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: VIM2-SELECTIVE_INH_PROBES_LATE STAGE PROBE DATA

Name: Late stage results from the probe development efforts to identify selective inhibitors of VIM-2 metallo-beta-lactamase: probe results

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).

Summary of Probe Development Effort:

Following nitrocefin-based primary HTS in singlicate to identify selective VIM-2 inhibitors (AID 1527), counterscreening in singlicate against the IMP-1 metallo-beta-lactamase (AID 1556), confirmation of hit activity in triplicate (AID 1860), counterscreening in triplicate against IMP-1 (AID 1856) and TEM-1 (AID 1866), VIM-2 CCF2-based counterscreening in triplicate to identify nitrocefin-based assay artifacts (AID1857), titration assays in triplicate to determine VIM-2 potency (AID 1919) and selectivity against IMP-1 (AID 1920 and 1927), TEM-1 (1925), and CCF2-based dose response counterscreening in triplicate to identify nitrocefin-based assay artifacts (AID 1926), certain compounds were identified as possible candidates for probe development. A promising lead compound (SID24790728) was order as a powder (SID 85856282) and tested in kinetic assays to determine the mode of inhibition and the inhibitory constant (Ki) against VIM2. SID 85856282 was also subsequently tested in VIM2 dose response assays, IMP1, and TEM1 counterscreens during probe development.

The results of these probe development efforts resulted in the identification of one compound (SID 85856282) as a selective, non-competitive inhibitor probe of the metallo-beta-lactamase VIM-2. This probe exhibits a submicromolar Ki value for VIM-2 with minimal activity against IMP-1 and TEM-1 enzymes, as determined using epi-absorbance and fluorescence-based biochemical assays.

Details of protocols, compound structures, and results from the original assays can be found in PubChem at the respective AIDs listed below. The results of our probe development efforts can be found at http://molscreen.florida.scripps.edu/probes.shtml. One manuscript has been published (14). Please also see Summary AID 1854.

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.

Keywords:

Late stage, probes, powder, VIM-2, beta-lactamase, antibiotic resistance, bacteria, primary, confirmation, HTS, high throughput screen, 1536, selective, inhibitor, epi-absorbance, fluorescence, Scripps, Scripps Florida, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Center Network, MLPCN.

Assays

Data Table(Active)    Data Table(All)

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PID§		Name	Substance		Panel Targets	Description	Additional Information
			Active	Inactive
1		VIM-2 Ki Assay	1		metallo beta-lactamase [Pseudomonas aeruginosa] [gi:7381449]		Taxonomy id: 287
2		Nitrocefin-based VIM2 Inhibition Assay	1		metallo beta-lactamase [Pseudomonas aeruginosa] [gi:7381449]		Taxonomy id: 287
3		Nitrocefin-based IMP1 Inhibition Counterscreen		1	metallo-beta-lactamase IMP-1 [Pseudomonas aeruginosa] [gi:27368096]		Taxonomy id: 287
4		Nitrocefin-based TEM-1 Inhibition Counterscreen	1		Beta lactamase [Pseudomonas aeruginosa] [gi:114881106]		Taxonomy id: 287 Gene id: 4290808

---

§ Panel component ID.

Please see AIDs 1527, 1556, 1856, 1857, 1860, 1866, 1919, 1920, 1925, 1926, 1927, 1854 and below for protocols performed in this probe development effort.

VIM-2 Ki Assay (Assay 1):

The purpose of this assay is to determine the inhibition constant (Ki) and modality of probe candidate molecules. Kinetic assays were conducted by incubating a range of substrate concentrations (100-8 muM) with varying inhibitor concentrations and 0.1 nM enzyme at room temperature in buffer containing 50mM HEPES, 50muM 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. Lineweaver-Burke best-fit lines are reported, along with Ki calculated from non-linear regression. The number of replicates for this assay is 3.

Nitrocefin-based VIM2 Inhibition Assay (Assay 2):

The purpose of this assay is to identify compounds that act as inhibitors of the VIM-2 beta-lactamase. This biochemical epi-absorbance-format assay employs the cephalosporin nitrocefin as the VIM-2 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 VIM-2 enzyme and nitrocefin in detergent-containing buffer at room temperature. The reaction is stopped by the addition of EDTA, followed by measurement of well fluorescence. As designed, compounds that inhibit VIM-2 will inhibit nitrocefin hydrolysis, inhibit generation of red product, and inhibit quenching of plate fluorescence, resulting in an increase in well fluorescence. Compounds are tested in triplicate using a dilution series starting at a nominal test concentration of 60 uM.

Nitrocefin-based IMP1 Inhibition Counterscreen (Assay 3):

The purpose of this assay is to identify compounds that act as inhibitors of the IMP-1 beta-lactamase. This assay also serves as a counterscreen to identify non-selective inhibitors. This biochemical epi-absorbance-format assay employs the cephalosporin nitrocefin as the IMP-1 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 IMP-1 enzyme and nitrocefin in detergent-containing buffer at room temperature. The reaction is stopped by the addition of EDTA, followed by measurement of well fluorescence. As designed, compounds that inhibit IMP-1 will inhibit nitrocefin hydrolysis, inhibit generation of red product, and inhibit quenching of plate fluorescence, resulting in an increase in well fluorescence. Compounds are tested in triplicate using a dilution series starting at a nominal test concentration of 60 uM.

Nitrocefin-based TEM-1 Inhibition Counterscreen (Assay 4):

The purpose of this assay is to determine whether 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" (PubChem AID 1527), were nonselective or assay artifact due to inhibition of TEM-1, a serine beta-lactamase that hydrolyzes ampicillin, has negligible activity against extended-spectrum cephalosporins, and is inhibited by clavulanic acid (14). This biochemical epi-absorbance-format assay employs the cephalosporin nitrocefin as the TEM-1 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 TEM-1 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 TEM-1 will inhibit nitrocefin hydrolysis, inhibit generation of red product, and inhibit quenching of plate fluorescence, resulting in an increase in well fluorescence. Compounds are tested in triplicate using a dilution series starting at a nominal test concentration of 60 uM.

This AID presents the results of assays that tested powder samples of a probe candidate. Probes were identified.

Grant Number: 1 R21 NS059451-01