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BioAssay: AID 2768

Late 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-lactamase

Name: Late 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-lactamase. ..more
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 Tested Compounds
 Tested Compounds
All(6)
 
 
Active(6)
 
 
 Tested Substances
 Tested Substances
All(6)
 
 
Active(6)
 
 
AID: 2768
Data Source: The Scripps Research Institute Molecular Screening Center (IMP-1NITRO_INH_EPIABS_1536_3XIC50 COMMON Round 0 SAR)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2010-04-19
Hold-until Date: 2011-04-16
Modify Date: 2013-01-04

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 6
Depositor Specified Assays
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AIDNameTypeProbeComment
1527Primary biochemical high throughput screening assay to identify inhibitors of VIM-2 metallo-beta-lactamasescreening Primary Assay (VIM-2, 1X%INH)
1556Epi-absorbance primary biochemical high throughput screening assay to identify inhibitors of IMP-1 metallo-beta-lactamasescreening Primary Assay (IMP-1, 1X%INH)
1854Summary of probe development efforts to identify selective inhibitors of VIM-2 metallo-beta-lactamasesummary1 Summary (TEM-1 inhibitors)
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 Counterscreen Assay (TEM-1, 1X%INH)
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 Counterscreen Assay (VIM-2, 1X%INH)
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 Confirmation Assay (IMP-1, 3X%INH))
2715Summary of probe development efforts to identify common inhibitors of VIM-2 and IMP-1 metallo-beta-lactamases (IMP-1 inhibitors)summary Summary AID
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 Confirmation Assay (VIM-2, 3XIC50)
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 Counterscreen Assay (TEM-1, 3XIC50)
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 Confirmation Assay (IMP-1, 3XIC50)
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
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
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
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: IMP-1NITRO_INH_EPIABS_1536_3XIC50 COMMON Round 0 SAR

Name: Late 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-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.
2. Gupta, V., An update on newer beta-lactamases. Indian J Med Res, 2007. 126(5): p. 417-27.
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.
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.
5. Koch, A.L., Bacterial wall as target for attack: past, present, and future research. Clin Microbiol Rev, 2003. 16(4): p. 673-87.
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.
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.
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.
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.
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.
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.
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.
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.

Keywords:

late stage, SAR, powders, purchased, IMP-1, VIM-2, beta-lactamase, antibiotic resistance, bacteria, dose response, HTS, high throughput screen, 1536, common, 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 determine dose response curves of powder samples of compounds for common IMP-1/VIM-2 inhibitor compounds identified as active in a previous set of entitled, "Primary biochemical high throughput screening assay to identify inhibitors of IMP-1 metallo-beta-lactamase" (AID 1556), and "Primary biochemical high throughput screening assay to identify inhibitors of VIM-2 metallo-beta-lactamase" (AID 1527), and that confirmed activity in a set of experiments entitled, "Epi-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" (AID 2187), and "Epi-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" (AID 2189). Compounds selected for testing also were inactive in a set of previous experiments entitled, "Epi-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" (AID 2184).

This biochemical epi-absorbance-format assay employs the cephalosporin nitrocefin as the IMP-1 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 IMP-1 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 IMP-1 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 triplicate using a dilution series starting at a nominal test concentration of 60 uM.

Protocol Summary:

Prior to the start of the assay, 2.5 uL of Assay Buffer (50 mM HEPES, 50 uM ZnSO4, 0.05% Brij 35, pH 7.1) containing 0.2 nM IMP-1 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 C.

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 C, 5.0 uL 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 = 480 nm, emission = 530 nm).

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:

%_Inhibition = 100 * ( 1 - ( Test_Compound - Median_Positive_Control ) / ( Median_Negative_Control - Median_Positive_Control ) )

Where:

Test_Compound is defined as wells containing IMP-1 in the presence of test compound.
Negative_Control is defined as wells containing IMP-1 in the presence of DMSO.
Positive_Control is defined as wells containing DMSO alone.

For each test compound, percent inhibition was plotted against compound concentration. A four parameter equation describing a sigmoidal dose-response curve was then fitted with adjustable baseline using Assay Explorer software (Symyx Technologies Inc). The reported IC50 values were generated from fitted curves by solving for the X-intercept value at the 50% inhibition level of the Y-intercept value. In cases where the highest concentration tested (i.e. 60 uM) did not result in greater than 50% inhibition, the IC50 was determined manually as greater than 60 uM.

PubChem Activity Outcome and Score:

Compounds with an IC50 greater than 10 uM were considered inactive. Compounds with an IC50 equal to or less than 10 uM were considered active.

Any compound with a percent activity value <50% at all test concentrations was assigned an activity score of zero. Any compound with a percent activity value >50% at any test concentration was assigned an activity score greater than zero. Activity score was then ranked by the potency, with the most potent compounds assigned the highest activity scores.

The PubChem Activity Score range for active compounds is 100-1, there are no inactives.

