Surface plasmon resonance

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How is surface plasmon resonance data displayed? Biacore systems ... This curve is displayed directly on the computer screen during the course of an analysis.
SCIENTIFIC ARTICLE

Comparison of p38 kinase inhibitor binding affinity and kinetics using Biacore Dave Casper, Marina Bukhtiyarova and Eric B. Springman Department of Biochemistry, Locus Pharmaceuticals, Inc., Four Valley Square, 512 Township Line Road, Blue Bell, PA 19422, USA

Protein kinases are ubiquitous in signal transduction pathways and perturbations in their function are frequently associated with human pathologies, including proliferative diseases like cancer. Kinase inhibitors, therefore, are attractive drug candidates. However, most kinase inhibitors characterized to date are competitive binders for the ATP/GTP binding site on the enzyme. As approximately 500 protein kinases are encoded within the human genome (Kostich et al., 2002), this presents considerable difficulties in the design of specific inhibitors.

M

ost frequently, protein kinase assays involve the use of radiochemicals such as 32P- or 33P-ATP for the detection of kinase activity. Biacore’s surface plasmon resonance (SPR)-based biosensor technology is a label-free alternative to radio-ATP assays and at the same time provides much more information on interaction parameters such as affinity and kinetic rate constants. However, in the course of our investigations into one therapeutically interesting protein kinase, p38α (also known as MAPK-14, SAPK2a or CSBP), we found that efficient immobilization on the sensor chip surface was compromised by the intrinsic instability of the protein during the coupling step. We show in this report that this instability could be overcome by the inclusion of a reversible structure-

stabilizing, kinase-binding molecule in the immobilization buffer during the coupling procedure (Figure 1), resulting in stable biosensor surfaces binding significantly greater amounts of protein (>90% of theoretical Rmax compared to approximately 10-15% of theoretical Rmax in the absence of inhibitor (Casper et al., 2004) (see Figure 2 and Table 1). DIRECT IMMOBILIZATION PROCEDURE

Sensor Chip CM5 was docked into Biacore’s automated instrument, Biacore® 3000, and preconditioned in water at a flow rate of 100 µl/minute by applying two consecutive 50 µl pulses of 50 mM NaOH and 1M NaCl, followed by 10 mM HCl, 0.1% SDS and finally water. The surfaces were prepared by standard amine coupling via exposed primary amines on

Bind Elute

Ligand Couple

Kinase

Biosensor Surface Figure 1. Immobilization procedures. The use of some protein kinases in Biacore assays may be limited by the instability of the proteins during the amine-coupling immobilization process. In the work described here, this technological problem was alleviated by including a reversible structure-stabilizing molecule during the coupling procedure, resulting in stable sensor chip surfaces.

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Biacore Journal – Number 3 2003

Procedure

Immobilized ligand (RU)

Theoretical Rmax (RU)

Experimental Rmax (RU)

Active surface

Standard

3000-4000

33

5

15%

Stabilized

3000-4000

34

32

94%

Table 1. Theoretical and experimental Rmax for immobilized p38α.

p38α in the absence or presence of a saturating concentration (10 µM) of the p38α kinase inhibitor, SB-203580 (see Figure 2). Immobilizations were conducted at 25ºC. Flow cells were activated for 7 minutes by injecting a 70 µl mixture of 50 mM NHS:200 mM EDC. 50 µl of a 50 µg/ml solution of p38α mixed with 10 µM inhibitor in sodium acetate at pH 5.5 was subsequently injected for 5 minutes followed by a 35 µl injection of ethanolamine to block any remaining activated ester groups. Typical immobilization levels ranged from 3000-5000 resonance units (RU) (see Figure 3). It is not possible to directly observe the removal of SB203580 from p38α on the biosensor surface during the post-immobilization wash steps. However, SB-203580 is known to be a reversible inhibitor of p38α, and subsequent Biacore experiments show the re-binding and reversible dissociation of SB-203580 to p38α immobilized on the biosensor surface in quantitative agreement with expected 1:1 binding. Non-derivatized flow cells served as reference surfaces. HIGH-RESOLUTION KINASE:INHIBITOR KINETIC STUDIES

Experiments were carried out in which binding of kinase inhibitors SKF-86002, SB-203580 and ALLO-1 to p38α were monitored (Figure 4 and Table 2). A 10 mM stock of inhibitor in DMSO was prepared and diluted directly into running buffer from which three-fold serial dilutions were made, typically spanning from 3 µM to 1 nM and were injected in triplicate for 1 minute, followed by a dissociation period of 2-5 minutes. All analyses were conducted at 100 µl/minute with a data collection rate of

2.5 Hz. The studies were repeated on newly immobilized surfaces. Typically, ten samples of experimental buffer were injected at the start of the analysis to ensure the instrument was fully equilibrated. In addition, blanks were injected after every eighth sample injection for double referencing. CLAMP™ software (Myszka and Morton, 1998) was used to derive association and dissociation rate constants from the binding profiles shown in Figure 4 and the data were plotted on a kinetic distribution plot (Figure 5). It is interesting to note that while the three p38α inhibitors span a 100-fold KD range, the differences are determined primarily by specific kinetic parameters. Comparing SKF-86002 and SB-203580, we see that the nearly 10-fold difference in KD derives exclusively from the faster dissociation rate constant (kd) for SKF-86002. In contrast, the 100-fold difference in KD between SB-203580 and ALLO-1 is entirely attributable to the slower association rate constant (ka) for ALLO-1. Since association and dissociation kinetics are related to different properties of the system, kinetic binding analysis of this type can help direct inhibitor optimization efforts. Thus, two inhibitors with identical KD can have very different binding kinetics (see diagonal lines in Figure 5). For a freely reversible simple bimolecular binding event, the association rate constant is directly related to the energy barrier to association. In turn, this energy barrier is closely associated with the pre-organization of the inhibitor and the protein, i.e. the preferred conformational and solvation states before and after the binding event. The dissociation rate constant is related to the depth of the energy well in the bound state, i.e.

