Bookmark and Share
BioAssay: AID 624097

Late 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)

Name: Late 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). ..more
_
   
 Tested Compounds
 Tested Compounds
All(8)
 
 
Probe(1)
 
 
Active(6)
 
 
Inactive(2)
 
 
 Tested Substances
 Tested Substances
All(8)
 
 
Probe(1)
 
 
Active(6)
 
 
Inactive(2)
 
 
AID: 624097
Data Source: The Scripps Research Institute Molecular Screening Center (IMP-1-ECOLI_INH_ABS_0384_SYNERGY MDCSRUN)
Depositor Category: NIH Molecular Libraries Probe Production Network, Assay Provider
BioAssay Version:
Deposit Date: 2012-04-12
Hold-until Date: 2013-04-12
Modify Date: 2013-04-13

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: Chemical Probe: 1    Active: 6
Depositor Specified Assays
Show more
AIDNameTypeProbeComment
1527Primary biochemical high throughput screening assay to identify inhibitors of VIM-2 metallo-beta-lactamasescreening Primary screen (VIM-2 inhibitors in singlicate)
1556Epi-absorbance primary biochemical high throughput screening assay to identify inhibitors of IMP-1 metallo-beta-lactamasescreening Primary screen (IMP-1 inhibitors in singlicate)
1854Summary of probe development efforts to identify selective inhibitors of VIM-2 metallo-beta-lactamasesummary1 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 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 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 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 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 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 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 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 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 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 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 Late stage results (VIM-2 selective inhibitors, prior art results)
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-1-ECOLI_INH_ABS_0384_SYNERGY MDCSRUN

Name: Late 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).

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, powder, synthesized, BL21, VIM-2, IMP-1, E. coli, beta-lactamase, metallo beta-lactamase, antibiotic resistance, bacteria, SAR, dose response, kinetic, nonselective, non-selective, inhibitor, inhibit, inhibition, absorbance, checkerboard assay, synergy, potentiation, 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 powder samples of a compounds identified as VIM-2 nonselective inhibitor probe candidates can reduce the growth of BL21 E. coli transformed with IMP-1. IMP-1 confers resistance to all carbapenems, penicillins, and cephalosporins, and this resistance cannot be overcome with any B-lactamase inhibitor. 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. BL21 IMP-1 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 Minimum Inhibitory Concentrations (MIC) 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 test compounds and imipenem. Next, 30 uL of CAMHB inoculated with BL21/IMP-1 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 media only (no bacteria).
Low_Control is defined as wells containing IMP1-transformed BL21 E.coli.
Test_Compound is defined as wells containing IMP1-transformed BL21 E.coli in the presence of test compounds.

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.

PubChem Activity Outcome and Score:

Compounds with a four fold reduction in MIC value greater than 10 uM were considered inactive. Compounds with an four fold reduction in MIC value equal to or less than 10 uM were considered active.

Activity score was then ranked by the potency of the compounds, with the most potent compounds assigned the highest activity scores.

The PubChem Activity Score range for active compounds is 100-90, and for inactive compounds 1-1.

List of Reagents:

384 well plates (Corning, part 3701)
Imipenem antibiotic (USP, Rockville MD, part 1337809)
IMP-1-transformed E. coli ( Assay Provider)
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, and 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.
Categorized Comment
BAO: version: 1.4b1090

BAO: bioassay specification: assay stage: confirmatory

BAO: bioassay specification: assay biosafety level: bsl1

BAO: assay format: cell-based format

BAO: bioassay specification: assay measurement type: kinetic assay

BAO: bioassay specification: assay readout content: assay readout method: regular screening

BAO: bioassay specification: assay readout content: content readout type: single readout

BAO: meta target: molecular target: protein target: enzyme: protease

BAO: meta target: biological process target: cell death

BAO: meta target detail: binding reporter specification: interaction: protein-small molecule

BAO: assay design: viability reporter: atp content

BAO: detection technology: spectrophotometry: absorbance

BAO: bioassay specification: bioassay type: functional: viability

BAO: bioassay specification: assay footprint: microplate: 384 well plate

BAO: bioassay specification: assay measurement throughput quality: concentration response multiple replicates

Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1MIC [4 fold reduction]*The minimum concentration of inhibitor observed to reduce by 4 fold the imipenem MIC (concentration at which no bacterial growth is observed) (Shown in micromolar).FloatμM
2PotentiationDescribes the maximum fold potentiation of imipenem observed by the inhibitor, distinct from the reported MIC4X value.String

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

Data Table (Concise)
PageFrom: