Substrate activity screening with kinases: Discovery of small-molecule substrate-competitive c-Src inhibitors

Article abstract of DOI:10.1002/anie.201311096

Substrate-competitive kinase inhibitors represent a promising class of kinase inhibitors, however, there is no methodology to selectively identify this type of inhibitor. Substrate activity screening was applied to tyrosine kinases. By using this methodology, the first small-molecule substrates for any protein kinase were discovered, as well as the first substrate-competitive inhibitors of c-Src with activity in both biochemical and cellular assays. Characterization of the lead inhibitor demonstrates that substrate-competitive kinase inhibitors possess unique properties, including cellular efficacy that matches biochemical potency and synergy with ATP-competitive inhibitors. SASsy inhibitors: Small-molecule substrate-competitive inhibitors of the tyrosine kinase c-Src were discovered through the application of substrate activity screening (SAS). Characterization of the lead inhibitor demonstrates that substrate-competitive kinase inhibitors possess unique properties, including cellular efficacy that matches biochemical potency and synergy with ATP-competitive inhibitors.

Full text of DOI:10.1002/anie.201311096

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DOI: 10.1002/anie.201311096
Kinase Inhibitors
Substrate Activity Screening with Kinases: Discovery of
Small-Molecule Substrate-Competitive c-Src Inhibitors**
Meghan E. Breen, Michael E. Steffey, Eric J. Lachacz, Frank E. Kwarcinski, Christel C. Fox, and
Matthew B. Soellner*
Abstract: Substrate-competitive kinase inhibitors represent
a promising class of kinase inhibitors, however, there is no
methodology to selectively identify this type of inhibitor.
Substrate activity screening was applied to tyrosine kinases.
By using this methodology, the first small-molecule substrates
for any protein kinase were discovered, as well as the first
substrate-competitive inhibitors of c-Src with activity in both
biochemical and cellular assays. Characterization of the lead
inhibitor demonstrates that substrate-competitive kinase inhib-
itors possess unique properties, including cellular efficacy that
matches biochemical potency and synergy with ATP-compet-
itive inhibitors.
itors. As a result, very few substrate-competitive kinase
inhibitors have been described.[6]
Herein, we describe a methodology for identifying
substrate-competitive kinase inhibitors. We utilized the sub-
strate activity screening (SAS) platform originally developed
for proteases and phosphatases by Ellman and co-work-
ers.[9,10] SAS first identifies weak binding non-peptide sub-
strates of an enzyme. The identified substrates are then
converted into inhibitors. While there are prior reports of
peptide substrates converted into peptide inhibitors for
kinases, no non-peptide substrates have been reported for
any protein kinase.[6,11–14]
Our SAS method for identifying substrate-competitive
kinase inhibitors consists of three steps (Scheme 1): 1) a
diverse library of low-molecular-weight phenols is screened to
identify non-peptidic kinase substrates; 2) the phenol sub-
strates are converted into inhibitors by replacement of the
P
rotein tyrosine kinases are heavily studied targets in drug
discovery.[1–3] All FDA-approved drugs targeting tyrosine
kinases inhibit kinase activity through competition with ATP.
Despite the popularity and success of ATP-competitive
inhibitors, there are limitations inherent to this class of
kinase inhibitors. First, owing to homology in the ATP
binding pocket across kinases, obtaining selective inhibition
for a particular kinase is exceptionally challenging.[4] Poor
selectivity leads to off-target toxicity and also limits the use of
most ATP-competitive kinase inhibitors in biological experi-
ments.[4] Additionally, ATP-competitive inhibitors must com-
pete with millimolar concentrations of ATP in cells, thus
necessitating difficult to obtain affinity (pm to nm) and/or very
high doses of inhibitor to obtain potent inhibition in vivo.[4]
Therefore, kinase inhibitors that bind outside of the ATP
pocket are of increasing interest.[5–7]
Targeting the substrate–kinase interaction is challenging
because it involves a flat protein–protein interaction surface
that lacks any obvious small molecule binding sites.[8]
Furthermore, there are no reported screening methods that
can selectively identify substrate-competitive kinase inhib-
Scheme 1. Overview of the SAS methodology for protein kinases.