Compound

ka (M-1s-1)

kd (s-1)

KD

SKF-86002

1.3 x 107

1.1

85 nM

SB-203580

1.2 x 107

1.2 x 10-1

10 nM

ALLO-1

5.6 x 104

5.6 x 10-2

1.0 µM

Table 2. Kinetic and affinity constants for binding of SKF-86002, SB-203580 and ALLO-1 to p38α at 25°C.

Biacore Journal – Number 3 2003

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SCIENTIFIC ARTICLE

% response

100

p38α, stabilized with SB-230580 during immobilization using amine coupling

75 50 25

p38α, standard amine coupling

0 30

0

60 Time (s)

90

120

Figure 2. Comparison of the surface binding capacity of unprotected vs. protected (stabilized) p38α immobilization. 1 µM SB-203580 was injected across two different immobilized p38α surfaces of approximately 3700 RU in surface density. One surface contained p38α that was immobilized by a standard amine coupling procedure (lower trace) while the second surface contained p38α that was stabilized by SB-230580 during immobilization (upper trace).

the stability of the bound complex. It has been shown that inhibitors with an allosteric binding mechanism similar to ALLO-1 require significant movement of an entire protein surface loop, which likely limits the association kinetics. Conversely, the other two inhibitors used in this study, SKF-86002 and SB-203580, are highly pre-organized for binding, and changes in the protein structure are relatively local. Thus, the energy barrier to binding is relatively low and the association rate is correspondingly fast. In both of these cases, the association kinetics are limited by structural changes in the protein. Optimization (decreasing KD), therefore, is largely driven by increasing the stability of the bound complex without adversely affecting the pre-organization of the

system related to the two different inhibition mechanisms. From the reverse perspective, it is also possible to use this type of kinetic binding analysis to relate the kinetics of a novel inhibitor with unknown binding mechanism to the kinetics of a comparator inhibitor with known binding mechanism. In these ways, the kinetic analysis of protein kinase inhibitors attainable using this Biacore method can greatly facilitate the inhibitor optimization process. CONCLUSIONS

By incorporating a structure-stabilizing, kinasebinding molecule in the immobilization step we have been able to achieve efficient direct and stable amine coupling of the protein kinase, p38α on Sensor Chip CM5. The technique allows the cap-

Ethanolamine

12

Response (kRU)

8 4

p38α

EDC/NHS

~ 3-5 kRU

0 -4 -8 0

5

10

15

20

25

Time (min) Figure 3. Sensorgram report of the biosensor response during kinase immobilization. The simulated sensorgram shows the response (RU) after injection of NHS:EDC (Sensor Chip CM5 surface activation), p38α kinase mixed with inhibitor and ethanolamine (blocking step).

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Biacore Journal – Number 3 2003

B

A

C

14

25

16

12

14

20

10

10 8 6

Response (RU)

Response (RU)

Response (RU)

12

15 10

4

6 4 2

5

2

8

0

0

0 0

10

20

30

40

50

60

0

70

20

40

60

80

100 120 140 160

0

Time (s)

Time (s)

20

40

60

100 80 Time (s)

120

140

160

180

Figure 4. Inhibitor binding to p38α surfaces. Biacore response data for (A) SKF-86002, (B) SB-203580 and (C) ALLO-1 binding at 25°C. Data from triplicate inejections are shown for each compound (black) along with kinetic curve fits (red).

Dissociation rate constant (s -1)

ture of greatly increased levels of active enzyme, resulting in biosensor surfaces that are useful for the study of small molecule binding to protein kinases. Recently, we have successfully applied this approach to the protein kinase GSK3β and we are currently investigating the utility of the approach for other protein kinases. In addition to protein kinases, this strategy may be applicable in many other applications involving labile ligands. By doing so, the resulting ligand surface may also be less susceptible to detrimental entropic changes that may be introduced during the coupling step. Development of a generally applicable immobilization procedure for protein kinases will facilitate the study of therapeutically important inhibitors and the implementation of emerging biosensor multiplexing technologies.

REFERENCES

Casper, D., Bukhtiyarova, M. and Springman, E.B. A Biacore biosensor method for detailed kinetic binding analysis of small molecule inhibitors of p38α MAP kinase. Anal Biochem: in press (2004) Kostich, M., English, J., Madison, V., Gheyas, F., Wang, L., Qiu, P., Greene, J. and Laz, T.M. Human members of the eukaryotic protein kinase family. Genome Biol 3: 43.1-.12 (2002) Myszka, D.G. and Morton, T.A. CLAMP: a biosensor kinetic data analysis program. Trends Biochem Sci 23: 149-50 (1998)

0.01

ALLO-1

0.1

SB-203580

1 nM

1

SKF-86002

10 nM

10 4 10

100 µM

10

10 µM 5

1 µM

10

6

100 nM

10 -1

7

10

8

-1

Association rate constant (M s ) Figure 5. Kinetic distribution plot for p38α inhibitors at 25°C. Dissociation rate constants (kd, y-axis) are plotted against association rate constants (ka, x-axis) for three p38α inhibitors. Diagonal lines of equal affinity (kd/ka = KD) are also shown.

Biacore Journal – Number 3 2003

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