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A Microtiter-based Assay for the Determination of ID50s of b-lactamase Inhibitors Employing Reporter Substrates Detected at UV or Visible Wavelengths (MaxLine Application Note #20)

β -lactamases are either plasmid or chromosomally encoded bacterial enzymes which hydrolyze β -lactam antibiotics. The majority of β -lactamases produced by clinical isolates are serine active site enzymes. One successful approach to combating the serine active site β -lactamases is the use of β -lactamase inhibitors, such as clavulanic acid, in combination with a β -lactam antibiotic4.

There is also a small group of β -lactamases which have a metal ion at their active site (metallo-β -lactamases). These enzymes are not sensitive to serine β lactamase inhibitors and have a much broader substrate profile than the serine enzymes. Metallo-β -lactamases (MBLs) are capable of hydrolyzing the majority of clinically important β -lactam antibiotics, including carbapenems.

To date, more than 190 different β -lactamases have been identified1 and we will continue to observe clinical isolates which produce new types of enzymes, such as MBLs. Therefore, to enable the clinical spectra of established β -lactamase inhibitor/β -lactam combinations to be defined, it is necessary to evaluate these inhibitors against all new β -lactamases. In addition, novel inhibitors with improved potency and spectra are required to combat the current plethora of enzymes (both metallo and serine active site β -lactamases) and such compounds require rapid and effective evaluation.

This application note describes a simple procedure to determine the inhibition (ID50 value) of β -lactamases by various agents using the chromogenic cephalosporin nitrocefin (λ max = 482 nm) and the carbapenem antibiotic imipenem (λ max = 299 nm) as reporter substrates. Imipenem was used for those carbapenemases which did not have significant hydrolytic activity against nitrocefin. The method described uses the UV capability of the SPECTRAmax 250 micro-plate spectrophotometer and can be readily modified to determine ID50 values of inhibitors of other enzymes requiring UV/Vis monitoring.


1. SPECTRAmax 250 microplate spectrophotometer
2.SOFTmax PRO software
3.Quartz microplates
4. Deep well microplate
5. Multichannel pipette
7. Imipenem and nitrocefin
8. β -lactamase preparation
9. Experimental compounds

Method for determining ID50 values following a 5 minute incubation of enzyme and inhibitor.

Step 1 Set the incubator temperature to 37C., then allow it to equilibrate for at least 30 minutes

Step 2 Set up SOFTmax PROs Instrument Settings dialog box for imipenem as shown in Figure 1 and for nitrocefin as shown in Figure 2.

Step 3 Dissolve the inhibitors in 25 mM piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES) buffer, pH 7.0 with no added Zn2+ to produce a 3 mM solution. 5% DMSO can be used if needed to aid in dissolving the inhibitors.

Step 4 In a deep-well 96 well plate, pipette 600 l of 25 mM PIPES buffer, pH 7.0 into each well in columns 1-11.

Step 5 Pipette 1 mL 3 mM inhibitor solution into each well in column 12.

Step 6 Using a multichannel pipette, make a 1:3 serial dilution of the 3 mM solution down to column 3: take 300 l of 3 mM solution from column 12 and dispense into column 11. Mix, then take 300 l of column 11 and dispense into column 10, and so on across the columns to column 3 which will then be 0.15 M.

Step 7 Aliquot 50 l of the contents of the deep wells into the corresponding wells on the assay microtiter plate to create a range of inhibitor concentrations between column 3 and column 12. If the reporter substrate requires UV monitoring (e.g. imipenem), UV transparent microtiter plates (e.g., plastic or quartz SPECTRAplates, Molecular Devices Cat. no: R9012 or R8024, respectively) must be used.

Table I shows the final concentration of inhibitor in each well, taking into account the 50 l reporter substrate and 50 l enzyme that will be added to each well. 100 l and 50 l of 25 mM PIPES buffer, pH 7.0 are added to the Blank and Control wells, respectively.

Step 8
Addition of enzyme: both pure and crude β -lactamase samples (prepared as described in reference 5) are compatible with this screening format. Some of the enzymes used are shown in Table 2. The enzyme preparations for the assay are diluted in PIPES buffer, pH 7.0, as required. 50 l of the diluted enzyme is added to columns 212 of the assay microtiter plate. For MBLs, CfiA, BcII and L-1, 150 M ZnSO4 is added to the dilution buffer. For CphA, 1.5 M ZnSO4 is added. These levels of zinc are used to achieve optimal enzyme activity. No ZnSO4 is added to the buffer used to dilute the serine active site enzymes.

Step 9 Using SOFTmax PRO, open the SPECTRAmax 250 drawer, place the plate onto the carriage and close the drawer. Incubate the assay microplate in the SPECTRAmax 250 at 37 C. After 5 minutes, open the drawer of the SPECTRAmax 250 and remove the plate from the carriage.

