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BioAssay: AID 463099

Late 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 imipenem

Name: Late 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 imipenem. ..more
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AID: 463099
Data Source: The Scripps Research Institute Molecular Screening Center (BL21-IMP1-ECOLI_INH_ABS_0384_SYNERGY SAR Round 0)
Depositor Category: NIH Molecular Libraries Probe Production Network, Assay Provider
BioAssay Version:
Deposit Date: 2010-08-18
Hold-until Date: 2011-08-16
Modify Date: 2013-01-04

Data Table ( Complete ):           View Active Data    View All Data
BioActive Compounds: 6
Related Experiments
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AIDNameTypeProbeComment
1527Primary biochemical high throughput screening assay to identify inhibitors of VIM-2 metallo-beta-lactamaseScreening depositor-specified cross reference: Primary Assay (VIM-2 inhibitors in singlicate)
1556Epi-absorbance primary biochemical high throughput screening assay to identify inhibitors of IMP-1 metallo-beta-lactamaseScreening depositor-specified cross reference: Primary Assay (IMP-1 inhibitors in singlicate)
1854Summary of probe development efforts to identify selective inhibitors of VIM-2 metallo-beta-lactamaseSummary1 depositor-specified cross reference: 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 depositor-specified cross reference: Counterscreen Assay (TEM-1 inhibitors in singlicate)
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: Counterscreen Assay (VIM-2 inhibitors in singlicate)
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: Confirmation Assay (IMP-1 inhibitors in triplicate)
2715Summary of probe development efforts to identify common inhibitors of VIM-2 and IMP-1 metallo-beta-lactamases (IMP-1 inhibitors)Summary depositor-specified cross reference: Summary (IMP-1 inhibitors)
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: Dose response (VIM-2 inhibitors in triplicate)
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: Dose response (TEM-1 inhibitors in triplicate)
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: Dose response (IMP-1 inhibitors in triplicate)
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: Dose response counterscreen (TEM-1 inhibitors in triplicate)
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: Dose response (IMP-1 inhibitors in triplicate)
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: Dose response (VIM-2 inhibitors in triplicate)
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: Dose response counterscreen MIC (Wildtype E. coli growth inhibition in triplicate)
1856Epi-absorbance-based counterscreen for selective VIM-2 inhibitors: biochemical high throughput screening assay to identify inhibitors of IMP-1 metallo-beta-lactamase.Screening same project related to Summary assay
1857FRET-based counterscreen assay for selective VIM-2 inhibitors: biochemical high throughput screening assay to identify epi-absorbance assay artifactsScreening same project related to Summary assay
1860Epi-absorbance-based confirmation biochemical high throughput screening assay to identify selective inhibitors of VIM-2 metallo-beta-lactamase.Screening same project related to Summary assay
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 same project related to Summary assay
1919Epi-absorbance-based dose response biochemical high throughput screening assay for selective inhibitors of VIM-2 metallo-beta-lactamaseConfirmatory same project related to Summary assay
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 same project related to Summary assay
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 same project related to Summary assay
1926FRET-based counterscreen for selective VIM-2 inhibitors: dose response biochemical high throughput screening assay to identify epi-absorbance assay artifacts.Confirmatory same project related to Summary assay
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 same project related to Summary assay
2128Late 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
2317Late stage results from the probe development efforts to identify selective inhibitors of VIM-2 metallo-beta-lactamase: Prior art resultsScreening same project related to Summary assay
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
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 same project related to Summary assay
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 same project related to Summary assay
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 same project related to Summary assay
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 same project related to Summary assay
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 same project related to Summary assay
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 same project related to Summary assay
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 same project related to Summary assay
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 same project related to Summary assay
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 same project related to Summary assay
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 same project related to Summary assay
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 same project related to Summary assay
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 same project related to Summary assay
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 same project related to Summary assay
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 same project related to Summary assay
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 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: BL21-IMP1-ECOLI_INH_ABS_0384_SYNERGY SAR Round 0

Name: Late 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 imipenem.

