FRET-based counterscreen assay for selective VIM-2 inhibitors: biochemical high throughput screening assay to identify epi-absorbance assay artifacts
Name: FRET-based counterscreen assay for selective VIM-2 inhibitors: biochemical high throughput screening assay to identify epi-absorbance assay artifacts ..more
BioActive Compounds: 290
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-2CCF2_INH_FRET_1536_3X%INH
Name: FRET-based counterscreen assay for selective VIM-2 inhibitors: biochemical high throughput screening assay to identify epi-absorbance assay artifacts
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.
VIM-2, beta-lactamase, antibiotic resistance, bacteria, counterscreen, HTS, high throughput screen, 1536, selective, inhibitor, CCF2, FRET, fluorescence, 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 confirm VIM-2 activity of compounds identified as active in a previous set of experiments entitled, "Primary biochemical high throughput screening assay to identify inhibitors of VIM-2 metallo-beta-lactamase" (AID 1527), and inactive in a set of experiments entitled, "Epi-absorbance primary biochemical high throughput screening assay to identify inhibitors of IMP-1 metallo-beta-lactamase" (AID 1556). This assay also serves as an orthogonal counterscreen to identify epi-absorbance assay artifacts. This biochemical FRET-based assay employs as the beta-lactamase substrate the cephalosporin scaffold CCF2 that has been tagged with a donor (7-hydroxycoumarin) and quencher/acceptor (fluorescein) FRET pair. VIM-2 mediated hydrolysis of the CCF2 beta-lactam bond releases the donor from the quencher, leading to increased donor fluorescence at 460nm and decreased acceptor fluorescence at 535nm. In this assay, test compounds are incubated with purified VIM-2 enzyme and CCF2 in detergent-containing buffer at room temperature. The reaction is stopped by the addition of EDTA, followed by measurement of well FRET. As designed, compounds that inhibit VIM-2 activity will prevent CCF2 hydrolysis, thereby preventing well FRET. Compounds were tested in triplicate at a final nominal concentration of 5.6 uM.
Prior to the start of the assay, 2.5 uL of Assay Buffer (50 mM HEPES, 50 uM ZnSO4, 0.05% Brij 35, pH 7.1) containing 0.2 nM VIM-2 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 20 uM CCF2 solution in Assay Buffer into all wells. After 25 minutes of incubation at 25 C, 5.0 uL of 500 mM EDTA 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 = 410 nm, emission = 460 nm and 530 nm).
For each well, a fluorescence ratio was calculated according to the following mathematical expression:
Ratio = I460nm / I535nm
I460nm represents the measured fluorescence emission at 460nm and I535nm represents the measured fluorescence emission at 535nm.
The percent inhibition for each compound was calculated as follows:
%_Inhibition = 100 * ( 1 - ( Ratio_Test_Compound - Ratio_Median_Positive_Control ) / ( Ratio_Median_Negative_Control - Ratio_Median_ Positive _Control ) )
Test_Compound is defined as wells containing VIM-2 in the presence of test compound,
Negative_Control is defined as wells containing VIM-2 in the presence of DMSO,
Positive_Control is defined as wells containing DMSO alone.
PubChem Activity Outcome and Score:
A mathematical algorithm was used to determine nominally inhibiting compounds in the counterscreen. Two values were calculated based on the plate that contained no control or test compounds in the sample field: (1) the average percent inhibition of all compounds tested, and (2) three times their standard deviation. The sum of these two values was used as a cutoff parameter, i.e. any compound that exhibited greater % inhibition than the cutoff parameter was declared active.
The reported PubChem activity score has been normalized to 100% of the highest observed inhibition value. Negative % inhibition values are reported as activity score zero.
The inactive compounds of this assay have an activity score range of 0 to 18 and the active compounds range of activity score is 19 to 100.
List of Reagents:
Recombinant VIM-2 (supplied by Assay Provider)
CCF2 (Invitrogen, part K1027)
1536-well plates (Greiner, part 789176)
HEPES (Invitrogen, part 15630)
Brij 35 (Sigma-Aldrich, part B4184)
Zinc Sulfate (Sigma-Aldrich, part 204986)
Due to the increasing size of the MLPCN compound library, this assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. In this case the results of each separate campaign were assigned "Active/Inactive" status based upon that campaign's specific compound activity cutoff value. 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 fluorescence. 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 by the MLSMR. The MLSMR was not able to provide all compounds selected for testing in this AID.
Categorized Comment - additional comments and annotations
** Test Concentration.
Data Table (Concise)