Late 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 AmpC
Name: Late 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 AmpC. ..more
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: AMPC_INH_ABS_1536_IC50 (Nonselective: run by AP)
Name: Late 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 AmpC.
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).
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. Manchanda V, Singh NP. Occurrence and detection of AmpC beta-lactamases among Gram-negative clinical isolates using a modified three-dimensional test at Guru Tegh Bahadur Hospital, Delhi, India. J Antimicrob Chemother. 2003 Feb;51(2):415-8.
ML302, analogs, Late stage, probe, powder, synthesis, purchased, VIM-2, beta-lactamase, antibiotic resistance, bacteria, 384, nonselective, non-selective, inhibitor, epi-absorbance, IC50, dose response, titration, Ki, Km, TEM-1, TEM, AmpC, Amp, clavulanate, cloxacillin, kinetic, nitrocefin, IMP-1, absorbance, Scripps, Scripps Florida, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Center Network, MLPCN.
The purpose of this assay is to determine whether powder samples of compounds identifed as nonselective VIM-2 inhibitor probe candidates and selected analogs can inhibit AmpC beta-lactamase. This assay also serves as a counterscreen to determine the non-selectivity of tested compounds. AmpC class of Beta-lactamases are cephalosporinases that are poorly inhibited by clavulanic acid. They can be differentiated from other extended-spectrum beta-lactamases (ESBLs) by their ability to hydrolyse cephamycins as well as other extended-spectrum cephalosporins. Plasmid-encoded AmpC beta-lactamases are produced by numerous pathogens, e.g. Klebsiella pneumoniae, Escherichia coli, Salmonella spp., Proteus mirabilis and Citrobacter freundii (14). AmpC-producing strains are typically resistant to oxyimino-beta lactams and to cephamycins and are susceptible to carbapenems; however, diminished porin expression can make such a strain carbapenem-resistant as well. This biochemical epi-absorbance-format assay employs the cephalosporin nitrocefin as the AmpC 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 AmpC enzyme and nitrocefin in detergent-containing buffer at room temperature. The reaction is stopped by the addition of potassium clavulanate, followed by measurement of well fluorescence. As designed, compounds that inhibit AmpC 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.
Prior to the start of the assay, 2.5 uL of Assay Buffer (PBS, 0.05% Brij 35, pH 7.4) containing 4 nM AmpC 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 200 uM nitrocefin substrate solution (2X) in Assay Buffer into all wells. After 25 minutes of incubation at 25 C, 5.0 uL of 4 uM Cloxacillin 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 )
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 ) )
Test_Compound is defined as wells containing 2 nM AmpC in the presence of test compound,
Negative_Control is defined as wells containing 2 nM AmpC in the presence of DMSO,
Positive_Control is defined as wells containing DMSO alone.
For this AmpC assay, the IC50 value for the Cloxacillin control compound was determined to be 1.879e-12 M.
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 (MDL Information Systems). 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.
Activity score was then ranked by the potency of the compounds with fitted curves, with the most potent compounds assigned the highest activity scores.
The PubChem Activity Score range for inactive compounds is 0-0. There are no active compounds.
List of Reagents:
Recombinant AmpC (Sigma, part P4524)
Nitrocefin (BD Diagnostic Systems, part 296289)
1536-well plates (Greiner SWSN, part 789175)
Phosphate Buffered Saline (Invitrogen, part 10010072)
Brij 35 (Sigma-Aldrich, part B4184)
Cloxacillin (Sigma, Part 27555)
These assays were performed by the assay provider. These assays 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 - additional comments and annotations
From BioAssay Depositor:
BAO: assay design: viability reporter: atp content
BAO: assay format: cell-based format
BAO: bioassay specification: assay biosafety level: bsl1
BAO: bioassay specification: assay footprint: microplate: 384 well plate
BAO: bioassay specification: assay measurement throughput quality: concentration response multiple replicates
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: bioassay specification: assay stage: confirmatory
BAO: bioassay specification: bioassay type: functional: viability
BAO: detection technology: spectrophotometry: absorbance
BAO: meta target detail: binding reporter specification: interaction: protein-small molecule
BAO: meta target: biological process target: cell death
BAO: meta target: molecular target: protein target: enzyme: protease
BAO: version: 1.4b1090
Assay Format: Biochemical
* Activity Concentration.
Data Table (Concise)