203_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

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. Binding kinetics 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.

Fig. 1: Assay principle for the determination of LasB activity.

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.


After addition of the detection mixture, the plate is sealed and incubated at room temperature for at least 1 hour. After 1-hour signal is stable for an extended period. Detection was performed with the PHERAstar FS using standard HTRF protocol settings.

Results & Discussion

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


Fig. 2: Km determination for the LasB substrate Abz-Ala-Gly-Leu-Ala-Nba.Fig. 3: Rate of substrate hydrolysis by LasB vs substrate concentration.Fig. 4: Km determined by Lineweaver-Burk plot. The double reciprocal of the data from figure 3 is used to linearise the data.

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.

Fig. 5: Progress Curves for hydrolysis of substrate by LasB in the presence of a range of concentrations of a typical LasB inhibitor.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).

Fig. 6: Linear transformation of progress curves for a typical LasB inhibitor.

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)
 

Basic

Aromatic

Large Aliphatic

Acidic

P’2

Lys

Arg

Phe

Trp

Val

Leu

Asp

Glu

P’1

 

 

 

 

 

 

 

 

His

332

(NI)

21

18

47

306

(NI)

(NI)

Arg

135

(NI)

224

125

(NI)

(NI)

650

(NI

Lys

433

(NI)

126

(NI)

555

123

971

(NI)

Ille

190

(NI)

(NI)

366

1.8

1.3

142

(NI)

Phe

76

(NI)

146

206

11

645

(NI)

(NI)

Leu

14

623

113

300

(NI)

53

587

(NI)

Trp

10

25

1.1

49

41

3.7

38

91

Ala

153

115

(NI)

395

51

21

316

(NI)

Met

3.9

6.6

867

204

98

(NI)

7.0

(NI)

Pro

766

56

(NI)

562

157

246

(NI)

(NI)

Cys

274

646

131

108

161

(NI)

(NI)

(NI)

Asn

289

280

37

70

180

503

(NI)

(NI)

Val

22

69

72

(NI)

10

69

(NI)

(NI)

Gly

451

641

51

122

457

138

(NI)

(NI)

Ser

(NI)

444

75

(NI)

229

510

(NI)

(NI)

Gln

380

217

(NI)

91

937

540

(NI)

(NI)

Tyr

8.5

3.0

6.5

14

0.77

33

5.5

2.7

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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