[*] M. E. Breen, M. E. Steffey, E. J. Lachacz, F. E. Kwarcinski, C. C. Fox,
Prof. M. B. Soellner
phenol with nonphosphorylatable surrogates; 3) the inhibi-
tors are optimized through analogue synthesis. We developed
our methodology by using c-Src, a eukaryotic protein tyrosine
kinase. c-Src was the first proto-oncogene described and
overexpression of c-Src has been linked to the progression of
many cancers.[15,16]
In the first step, a library of 88 phenols was selected by
using computational clustering analysis from thousands of
commercially available phenols with molecular weights below
300 Da (see the Supporting Information for a complete list of
phenols screened). The phenol library was initially screened
at 100 mm in the presence of 1 mm ATP and 100 nm c-Src.
After 30 min incubation, the production of ADP (a byproduct
Departments of Medicinal Chemistry and Chemistry
University of Michigan
930 N. University Avenue, Ann Arbor, MI 48109 (USA)
E-mail: soellner@med.umich.edu
[**] Funding for this research was provided by NIH grant R01GM088546
to M.B.S. and by the University of Michigan College of Pharmacy.
M.E.B. was supported, in part, by a Pharmacological Sciences
Training Program NIH training grant (GM007767). We would like to
thank Markus Seeliger (Stony Brook) and John Kuriyan (UC
Berkeley) for providing expression plasmids for c-Src, c-Abl and
Hck. We would like to thank Kristin Ko for synthesis of PP5.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201311096.
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of the kinase reaction) was measured by using ADP-Glo,
a luciferase-based assay.[17]
From the phenol library, nine non-peptidic substrates
were found that gave more than 2.5% ADP formation (more
than two standard deviations from the mean of the entire
phenol library). Phosphorylation of the substrate was verified
by HPLC (see the Supporting Information). We next
obtained KM values for the nine selected substrates and
found KM values that spanned 15–522 mm (Scheme 2). Sig-
nificantly, these represent the only non-peptidic substrates
known for any protein kinase, and five of the phenols gave KM
values lower than an optimal pentapeptide substrate for c-Src
(Ac-AIYAA-NH2, KM = 60 mm; Table S2 in the Supporting
Information).
Scheme 3. Conversion into inhibitors by using nonphosphorylatable
surrogates of phenol.
modest inhibition (Ki = 478 and 552 mm, respectively). Each
inhibitor was found to be substrate-competitive in biochem-
ical assays (Table S4) and none were phosphorylated by c-Src
(see the Supporting Information).
We found that tetrafluorophenol 7 is selective for c-Src
over Hck, while both pyridine N-oxide 8 and hydroxypyridine
9 inhibited c-Src and Hck with comparable potencies (see the
Supporting Information). These results suggest that the
optimal inhibitor pharmacophore is likely different for each
kinase. While this necessitates additional compound syn-
thesis, it also affords an opportunity to refine the selectivity of
the inhibitor.
In the third step, we synthesized a small focused library of
tetrafluorophenol diphenyl amines (Table S3). The tetrafluo-
rophenol inhibitor pharmacophore was chosen for inhibitor
optimization on the basis of its superior potency and
selectivity. Three members of the library gave improved Ki
values relative to the parent inhibitor 7 (Scheme 4). The most
potent analogue, compound 12, gave a Ki value of 16 mm and
represents one of the most potent substrate-competitive
kinase inhibitors reported to date,[6] on par with other small-
molecule inhibitors of protein-protein interactions.[19]
Scheme 2. Phenol substrates of c-Src and their corresponding KM
values.
As a measure of substrate selectivity, we determined the
KM value for each phenol against two kinases with homology
to c-Src: Hck and c-Abl (85% and 70% similarity, respec-
tively, to the c-Src kinase domain). Of the nine best c-Src
phenolic substrates, four phenols were also Hck substrates
and none were phosphorylated by c-Abl. This suggests that
despite high sequence conservation and nearly identical ATP-
binding sites, homologous kinases have divergent substrate-
binding preferences, even for small molecule substrates.
The second step built on prior work by Graves and co-
workers, who showed that fluorination of a substrate tyrosine
yielded a peptide inhibitor for insulin receptor kinase.[11,18]
The tetrafluorotyrosine peptide bound the substrate site,
however, it was not phosphorylated at neutral pH values.[11,18]
In addition to tetrafluorophenol, we hypothesized that
pyridine N-oxide (8) and hydroxypyridine (9) might also
serve as mimics of a phenol without being phosphorylatable
(Scheme 3).
