R. Krishnamurty et al. / Bioorg. Med. Chem. Lett. 21 (2011) 550–554
553
Figure 3. Pull-down assays. (a) Procedure for generating affinity resin 18. (b) Enrichment of protein kinases from E. coli lysate using affinity resin 18. His6-PAK5 and
His6-CLK1 were added to bacterial lysate and enriched using a standard pull-down protocol. Collected fractions were subjected to SDS–PAGE, transferred to nitrocellulose and
analyzed using HisProbe-HRP (Pierce). Lane 2 contains a purified standard for each kinase tested. (c) Eluted fractions from CLK1, PAK5, and PAK4 pull-down experiments were
subjected to SDS–PAGE and analyzed by silver staining (SilverXpress, Invitrogen).
immobilization was monitored by analytical HPLC in the presence
of an internal standard. Optimized coupling conditions resulted in
63% of inhibitor 17 being immobilized. Final affinity resin 18 was
generated by quenching any unreacted sites with ethanolamine.
To characterize resin 18 in a complex protein mixture, affinity
pull-down assays were performed with purified protein kinases
that had been added to Escherichia coli (E. coli) lysate. This is an
attractive model for enrichment studies because E. coli lack endog-
enous eukaryotic protein kinases and only exogenously added ki-
nases should be retained. The protein kinases PAK4, PAK5 and
CLK1 were each added to E. coli lysate and the resultant kinase/ly-
sate mixtures were incubated with resin 18. After 4 h of incuba-
tion, the resin was washed multiple times, and bound protein
was eluted with an excess of free inhibitor 7. Collected fractions
were subjected to SDS–PAGE and analyzed by Western Blot
Analysis or silver staining. Figure 3B shows Western Blot Analysis
(HisProbe-HRP) of the pull-down experiments performed with
PAK5 and CLK1. In both kinases, a majority of the kinase is cap-
tured by resin 18 and very little protein is detected in the washes.
Furthermore, an ATP-competitive ligand is able to efficiently elute
both kinases from the resin. Silver staining of the eluted fractions
from each enrichment experiment show that only the exogenously
added kinase is present (Fig. 3C). This demonstrates that probe 18
is able to selectively enrich protein kinases over other highly abun-
dant ATP-binding proteins and enzymes present in E. coli, including
metabolic enzymes and heat shock proteins.
In conclusion, compounds based on the 5-aminoindazole scaf-
fold appear to be general ligands for protein kinases. This scaffold
can readily be modified with a wide array of binding elements that
mediate inhibitor potency and selectivity. The ability to rapidly
generate diverse affinity reagents based on a general protein kinase
ligand should allow extensive coverage of the kinome. In addition,
specific subsets of kinases can be targeted by using more selective
indazole reagents. Importantly, the 5-aminoindazole scaffold can
be derivatized with flexible linkers that allow immobilization on
a solid support or reporter attachment. In vitro activity assays
demonstrate that linker-derivatized versions of 5-aminoindazoles
do not have reduced potencies against most kinases. Furthermore,
an immobilized version of these inhibitors serves as an effective
affinity reagent in kinase enrichment experiments. The use of affin-
ity reagents based on the 5-aminoindazole scaffold to study kinase
function will be reported in due course.
Acknowledgments
The authors acknowledge financial support from the National
Institutes of Health (NIGMS R01GM086858). We thank J. Kuriyan
(University of California, Berkeley) for providing an expression
plasmid for SRC kinase. We thank S. Knapp (Structural Genomics
Consortium) for providing expression plasmids for His6-CLK1,
GST-PAK4, and His6-PAK5.
Supplementary data
Supplementary data associated with this article can be found, in
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