Step 10 Add 50 l of reporter substrate to all 96 wells. 150 M ZnSO4 is added to the reporter substrate solutions used for assays with BcII, CfiA and L-1 metallo-β -lactamases. 1.5 M is added to CphA and no ZnSO4 is added to the reporter substrate solutions used to assay the serine β -lactamases. With the exception of the CphA β -lactamase, all ID50s were measured using a concentration of reporter substrate which was approximately 5 times the Km of the reporter substrate for each of the enzymes (Table 2 ). This enables more accurate comparisons of ID50s for the different enzymes. This is particularly relevant if competitive inhibitors are being evaluated. Table 3 summarizes the contents of each well in the assay microtiter plate.

Step 11 Rapidly place the plate on the carriage of the SPECTRAmax 250 and click on the READ button.

Step 12 The axis of the well graphs can be changed while reading by clicking the reduction button in the plate window. Reading can be stopped at any point before the completion of the test by clicking on the STOP button in the control panel of the software. (This will save the data already collected.)

This protocol can also be used to measure ID50s following different preincubation times of enzyme and inhibitor. In addition, where no preincubation is required, the enzyme solution should be added last.

Figure 3
shows an assay microtiter plate set up to record the ID50s of 8 compounds. The rate of hydrolysis of reporter substrate is shown for each well (column 1 = blank, column 2 = control, columns 3 - 12 = range of inhibitor concentrations). This particular protocol has been set up to calculate the% inhibition of the enzyme at each concentration of inhibitor compared to the control. The blank value for each compound is automatically subtracted within the software program.

SOFTmax PRO will plot percent inhibition vs. concentration on a 4 Parameter logistic curve fit, and will then calculate the ID50 for each compound, in this case taken as the concentration of inhibitor (M) required to inhibit the enzyme by 50%.

Examples of the results produced are shown in Figure 3 and Figure 4.

Table 4 shows the SOFTmax PRO printout of ID50s for inhibitors A-H.

Using SPECTRAmax 250 to determine ID50 has three major advantages:
1. High throughput. The protocol described enables 16 ID50s to be determined for 5 different enzymes in 3-4 hours (80 ID50s in total). SOFTmax PRO software rapid and accurate determination of ID50s without having to download data into other curve fitting software.

2. Flexibility of reporter substrate. β -lactamase assays performed on other plate readers are limited to using chromogenic substrates such as nitrocefin. However, as the SPECTRAmax 250 reads in the UV, other substrates can be utilized. This means that assays requiring UV monitoring were previously limited to standard spectrophotometers, but can now be converted to microtiter plate formats for high throughput. For example, nitrocefin is an extremely poor substrate for the CphA enzyme and an inappropriate reporter substrate. The UV capability of the SPECTRAmax 250 means that imipenem can be utilized as the substrate, enabling this enzyme to now be evaluated in the micro-titer plate format.

3. Use of small quantities of reagents. This system enables the β -lactamase ID50 assays to be performed on a microtiter plate system. If equivalent data was determined with a spectrophotometer using a method similar to that reported by Holt et al (1983), 20 times more reagent would be required.

This methodology could be utilized to measure the inhibition of a variety of other enzymes.

The introduction of the SPECTRAmax 250 spectrophotometric microplate reader together with its powerful data analysis software, SOFTmax PRO, has streamlined and greatly simplified the inhibitory screening of test compounds, allowing the rapid determination of ID50 values using limited amounts of reagents.

1. Bush, K., Jacoby, G. A. and Medeiros, A. A. A functional classification scheme for β -lactamases and its correlation with molecular structure. Antimicrobial Agents and Chemotherapy 39:1211-1233 (1995).

2. Holt, J., Simpson, I.N. and Harper, P.B. Quantitative methods for assessing the stability of β -lactam antibiotics to cell-free b -lactamase extracts. Antibiotics: Assessment of Antimicrobial Activity and Resistance (Russell, A.D. and Quesnel, L.B., Eds.) pages 127-139 (1983).

3. Khushi, T., Payne, D.J., Fosberry, A. and Reading. Production of metal dependent β -lactamases by clinical strains of B.fragilis isolated before 1987. Journal of Antimicrobial Chemotherapy 37:345-350 (1996).

4. Neu, H.C., Wilson, A.P.R., Gruneberg, R.N. Amoxycillin/clavulanic acid: a review of its efficacy in over 38,500 patients from 1979-1992. Journal of Chemotherapy 5:67-93 (1993).

5. Payne, D.J., Cramp, R., Winstanley, D.J. and Knowles, D.J.C. Comparative activities of clavulanic acid, sublactam and tazobactam against clinically important β -lactamases. Antimicrobial Agents and Chemotherapy 38, 767-772 (1994).

We thank Dr. David Livermore of the Department of Medical Microbiology, London Hospital Medical College, for the bacterial strain producing SME-1.

Dr. David J. Payne and Sarbendra Pradhananga
Department of Molecular Microbiology, SmithKline Beecham Pharmaceuticals, 1250 S. Collegeville Rd., PO Box 5089, Collegeville PA 19426-0989, USA

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