Scheme 4. Optimized substrate-competitive c-Src inhibitors.
Inhibitor 12 is selective for c-Src over homologous
kinases, which is in stark contrast to PP2, an ATP-competitive
inhibitor of c-Src (Table 1).[20,21] We tested both 12 and PP2
against the Src family of kinases (nine members) and found
that while PP2 showed no selectivity across the family,
compound 12 showed an average selectivity of six-fold.
Within the homologous Src family, compound 12 is the most
selective inhibitor for c-Src reported to date. Notably,
compound 12 shows more than five-fold selectivity for c-Src
over c-Yes, a Src family kinase with 95% sequence similarity
and 90% sequence identity to c-Src in the kinase domain. A
For conversion from substrate to inhibitor, we selected the
p-aniline phenol 5 (KM = 120 mm) because of its affinity and
the synthetic feasibility of evaluating each of the three
potential phenol isosteres. Fluorination of the phenol led to
inhibitor 7, which gave a Ki value of 257 mm. Meanwhile,
pyridine N-oxide (8) and hydroxypyridine (9) each provided
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Table 1: Biochemical selectivity data for homologous kinases.
Table 2: Comparison of cellular data for substrate-competitive inhibitor
12 and ATP-competitive inhibitor PP2.
Kinase
12 (selectivity ratio)
PP2 (selectivity ratio)
12
PP2
c-Src
Yes
Hck
Blk
Fgr
Frk
Fyn
Lck
Lyn
c-Abl
16 mm
0.05 mm
82 mm (5)
325 mm (20)
52 mm (3)
51 mm (3)
83 mm (5)
61 mm (4)
63 mm (4)
60 mm (4)
1.0 mm (63)
0.05 mm (1)
0.09 mm (2)
0.07 mm (2)
0.03 mm (1)
0.02 mm (1)
0.02 mm (1)
0.01 mm (1)
0.02 mm (1)
0.4 mm (9)
Biochemical Ki
Cellular phosphorylation (IC50)
Ratio (cell/biochemical)
16 mm
15 mm
0.9
0.05 mm
2.2 mm
44
the non-Src-dependent cancer cell lines MCF7 and T47D.
Finally, we examined the activation of c-Src dependent (Jnk
and STAT3) and c-Src independent (Akt and Erk) signaling
pathways in SK-BR-3 cells. We found that compound 12
inhibited the activation only of Src-dependent pathways,
while PP2 was active against all four signaling pathways.
Together, these data demonstrate that inhibitor 12 acts as
a highly selective c-Src inhibitor in cellulo.
recent comprehensive survey of ATP-competitive kinase
inhibitor selectivity found no ATP-competitive kinase inhib-
itors with this level of selectivity for c-Src over c-Yes.[22] These
results highlight the unprecedented selectivity that can read-
ily be obtained with substrate-competitive kinase inhibitors.
Inhibitors identified from SAS should inherently be
substrate-competitive. However, because multiple binding
sites exist on protein kinases, we wanted to confirm the mode
of action for inhibitor 12. We found that the IC50 values were
sensitive to peptide substrate concentration but not to ATP
concentration (see the Supporting Information). Further-
more, Lineweaver–Burk and KM analyses are consistent with
a substrate-competitive and ATP-noncompetitive mode of
action (see the Supporting Information).
To provide further insight into the binding mode, we
performed induced-fit docking to flexibly dock 12 into c-Src
(see the Supporting Information).[23] In the docked model, an
interaction was predicted between Arg388 and inhibitor 12.
This arginine residue is replaced by an alanine in c-Abl
(Arg365 in c-Abl replaces Ala390 in c-Src). We produced
R388A/A390R c-Src and found that inhibitor 12 was a weak
inhibitor of this enzyme (Ki = 184 mm) compared to wild-type
c-Src (Ki = 16 mm). These data are consistent with the
proposed binding model and, together with the biochemical
analyses, strongly support a substrate-competitive mode of
action.
