Trp115Phe114

Phe130

Leu202

Leu133

Val139Tyr157

His146

Zn2+

His231Ile188

Glu166

Arg203

Glu143His142

Ala113

Asn112

Asn111

Asn116

N

N

NNH

N

NH

OS1

S2'

P2'

S1'

NH

O

P

HN

HN

O

O-O

H2N

O

NH

HN

HN

HO

O

OOH

O

OH

2N

NH2

H2N

OH

3

4

5

6

-3 -2 -1 0

Log

k a (s-1

M-1

)

Fast

er a

ssoc

iatio

n

Log kd (s-1)

Slower dissociation

A1A2A3A4A5A6A7

B1B2B3B4

C1C2C3C4C5

D1D2D3

100 nM10 nM

1 µM

10 µM

100 µM

Kd

Biotin CAPture reagent

Biotinylated Thermolysin

Inhibitor

Sensor chip CAP

Generic regeneration

(S) (S) (R) (R)

OH

O

OH

O

OH

O

OH

O

OH

O

NH2

NH2

OH

O

OH

O

NH2

O

Resp

onse

(RU

)

Time (s)

-3

1

5

9

13

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Resp

onse

(RU

)

Time (s)

-3

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Resp

onse

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-100 0 100 200 300 400 500 600 700 800 900 1000Time (s)

-3

1

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0 100 200 300 400 500 600 700 800 900 1000

Resp

onse

(RU

)

1

-3

5

9

13

-200 0 200 400 600 800 1000 1200 1400 1600 1800

Resp

onse

(RU

)

Time (s)

Resp

onse

(RU

)

Time (s)

-3

1

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1

-3

5

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onse

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)

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onse

(RU

)

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-3

1

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-100 0 100 200 300 400 500 600 700 800 900 1000

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Introduction Results SPR analysis

Structure-kinetic relationship

Assay overview

Thermolysin as a model system for SAR and SKR

References

Conclusions

Binding kinetics by Surface Plasmon Resonance: Insight into Structure-kinetics/thermodynamics relationshipsVeronica Fridh1, Robert Karlsson1, Stefan Krimmer2, Jonathan Cramer2, and Gerhard Klebe2

1GE Healthcare Bio-Sciences AB, SE-751 84 Uppsala, Sweden. 2Institute of Pharmaceutical Chemistry, Philipps-University Marburg, Marbacher Weg 6, D 35032 Marburg, Germany

Biacore™ systems are highly sensitive and robust instruments capable of characterizing binding kinetics of biochemical interactions over a wide dynamic range and at very low response levels. This is a prerequisite when studying structure-kinetics relationships for small molecule series with large variation in their kinetic behaviors and when working with difficult protein targets.

In an on-going collaboration with Philipps-University Marburg we have been determining the binding kinetics between Thermolysin and a range of inhibitors with known thermodynamic and structural properties. These inhibitors are part of congeneric, systematic series initially designed for Structure-Activity Relationships by ITC and crystallography. However, Structure-Kinetics-Thermodynamic relationship of such a set of closely related compounds has not been reported so far. By thorough analysis of this system we aim to deepening our understanding about the importance of binding kinetics in small molecule-protein interactions.

Here we present novel data from the kinetic characterization of the thermolysin inhibitors using Biacore T200 and some early conclusions around the relation between kinetics, thermodynamic and structure.

20 available thermolysin inhibitors from the congeneric, systematic series provided by the group of Prof Klebe, were analyzed by SPR. The sensorgrams showed clear differences in binding characteristics within and/or between series. Representative sensorgrams are shown below.

Series A. Bulkier P2' substituent.

A1

A1

B2

C2

D1

A5

B3

C5

D3

A5A3 A7A2

B1

D1

C1

A6

B3

D3

C3

A4

B2

D2

C2

A8** No kinetic data

obtained due to precipitation.

B4

C4 C5

Series B. Steroisomers from A4.

Series C. Addition of carboxy group + bulkier P2' substituent.

Series D. Addition of an amine or amide.

Thermolysin (Mw = 37.5 kDa, Calbiochem) was biotinylated and

immobilized using the Biotin CAPture Kit (GE Healthcare) enabling reversible capture. Inhibitors (M

w average 454 Da) were analyzed

using Single Cycle Kinetics. Experiments were performed on Biacore T200 and data was fitted to a 1:1 binding model.

Binding kinetic parameters were determined in duplicate for all inhibitors except for A8 (no data) and D2 (single data) due to precipitation issues. To view significant changes in binding kinetic properties, k

d values were plotted agains k

a values with highlighted 95% confidence intervall (2 × SD).

• Thermolysin, a zink metalloprotease, is a proven model system for Structure Activity Relationships (SAR) based on thermodynamic and crystallographic studies (1,2)

• The binding site consists of three specificity pockets; S1, S

1' and S

2'

• Inhibitor series were designed by varying the P2' position in the S

2' site

1. A. Biela, N. N. Nasief, M. Betz, A. Heine, D. Hangauer, G. Klebe, Angew. Chem. Int. Ed. 2013, 52, 1822 –1828; Angew. Chem. 2013, 125, 1868 –1876.

2. Krimmer SG, Betz M, Heine A, Klebe G. ChemMedChem. 2014 May;9(5):877

The binding pocket of Thermolysin. Highly variable water patterns characterize the binding of molecular portions to the least buried site, the S

2’ site.

• Kinetic parameters for thermolysin inhibitors from closely related series, were determined in Biacore T200 using reversible capture of biotinylated Thermolysin

• The small modifications of the inhibitor structures resulted in a variation in dissociation with 2 orders of magnitude and a variation in association with 1 order of magnitude

• The most pronounced kinetic effect was achieved by altering the electrostatics of the compounds as seen by the addition of a carboxy group (series C) resulting in a significant decrease in k

d

• Ongoing analysis of the Biacore results combined with existing thermodynamic and high resolution crystallographic data aim to further our understanding of Structure-Kinetics-Thermodynamic relationships in general

• Modifications in P2' resulted in

kd values from 6.7 × 10-1 to 6.0 × 10-3 s-1 and

ka values from 1.3 × 104 to 2.2 × 105 s-1M-1.

• Most compounds within the same series distribute along the same isoaffinity lines; a structural change that affects k

d seem to be compensated for by a

change in ka.

• Series C, with added carboxy group, displayed significantly more stable complex formations compared to series A, without the carboxy group.

• The exception is C5, the bulkiest inhibitor in series C, indicating that large, apolar residues may affect dissociation.

• Highest affinity was seen for C2 but further increase of the P2

' in series C does not favour the ka.

• B1 and B3 have similar kinetics to A4. Further addition of a methyl group (B2 and B4) results in significantly slower k

a. Notably, this pairing is on

contrast to the thermodynamic/crystal structure data where the compounds paired according to stereochemistry: B1 and B2 (ΔΔH = 1.0 kJ/mol; -TΔΔS = 2.1 kJ/mol); B3 and B4 (ΔΔH = 0.5 kJ/mol; -TΔΔS = 0.2 kJ/mol).