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 IMP-1
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 IMP-1. ..more
Depositor Specified Assays
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: IMP1_INH_ABS_384_IC50_KI (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 IMP-1.
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.
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 the IMP-1 inhibition constant (Ki) and mode of inhibition of powder samples of compounds identifed as nonselective VIM-2 inhibitor probe candidates and selected analogs. This assay serves as a counterscreen to assess compound selectivity.
Kinetic assays were conducted by incubating the nitrocefin substrate with varying inhibitor concentrations and 0.1 nM IMP-1 enzyme at room temperature in buffer containing 50 mM HEPES, 50 uM ZnSO4, 0.05% Brij 35, pH 7.1. Absorbance was measured on a Tecan Safire2 monochromatic microplate reader at 495 nm. Initial velocities were obtained from plots of absorbance at 495 nm versus time, using data points from only the linear portion of the hydrolysis curve. Substrate hydrolysis was continuously monitored. Initial velocities were plotted vs. substrate concentration and kinetic parameters were calculated using Graphpad Prism version 5.01 suite of programs. All Ki values were determined by non-linear regression (hyperbolic equation) analysis using the mixed inhibition model which allows for simultaneous determination of mechanism of inhibition. Mechanism of inhibition was determined using the "alpha" parameter derived from a mixed-model inhibition by GraphPad Prism. The mechanism of inhibition was additionally evaluated by Hanes-Woolf and Lineweaver-Burke plots. Ki values were calculated from non-linear regression. The number of replicates for this assay is 2.
For this IMP1 assay, the final reaction volume was 44 uL, the final nominal concentration of IMP-1 enzyme was 0.1nM, the concentration of nitrocefin varied, and 384-well SWCN plates (Greiner, Part 789005) were used. Well absorbance was read using a Tecan platereader for 6 minutes at 495 nm.
PubChem Activity Outcome and Score:
Compounds with an Ki greater than 10 uM were considered inactive. Compounds with an Ki 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 active compounds is 100-100. There are no active compounds.
List of Reagents:
Recombinant IMP-1 (supplied by Assay Provider)
Nitrocefin (BD Diagnostic Systems, part 296289)
1536-well plates (Greiner SWSN, part 789175)
HEPES (Invitrogen, Carlsbad, CA, part 15630)
Brij 35 (Sigma-Aldrich, St. Louis, MO, part B4184)
Zinc Sulfate (Sigma-Aldrich, St. Louis, MO, part 204986)
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.
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
* Activity Concentration.
Data Table (Concise)