Protein kinase affinity reagents based on a 5-aminoindazole scaffold

Article abstract of DOI:10.1016/j.bmcl.2010.10.069

Affinity reagents that target protein kinases are powerful tools for signal transduction research. Here, we describe a general set of kinase ligands based on a 5-aminoindazole scaffold. This scaffold can readily be derivatized with diverse binding elements and immobilized analogs allow selective enrichment of protein kinases from complex mixtures.

Full text of DOI:10.1016/j.bmcl.2010.10.069

Bioorganic & Medicinal Chemistry Letters 21 (2011) 550–554
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Protein kinase affinity reagents based on a 5-aminoindazole scaffold
⇑
Ratika Krishnamurty, Amanda M. Brock, Dustin J. Maly
Department of Chemistry, Box 351700, University of Washington, Seattle, WA 98195-1700, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Affinity reagents that target protein kinases are powerful tools for signal transduction research. Here, we
describe a general set of kinase ligands based on a 5-aminoindazole scaffold. This scaffold can readily be
derivatized with diverse binding elements and immobilized analogs allow selective enrichment of pro-
tein kinases from complex mixtures.
Received 7 September 2010
Revised 12 October 2010
Accepted 14 October 2010
Available online 21 October 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Protein kinase
Affinity reagent
Structure–activity relationship
Indazole
Pharmacophore
Protein kinases are a key family of enzymes that regulate
reversible protein phosphorylation cascades in cells. Protein ki-
nases catalyze the transfer of a phosphate group from ATP to a tar-
get protein, serving as regulatory elements in signal transduction
pathways.1 Proper control of protein kinase activity is essential
for normal cellular behavior and aberrant kinase function has been
correlated with a number of diseases.2 Currently there are 11 small
molecule protein kinase inhibitors approved for clinical use, with
dozens of others undergoing clinical evaluation.3,4
Due to the promise of protein kinases as drug targets, significant
efforts have been made to investigate this large enzyme family.
However, only a small percentage of the kinase superfamily
(>500 members in humans) has adequately been functionally ana-
lyzed. Characterization of protein kinase expression and function
in diverse cellular contexts provides important insight into these
enzymes but global profiling remains challenging due to their gen-
erally low cellular abundance compared to other proteins. Studies
performed on the yeast proteome of S. cerevisiae found that 63% of
protein kinases were expressed at concentrations ranging from
1 nM to 50 nM, which is significantly lower than the average pro-
tein concentration in cells.5,6 A valuable technique that has aided
the global analysis of protein kinases is the use of immobilized ki-
nase inhibitors that selectively enrich these low abundance en-
zymes. These affinity reagents have been used to identify the
cellular targets of small molecule inhibitors, provide exhaustive
analysis of kinase inhibitor selectivity, and delineate phosphoryla-
tion profiles of protein kinases and their substrates.7–19 While
currently existing affinity reagents have proven to be very useful,
there still remains a need for new and diverse chemical tools for
studying kinase function. Affinity reagents based on inhibitors that
are tuned to conserved binding elements in the ATP-binding sites
of protein kinases allow for unbiased profiling of these enzymes
and increase the sensitivity of currently existing analytical tech-
niques. Furthermore, a general scaffold that that can be readily
modified should allow subsets of kinases to be enriched and ana-
lyzed. Here we describe the synthesis and biochemical character-
ization of an indazole pharmacophore, that is, a general protein
kinase ligand. In addition to possessing selectivity for protein ki-
nases over other cellular enzymes, this indazole core can be easily
derivatized for proteomics applications using a modular synthetic
approach.
An inhibitor based on the 5-aminoindazole scaffold was previ-
ously identified as a promiscuous inhibitor of protein kinases.20
We selected the indazole scaffold as a general protein kinase ligand
due to its ability to exploit conserved binding elements in the
ATP-binding site. Furthermore, the modular nature of this scaffold
allows for diverse functionalities to be introduced at multiple posi-
tions. Figure 1 shows how the 5-aminoindazole core interacts with
the ATP-binding sites of protein kinases. The indazole pharmaco-
phore lies in the narrow ATP-binding pocket, forming similar
hydrophobic interactions as the adenine ring of ATP.21 Further-
more, the indazole scaffold is able to make three hydrogen bonds
with the conserved hinge region, which is one more than the ade-
nine ring of ATP. This binding orientation projects substituents at
the C-5 amine of the indazole core into a deep hydrophobic pocket
and the amine at the C-3 position out of the binding pocket into
solvent. The solvent accessibility of the C-3 position makes this site
an ideal position for linker or reporter attachment.
⇑
Corresponding author. Tel.: +1 206 543 1653; fax: +1 206 685 8665.
E-mail address: Maly@chem.washington.edu (D.J. Maly).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.10.069