List of Reagents:

Recombinant IMP-1 (supplied by Assay Provider)
Nitrocefin (BD Diagnostic Systems, part 296289)
1536-well plates (Greiner, part 789173)
HEPES (Invitrogen, part 15630)
Brij 35 (Sigma-Aldrich, part B4184)
Zinc Sulfate (Sigma-Aldrich, part 204986)
Comment
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.
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1QualifierActivity Qualifier identifies if the resultant data IC50 came from a fitted curve or was determined manually to be less than or greater than its listed IC50 concentration.String
2IC50*The concentration at which 50 percent of the activity in the inhibitor assay is observed; (IC50) shown in micromolar.FloatμM
3LogIC50Log10 of the qualified IC50 (IC50) from the inhibitor assay in M concentration.Float
4Hill SlopeThe variable HillSlope describes the steepness of the curve. This variable is called the Hill slope, the slope factor, or the Hill coefficient. If it is positive, the curve increases as X increases. If it is negative, the curve decreases as X increases. A standard sigmoid dose-response curve (previous equation) has a Hill Slope of 1.0. When HillSlope is less than 1.0, the curve is more shallow. When HillSlope is greater than 1.0, the curve is steeper. The Hill slope has no units.Float
5Hill S0Y-min of the curve.Float
6Hill SinfY-max of the curve.Float
7Hill dSThe range of Y.Float
8Chi SquareA measure for the 'goodness' of a fit. The chi-square test (Snedecor and Cochran, 1989) is used to test if a sample of data came from a population with a specific distribution.Float
9RsquareThis statistic measures how successful the fit explains the variation of the data; R-square is the square of the correlation between the response values and the predicted response values.Float
10Number of DataPointsOverall number of data points of normalized percent inhibition that was used for calculations (includes all concentration points); in some cases a data point can be excluded as outlier.Integer
11Inhibition at 3.0 nM (0.003μM**)Value of %inhibition at 3.0 nanomolar inhibitor concentration; average of triplicate measurement.Float%
12Inhibition at 9.1 nM (0.009μM**)Value of %inhibition at 9.0 nanomolar inhibitor concentration; average of triplicate measurement.Float%
13Inhibition at 27.3 nM (0.0273μM**)Value of %inhibition at 27.3 nanomolar inhibitor concentration; average of triplicate measurement.Float%
14Inhibition at 81.8 nM (0.0818μM**)Value of %inhibition at 81.8 nanomolar inhibitor concentration; average of triplicate measurement.Float%
15Inhibition at 245.4 nM (0.245μM**)Value of %inhibition at 245 nanomolar inhibitor concentration; average of triplicate measurement.Float%
16Inhibition at 736.3 nM (0.736μM**)Value of %inhibition at 736 nanomolar inhibitor concentration; average of triplicate measurement.Float%
17Inhibition at 2.2 uM (2.2μM**)Value of %inhibition at 2.2 micromolar inhibitor concentration; average of triplicate measurement.Float%
18Inhibition at 6.6 uM (6.6μM**)Value of %inhibition at 6.6 micromolar inhibitor concentration; average of triplicate measurement.Float%
19Inhibition at 19.9 uM (19.9μM**)Value of %inhibition at 19.9 micromolar inhibitor concentration; average of triplicate measurement.Float%
20Inhibition at 59.6 uM (59.6μM**)Value of %inhibition at 59.6 micromolar inhibitor concentration; average of triplicate measurement.Float%
21Inhibition at 7.6 nM (0.0076μM**)Value of %inhibition at 7.6 nanomolar inhibitor concentration; average of triplicate measurement.Float%
22Inhibition at 22.7 nM (0.0227μM**)Value of %inhibition at 22.7 nanomolar inhibitor concentration; average of triplicate measurement.Float%
23Inhibition at 68.2 nM (0.0682μM**)Value of %inhibition at 68.2 nanomolar inhibitor concentration; average of triplicate measurement.Float%
24Inhibition at 204.5 nM (0.2045μM**)Value of %inhibition at 204.5 nanomolar inhibitor concentration; average of triplicate measurement.Float%
25Inhibition at 613.6 nM (0.6136μM**)Value of %inhibition at 613.6 nanomolar inhibitor concentration; average of triplicate measurement.Float%
26Inhibition at 1.8 uM (1.8μM**)Value of %inhibition at 1.8 micromolar inhibitor concentration; average of triplicate measurement.Float%
27Inhibition at 5.5 uM (5.5μM**)Value of %inhibition at 5.5 micromolar inhibitor concentration; average of triplicate measurement.Float%
28Inhibition at 16.6 uM (16.6μM**)Value of %inhibition at 16.6 micromolar inhibitor concentration; average of triplicate measurement.Float%
29Inhibition at 49.7 uM (49.7μM**)Value of %inhibition at 49.7 micromolar inhibitor concentration; average of triplicate measurement.Float%
30Inhibition at 149.1 uM (149.1μM**)Value of %inhibition at 149.1 micromolar inhibitor concentration; average of triplicate measurement.Float%

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

Data Table (Concise)
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