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Enzyme kinetic measurements for a combinatorial library of inhibitors of Pseudomonas elastase

George Cathcart, Brendan Gilmore, Brian Walker School of Pharmacy, QUB, Belfast 10/2009
  • Pseudomonas elastase activity monitored using Abz-Nba internally quenched substrate
  • Km and Vmax calculated using MARS Data Analysis Software
  • Michaelis Menten kinetics determined for a library of 160 elastase inhibitors

Table of contents

Introduction

Pseudomonas elastase (pseudolysin, LasB) is a metalloprotease virulence factor secreted by the opportunistic pathogen Pseudomonas aeruginosa. As one of the main virulence factors of this bacterium, it contributes to chronic and intractable infection in  various disease states from the cystic fibrosis lung, to chronic ulcers of the skin.


The central role of LasB makes it a key drug target  in this process, and so a library of inhibitor candidates was developed for screening against this enzyme.  Assays were performed using a filter-based microplate reader from BMG LABTECH, which allowed highly adaptable data capture, and screening of multiple compounds in parallel. Data was analysed directly  within the MARS software, which allowed extraction of subsets of data post-assay.


Assay Principle

The assay principle is shown in figure 1.

The internally quenched protease substrate Abz-peptide-Nba (2-aminobenzoyl-Ala-Gly-Leu-Ala-4-nitro- benzylamide) gives only a low fluorescence signal. After cleavage of the peptide bound by LasB the fluorescent donor group cannot transfer the energy to the quenching acceptor group resulting in a high fluorescence signal which is directly related to the enzymatic activity.


Materials & Methods

  • Abz-Ala-Gly-Leu-Ala-Nba (Peptides International, US)
  • Library of LasB inhibitors, synthesised at The School  of Pharmacy, Queens’ University, Belfast

 

Kinetic measurement
LasB was prepared at 1 in 1000 dilution from 100 μg/mL stock, and used at 10 μL per well, giving a working concentration of 1 ng of LasB per well.


The Km of the substrate was first calculated by assay  of a series of concentrations of substrate from 20 μM to 1000 μM, against a fixed concentration of LasB.


Inhibitor studies
Stock solutions of inhibitors were prepared in DMF  at 10 mM, and further diluted when required.  Instrument settings employed were as follows:


No. of flashes per well: 10


Target temperature: 37 °C


Ex filter: 310/10 nm and Em filter: 460/10 nm


All assays were performed in buffer containing  0.05 M TRIS HCL, 2.5 mM CaCl2, 1 % DMF, pH 7.2, across a range of concentrations of inhibitor.


Results & Discussion

The results can be seen in figure 2, followed by graphical display of the rate of hydrolysis vs substrate con- centration (figure 3), and a double reciprocal or Lineweaver-Burk plot, figure 4.

The slope of the line on the Lineweaver-Burk plot  gives Km / Vmax, while the X- intercept gives - 1 / Km, and  the Y- intercept, 1 / Vmax. The data from figure 4 can therefore be used to calculate Km by solving the equation of the line Y = mX + c, where m = slope.

Linear transformation provides a value for the slope of the line, according to the equation y = mx + c. The Ki could be determined for each inhibitor in turn, via the Michael Menten equation (figure 6 and table 1).

Table 1: Ki Values (μM) for inhibitor library. ‘NI’ (No Inhibition) has been stated for values over 1000 μM. Values in grey identify a  general trend for low Ki values in inhibitors containing P’1 Trp  and Tyr residues.

 

Ki (μM)
 BasicAromaticLarge AliphaticAcidic
P’2LysArgPheTrpValLeuAspGlu
P’1        
His332(NI)211847306(NI)(NI)
Arg135(NI)224125(NI)(NI)650(NI)
Lys433(NI)126(NI)555123971(NI)
Ille190(NI)(NI)3661.81.3142(NI)
Phe76(NI)14620611645(NI)(NI)
Leu14623113300(NI)53587(NI)
Trp10251.1494.13.73891
Ala153115(NI)3955121316(NI)
Met3.96.686720498(NI)7.0(NI)
Pro76656(NI)562157246(NI)(NI)
Cys274646131108161(NI)(NI)(NI)
Asn2892803770180508(NI)(NI)
Val226972(NI)1069(NI)(NI)
Gly45164151122457138(NI)(NI)
Ser(NI)44475(NI)229510(NI)(NI)
Gln380217(NI)91937540(NI)(NI)
Tyr8.53.06.5140.77335.52.7

 

 

Conclusion

 

The microplate readers from BMG LABTECH offer convenient calculation of Km, adaptable assay optimization, parallel assay of multiple inhibitors, and isolation of subsets of data post-assay.

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