Opin Drug Deliv, 2008. 5(9): p. 931-49. PMID: 18754746.
8. Wang, Z., Fast, W., Valentine, A.M., and Benkovic, S.J., Metallo-beta-lactamase: structure and mechanism. Curr Opin Chem Biol, 1999. 3(5): p. 614-22. PMID: 10508665.
9. Walsh, T.R., Toleman, M.A., Poirel, L., and Nordmann, P., Metallo-beta-lactamases: the quiet before the storm? Clin Microbiol Rev, 2005. 18(2): p. 306-25. PMID: 15831827.
10. Hirakata, Y., Izumikawa, K., Yamaguchi, T., Takemura, H., Tanaka, H., Yoshida, R., Matsuda, J., Nakano, M., Tomono, K., Maesaki, S., Kaku, M., Yamada, Y., Kamihira, S., and Kohno, S., Rapid detection and evaluation of clinical characteristics of emerging multiple-drug-resistant gram-negative rods carrying the metallo-beta-lactamase gene blaIMP. Antimicrob Agents Chemother, 1998. 42(8): p. 2006-11. PMID: 9687398.
11. Lauretti, L., Riccio, M.L., Mazzariol, A., Cornaglia, G., Amicosante, G., Fontana, R., and Rossolini, G.M., Cloning and characterization of blaVIM, a new integron-borne metallo-beta-lactamase gene from a Pseudomonas aeruginosa clinical isolate. Antimicrob Agents Chemother, 1999. 43(7): p. 1584-90. PMID: 10390207.
12. Wang, C.X. and Mi, Z.H., Imipenem-resistant Pseudomonas aeruginosa producing IMP-1 metallo-beta-lactamases and lacking the outer-membrane protein OprD. J Med Microbiol, 2006. 55(Pt 3): p. 353-4. PMID: 16476803.
13. Zuck P, O'Donnell GT, Cassaday J, Chase P, Hodder P, Strulovici B, Ferrer M. Miniaturization of absorbance assays using the fluorescent properties of white microplates. Anal Biochem. 2005 Jul 15;342 (2):254-9. PMID: 15949786.


Keywords:

VIM-2, beta-lactamase, antibiotic resistance, bacteria, primary, primary screen, HTS, high throughput screen, 1536, inhibitor, epi-absorbance, fluorescence, Scripps, Scripps Florida, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Center Network, MLPCN.
Protocol
Assay Overview:

The purpose of this assay is to identify compounds that act as inhibitors of the VIM-2 beta-lactamase. This biochemical epi-absorbance-format assay employs the cephalosporin nitrocefin as the VIM-2 substrate, and takes advantage of the fluorescent properties of white microtiter plates (13). Nitrocefin is a yellow chromogenic substrate (Imax = 395 nm) that is hydrolyzed by beta-lactamases to yield a red product with increased absorbance properties (Imax = 495 nm) that quenches plate fluorescence by absorbing the plate's emission light (13). In this assay, test compounds are incubated with purified VIM-2 enzyme and nitrocefin in detergent-containing buffer at room temperature. The reaction is stopped by the addition of EDTA, followed by measurement of well fluorescence. As designed, compounds that inhibit VIM-2 will inhibit nitrocefin hydrolysis, inhibit generation of red product, and inhibit quenching of plate fluorescence, resulting in an increase in well fluorescence. Compounds were tested in singlicate at a final nominal concentration of 5.6 uM.

Protocol Summary:

Prior to the start of the assay, 2.5 ul of Assay Buffer (50mM HEPES, 50mM ZnSO4, 0.05% Brij 35, pH 7.1) containing 0.13 nM VIM-2 protein were dispensed into a 1536 microtiter plate. Next, 30 nL of test compound in DMSO or DMSO alone (0.45% final concentration) were added to the appropriate wells. The plates were then incubated for 15 minutes at 25 degrees Celsius.

The assay was started by dispensing 2.5 ul of 120 uM nitrocefin solution in Assay Buffer into all wells. After 25 minutes of incubation at 25 degrees Celsius, 5.0 microliters of 500 mM EDTA were added to each well to stop the reaction. Next, the plates were centrifuged briefly and well fluorescence was read on a Viewlux microplate reader (PerkinElmer, Turku, Finland) (excitation = 480nm, emission = 530nm).

The Optical density (OD) for each well was calculated according to the following equation:

OD = -log(RFU_SampleWell/ RFU_BlankWell)

Where:
RFU_SampleWell is defined as the raw fluorescence value obtained from test compound wells
RFU_BlankWell is defined as the raw fluorescence value obtained from wells containing Assay Buffer

The percent inhibition for each compound was calculated as follows:
Percent inhibition = 100*(1- (Test_Compound - Median_Positive_Control) / (Median_Negative_Control - Median_ Positive _Control))

Where:
Negative_Control is defined as wells containing VIM-2 in the presence of DMSO,
Test_Compound is defined as wells containing VIM-2 in the presence of test compound,
Positive_Control is defined as wells containing DMSO alone.

A mathematical algorithm was used to determine nominally inhibiting compounds in the Primary screen. Two values were calculated: (1) the average percent inhibition of all compounds tested, and (2) three times their standard deviation. The sum of these two values was used as a cutoff parameter, i.e. any compound that exhibited greater % inhibition than the cutoff parameter was declared active.

The reported PubChem Activity Score has been normalized to 100% observed primary inhibition. Negative % inhibition values are reported as activity score zero.

The inactive compounds of this assay have activity score range of 0 to 5 and active compounds range of activity score is 5 to 100.

List of Reagents:

Recombinant VIM-2 (supplied by Assay Provider)
Nitrocefin (BD Diagnostic Systems, part 296289)
1536-well plates (Greiner, part 789173)
HEPES (Invitrogen, Carlsbad, CA, part 15630)
Brij 35 (Sigma-Aldrich, St. Louis, MO, part B4184)
Zinc Sulfate (Sigma-Aldrich, St. Louis, MO, part 204986)
Comment
Due to the increasing size of the MLPCN compound library, this assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. In this case the results of each separate campaign were assigned "Active/Inactive" status based upon that campaign#s specific compound activity cutoff value. All data reported were normalized on a per-plate basis. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, compounds that modulate well fluorescence. All test compound concentrations reported above and below are nominal; the specific test concentration(s) for a particular compound may vary based upon the actual sample provided by the MLSMR.
Categorized Comment - additional comments and annotations
From PubChem:
Assay Format: Biochemical
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Inhibition (5.6μM**)Normalized percent inhibition of the primary screen at a compound concentration of 5.6 micromolar.Float%

** Test Concentration.
Additional Information
Grant Number: 1 R21 NS059451-01

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
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