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.
14. Blizzard, T., Chen, H., Kim, S., Wu, J., Young, K., Park, Y.-W., Ogawa, A., Raghoobar, S., Painter, R., Hairston, N., Lee, S., Misura, A., Felcetto, T., Fitzgerald, P., Sharma, N., Lu, J., Ha, S., Hickey, E., Hermes, J., and Hammond, M. Side chain SAR of bicyclic B-lactamase inhibitors (BLIs). 1. Discovery of a class C BLI for combination with imipenem. Bioorganic & Medicinal Chemistry Letters 20 (2010) 918-921.
15. CLSI. In M7-A7: Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grows Aerobically; Approved Standard, 7th ed.; C. a. L. S. Institute, Ed.; CLSI:Wayne, 2006; Vol. 2.

Keywords:

late stage, SAR, powders, purchased, imipenem, carabapenem, IMP-1, VIM-2, TEM-1, metallo beta-lactamase, MBL, beta-lactamase, antibiotic resistance, bacteria, dose response, counterscreen, synergy, checkerboard, MIC, minimum inhibitory concentration, potentiate, transformed, E. coli, 384, common, inhibitor, absorbance, assay provider, 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 whether powder samples of test compounds identified as possible VIM-2/IMP-1 common inhibitor probe candidates can increase the antibacterial activity of imipenem in E. coli transformed with IMP-1. In-vitro synergy assays were performed in 384 well plates in 0.06 mL final volume. Imipenem was titrated in CAMHB using 13 point two-fold serial dilutions down the plate while at the same time test compound was titrated using 10 point two-fold serial dilutions across the plate immediately prior to testing. These dilutions yield a checkerboard pattern such that each well in the microtiter plate contains a different combination of concentrations of imipenem and test compound. BL21 IMP-1 E. coli previously grown in CAMHB to log phase OD600 of 0.5-0.7 is used to inoculate each well with 0.03 mL of bacterial inoculums of 1E6 CFU/mL. The plates were incubated for at least 18 hours at 37 C under aerobic conditions. Synergy between the antibiotic and test compound is reported as the minimum concentration of test compound required to restore or enhance imipenem susceptibility (14).
Protocol Summary:
Prior to the start of the assay, compounds were titrated in 15 uL of CAMHB in a 384 well microtiter plate. Imipenem that was previously titrated in CAMHB is subsequently overlaid onto the compound dilution series. Next, 30 uL of CAMHB inoculated with BL21 IMP-1 E. Coli at 1E6 CFU/mL was added to wells.
All plates were centrifuged briefly and were incubated for 18 hours at 37 C in a humidified environment. Absorbance (OD590) was read on a Tecan Spectraflour Plus plate reader (Tecan U.S., Inc.) using 4 reads per well.
In order to determine synergy, the CLSI method of Minimum Inhibitory Concentration (MIC) determination was used to identify the MIC of imipenem in the presence of compounds (15). MIC values were generated by identifying the lowest concentration of inhibitor in combination with imipenem that yields no observable bacterial growth. Previously, the MIC of imipenem (PubChem CID 5282372) alone was determined for BL21 wild type E. coli and was reported in AID 449774. Here we determined the minimum concentration of compound required to restore the MIC of imipenem in resistant bacteria back to the wild type MIC.
PubChem Activity Outcome and Score:
Any compound with a MIC less than 10 uM was considered active.
Compounds were then sorted by MIC value, such that compounds exhibiting lower MIC were assigned a higher activity score.
The PubChem Activity Score range for active compounds is 100-1. There are no inactive compounds.
List of Reagents:
384 well plates (Corning, part 3701)
Imipenem antibiotic (USP, Rockville MD, part 1337809)
IMP-1-transformed E. coli (DE3) (BL21, Novagen, Gibbstown, NJ)
Mueller Hinton II Broth Cation-Adjusted (CAMHB; Becton Dickinson, part 297963).
Comment
This assay was performed by the assay provider. All compounds were tested on at least two separate occasions. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, or compounds that modulate well absorbance. 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
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1MICThe minimum concentration at which the activity of the imipenem in combination with inhibitor produces no observable growth and restores the imipenem MIC to wild type; (MIC) shown in micromolar.FloatμM
2Standard DeviationStandard deviation of the counterscreen assay derived from the normalized percent activation of the triplicate data for each compound.Float
3Log(MIC)Log10 of the qualified MIC from the inhibitor assay in M concentrationFloat
Additional Information
Grant Number: 1 R21 NS059451-01

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