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BioAssay: AID 504620

Late 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)

Name: Late 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). ..more
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AID: 504620
Data Source: The Scripps Research Institute Molecular Screening Center (VIM-2-ECOLI_INH_ABS_0384_SYNERGY)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network, Assay Provider
BioAssay Version:
Deposit Date: 2011-03-30
Modify Date: 2013-01-04

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compound: 1
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 screen (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 screen (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 (VIM-2 inhibitors)
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: Counterscreen (IMP-1 inhibitors in triplicate)
1857FRET-based counterscreen assay for selective VIM-2 inhibitors: biochemical high throughput screening assay to identify epi-absorbance assay artifactsScreening depositor-specified cross reference: Counterscreen (epi-absorbance assay artifacts in triplicate)
1860Epi-absorbance-based confirmation biochemical high throughput screening assay to identify selective inhibitors of VIM-2 metallo-beta-lactamase.Screening depositor-specified cross reference: Confirmation screen (VIM-2 inhibitors in triplicate)
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: Counterscreen (TEM-1 inhibitors in triplicate)
1919Epi-absorbance-based dose response biochemical high throughput screening assay for selective inhibitors of VIM-2 metallo-beta-lactamaseConfirmatory depositor-specified cross reference: Dose response (VIM-2 inhibitors in triplicate)
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: Dose response counterscreen (IMP-1 inhibitors in triplicate)
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: Dose response counterscreen (TEM-1 inhibitors in triplicate)
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: Dose response counterscreen (epi-absorbance assay artifacts in triplicate)
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: Dose response counterscreen (IMP-1 inhibitors in triplicate)
2128Late stage results from the probe development efforts to identify selective inhibitors of VIM-2 metallo-beta-lactamase: probe resultsOther depositor-specified cross reference: Late stage results (VIM-2 selective inhibitors, probe results)
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: Late stage results (VIM-2 selective inhibitors, prior art results)
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 same project related to Summary assay
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 same project related to Summary assay
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 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
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
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 same project related to Summary assay
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 same project related to Summary assay
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 same project related to Summary assay
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 same project related to Summary assay
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 same project related to Summary assay
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 same project related to Summary assay
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 same project related to Summary assay
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 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
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
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-ECOLI_INH_ABS_0384_SYNERGY

Name: Late 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).

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. Minond D, Saldanha SA, Subramaniam P, Spaargaren M, Spicer T, Fotsing JR, Weide T, Fokin VV, Sharpless KB, Galleni M, Bebrone C, Lassaux P, Hodder P. Inhibitors of VIM-2 by screening pharmacologically active and click-chemistry compound libraries. Bioorg Med Chem. 2009 Jul 15;17(14):5027-37.
15. 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.
16. Bonapace, C.R., J.A. Bosso, L.V. Friedrich, and R.L. White, Comparison of methods of interpretation of checkerboard synergy testing. Diagn Microbiol Infect Dis, 2002. 44(4): p. 363-6.
17. Bajaksouzian, S., M.A. Visalli, M.R. Jacobs, and P.C. Appelbaum, Activities of levofloxacin, ofloxacin, and ciprofloxacin, alone and in combination with amikacin, against acinetobacters as determined by checkerboard and time-kill studies. Antimicrob Agents Chemother, 1997. 41(5): p. 1073-6.
18. Wayne, M26-A: Methods for Determining Bactericidal Activity of Antimicrobial Agents; Approved Guideline, E. C. a. L. S. Institute, Editor. 1999, National Committee on Clinical Laboratory Standards

Keywords:

Late stage, probes, powder, synthesized, VIM-2, E. coli, beta-lactamase, metallo beta-lactamase, antibiotic resistance, bacteria, SAR, dose response, selective, inhibitor, inhibit, inhibition, absorbance, synergy, checkerboard assay, assay provider, growth, viability, cytotoxicity, minimum inhibitory concentration, MIC, 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 if a powder sample of a compound identified as possible VIM-2 selective inhibitor probe candidate can increase the antibacterial activity of imipenem in E. coli transformed with VIM-2. Synergy assays were conducted using two-fold serial broth dilution method as recommended by Clinical and Laboratory Standards Institute (CLSI). All testing was performed in 384 well plates in 0.06 mL final volume. Imipenem or test compound was titrated in Cation Adjusted Mueller-Hinton Broth (CAMHB) immediately prior to testing. Imipenem and inhibitor were diluted down columns and across rows respectively. E. coli previously grown in CAMHB to log phase OD600 of 0.5 - 0.7 was used to inoculate each well with 0.03 mL of bacterial inoculums of 1 x 10^6 CFU/mL. The plates were incubated for at least 18 hours at 37 C under aerobic conditions. The results of synergy testing were determined in manners previously reported (14-17).Turbidity was determined using OD590 and MICs were determined per CLSI methods (18). As designed, compounds with antibacterial activity will inhibit bacterial growth, leading to reduced well absorbance. 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 or imipenem was titrated as described above, in separate plates, in 15 uL of CAMHB in a 384 well microtiter plate. Next, these titrations were combined into one assay plate for a total volume of 30 uL yielding unique combinations of ML121 and imipenem. Next, 30 uL of CAMHB inoculated with BL21/VIM-2 E.coli at 1 x 10^6 CFU/mL was added to wells. Next, the 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. The percent inhibition for each compound was calculated as follows:

%_Inhibition = 100 * ( ( Test_Compound - Median_Low_Control ) / ( Median_High_Control - Median_Low_Control ) )

Where:

High_Control is defined as wells containing Imipenem (100 uM).
Low_Control is defined as wells containing VIM2-transformed BL21 E.coli.
Test_Compound is defined as wells containing VIM2-transformed BL21 E.coli in the presence of test compound.

A mathematical algorithm was used to determine the MIC of compounds. Two values were calculated: (1) the average percent inhibition of all high control wells, and (2) three times their standard deviation. The sum of these two values was used as a cutoff parameter. The reported MIC values were generated by identifying the lowest concentration of inhibitor that yields a value that is less than the cutoff value; i.e. no bacterial growth. In cases where the highest concentration tested (i.e. 100 uM) did not result in a value that was less than the cutoff, the MIC was determined as greater than the highest concentration tested.

Using the method described above, the probe candidate, ML121, was tested for its ability to potentiate the efficacy of imipenem on VIM-2-expressing E. coli (BL21/VIM-2). ML121 partially restored susceptibility of BL21/VIM2 bacteria to imipenem. The synergistic effect of ML121 is described as a factor or fold effect. The amount ML121 lowers the MIC of imipenem compared to that of imipenem alone yields this result. The resynthesized probe ML121 recapitulated former results and exhibited synergy with imipenem when present at concentrations as low as 12.5 uM.

PubChem Activity Outcome and Score:

This is the molecular probe ML121 and as such represents an active compound.

The PubChem Activity Score range for active compounds is 100-100. There are no inactive compounds.

List of Reagents:

384 well plates (Corning, part 3701)
Imipenem antibiotic (USP, Rockville MD, part 1337809)
VIM-2-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. This assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. 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 absorbance. All test compound concentrations reported 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
1QualifierActivity Qualifier identifies if the resultant data MIC was determined manually to be less than or greater than its tested concentration range.String
2MIC*The lowest concentration at which no bacterial growth is observed; (MIC) shown in micromolar.FloatμM
3Minimum SynergisticThe minimum concentration of inhibitor to reduce the imipenem MIC 4 fold. Shown in micromolar.FloatμM

* Activity 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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