R. Krishnamurty et al. / Bioorg. Med. Chem. Lett. 21 (2011) 550–554
551
Figure 1. A schematic representation of ATP (left) and the 5-aminoindazole scaffold (right) bound to the ATP-binding pocket of a protein kinase. The indazole scaffold
occupies the adenine binding region of the pocket and makes three hydrogen bonds (dashed lines) to the amide backbone of the protein kinase hinge region.
To probe the generality of the indazole scaffold and the poten-
tial of this core to serve as a versatile affinity reagent, two flexible
synthetic schemes were developed (Scheme 1). Both synthetic
routes were used to generate a small panel of compounds based
on the indazole core (Fig. 2). The key in synthesizing this panel is
the ability to selectively modify the C-3 and C-5 amines of the inda-
zole core. Indazole scaffold 1, which was used in both synthetic
routes, was generated in two-steps. 2-Fluoro-5-nitrobenzonitrile
and hydrazine monohydrate were refluxed in ethanol to generate
the heterocyclic indazole core. Selective Boc-protection of the
N-1 position yielded the starting scaffold for derivatization. In
the first synthetic route (Route 1), the C-3 amine of nitroindazole
1 was acylated with an acid chloride of interest to give 2. Subse-
quent reduction of the nitro group via palladium-catalyzed hydro-
genation yielded the reduced indazole to which various carboxylic
acids were coupled using standard amide bond-forming condi-
tions. Final compounds 4–15 were generated by deprotection with
TFA. Synthetic route 2 allows increased diversity to be introduced
at the 3-position. First, nitroindazole 1 was reduced via palladium-
catalyzed hydrogenation to generate aniline 3. Next, the C-5 amine
was selectively acylated with carboxylic acids that had been acti-
vated with carbodiimide coupling reagents. Subsequent acylation
of the C-3 position with acid chlorides containing linkers for immo-
bilization or tag attachment yielded fully derivatized indazole
intermediates. The coupling reagent chlorodipyrroli dinocarbenium
hexafluorophosphate (PyClU) was found to be an extremely mild
and efficient means for generating acid chloride coupling partners
for this reaction. Final compounds 16 and 17 were generated by
deprotection with TFA.
To determine which substituents are readily accommodated in
the ATP-binding sites of protein kinases, indazole-based inhibitors
4–15 were tested for their ability to inhibit three diverse protein
kinases: SRC (a tyrosine kinase), PKA (an AGC kinase), and CLK1
(a CMGC kinase). The percent inhibition of each kinase at a 1 lM
concentration of inhibitor is shown in Supplementary Table 1.
Most compounds based on the indazole core were found to be
sub-micromolar inhibitors of these three kinases. We observed
that compounds containing a phenylacetic acid group at the 5-po-
sition, particularly with substitution at the meta-position, are po-
tent inhibitors of the protein kinases tested. Additionally,
Scheme 1. Functionalization of the indazole scaffold.

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R. Krishnamurty et al. / Bioorg. Med. Chem. Lett. 21 (2011) 550–554
Figure 2. Indazoles that were generated and tested for their ability to inhibit the protein kinases SRC, PKA, and CLK1.
substituents at the
a-position of the phenylacetic moiety improve
which contains an ethylamino group at the a-position of the phen-
inhibitor potency across the protein kinase panel.
ylacetic moiety, displayed the broadest inhibitory profile. Most
importantly, linker-containing compounds 16 and 17 are similarly
effective inhibitors as their parent compounds (7 and 8) against
all of the kinases tested. These results confirm that the 3-position
of the indazole scaffold can be used as a site for immobilization
and that modification with a long tether will not disrupt the inter-
action of this pharmacophore with most kinases.
To demonstrate the utility of indazole-based affinity reagents, a
series of pull-down experiments were performed. Compound 17
was selected for immobilization on solid support. ECH Sepharose
resin, which contains free carboxylic acid groups, was coupled
to the free amine of 17 (Fig. 3A). The efficiency of inhibitor
In order to further confirm that the indazole scaffold is a general
kinase binder, a smaller panel of five compounds was assayed
against an expanded set of protein kinases (SRC, CLK1, CDK2 (CMGC
kinase), Aurora A (CAM kinase), and PAK4 (STE kinase)). Promiscu-
ous inhibitors 7, 8, and 9 were selected from the original panel. In
addition, a modified version of 7, which contains an amino-polyeth-
ylene glycol (PEG) linker (analog 17), and a modified version of 8,
which contains an azidopropyl linker (analog 16), were also tested.
Gratifyingly, each of these compounds was found to be a fairly
potent inhibitor of the kinases tested, with several compounds dis-
playing IC50s in the low nanomolar range (Table 1). Compound 9,
Table 1
IC50 values (nM) of compounds 7, 8, 9, 16 and 17 against the kinases SRC, CLK1, CDK2, Aurora A, and PAK4. Values shown are the average of
three assays SEM
Compound
SRC
CLK1
CDK2
Aurora A
PAK4
7
8
9
16
17
530 70
140 10
27
<10
<10
Not tested
3
150 20
1800 300
360 30
2700 600
230 40
17
30
8
18
19
1
4
1
1
1
140
64
24
22
410 10
8
6
1
2
45
6
150 20
420 40
21
3

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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
the online version, at doi:10.1016/j.bmcl.2010.10.069.
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