We next evaluated compound 12 in a cellular context. In
an enzyme-linked immunosorbent assay (ELISA)-based
assay, compound 12 was found to have an IC50 value of
15 mm for cellular c-Src autophosphorylation. This result
demonstrates that compound 12 is both cell permeable and
capable of inhibiting c-Src in cells. We then tested the ability
of compound 12 to inhibit the growth of SK-BR-3 and HT-29
cells, cancer cell lines previously shown to be c-Src growth
dependent.[20,24] In this assay, compound 12 produced growth
inhibition with GI50 = 15 mm for SK-BR-3 and GI50 = 37 mm
for HT-29. Of note, compound 12 is significantly more potent
than PP2 against both SK-BR-3 and HT-29 cells (Table 2).[20]
In fact, compound 12 shows SK-BR-3 antiproliferative
activity similar to the most potent c-Src inhibitors known,
including the FDA-approved c-Src inhibitors dasatinib and
bosutinib.[25,26]
A
long-standing hypothesis of substrate-competitive
kinase inhibitors posits that no significant loss in cellular
potency should be observed for substrate-competitive inhib-
itors because kinase substrates are present in concentrations
at or below their KM values.[6] This is in stark contrast to ATP
competitive inhibitors, where the KM values are often low
micromolar while ATP is present in millimolar concentra-
tions.[4,27] Inhibitor 12 represents one of very few substrate-
competitive tyrosine kinase inhibitors that shows activity in
both biochemical and cellular assays and is the only such
inhibitor of c-Src.[6] The biochemical Ki value of 12 for c-Src is
nearly identical to the IC50 value for cellular autophosporyl-
ation of c-Src. In contrast, PP2 has a biochemical Ki of 45 nm
and an IC50 for c-Src autophosphorylation of 2.2 mm. Thus,
while our substrate-competitive inhibitor loses no efficacy,
a classic ATP-competitive inhibitor is 44-fold less active
in cellulo (Table 2).
We also hypothesized that our substrate-competitive
inhibitor could be used simultaneously with an ATP-compet-
itive kinase inhibitor. To test this hypothesis, we used IC35
concentrations of compound 12 in combination with PP2 or
PP5. PP2 and PP5 are well-established ATP-competitive
inhibitors of c-Src that bind the active and inactive con-
formations, respectively.[21,28] We found that both PP2 and PP5
were synergistic (hyper-additive) when combined with inhib-
itor 12 (Figure 1).[29] Together, these data show for the first
time the ability of substrate-competitive inhibitors to bind
simultaneously with ATP-competitive inhibitors.
Herein, we have described the first methodology that
enables the discovery of small molecule substrate-competitive
kinase inhibitors. This class of compounds has been proposed
to have several advantages, however, a dearth of compounds
prevented proper evaluation of their potential. We applied
our methodology to c-Src and identified inhibitor 12 (Ki =
16 mm). Biochemical, computational, and mutagenesis studies
support a substrate-competitive mode of action. When using
compound 12, we observed nearly identical cellular efficacy
compared to biochemical potency, a feature not found with
ATP-competitive inhibitors. Unlike ATP-competitive inhib-
itors, we demonstrated that biochemical and cellular selec-
tivity is inherent in this class of compounds. Finally, we
demonstrated that substrate-competitive inhibitors can be
used simultaneously with ATP-competitive inhibitors to
We observed excellent correlation between the ability of
compound 12 to inhibit c-Src activity and the growth of
a cancer cell line dependent upon c-Src activity (Table 2). In
addition, compound 12 was inactive (GI50 > 100 mm) against
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[8] R. Bose, M. A. Holbert, K. A. Pickin, P. A. Cole, Curr. Opin.
Struct. Biol. 2006, 16, 668 – 675.
[9] W. J. Wood, A. W. Patterson, H. Tsuroka, R. K. Jain, J. A.
Ellman, J. Am. Chem. Soc. 2005, 127, 15521 – 15527.
[10] M. B. Soellner, K. A. Rawls, C. Grundner, T. Alber, J. A.
Ellman, J. Am. Chem. Soc. 2007, 129, 9613 – 9615.
[11] C. J. Yuan, S. Jakes, S. Elliott, D. J. Graves, J. Biol. Chem. 1990,
265, 16205 – 16209.
[12] F. A. al-Obeidi, J. J. Wu, K. S. Lam, Biopolymers 1998, 47, 197 –
223.
[13] J. Niu, D. S. Lawrence, J. Biol. Chem. 1997, 272, 1493 – 1499.
[14] J. Chapelat, F. Berst, A. L. Marzinzik, H. Moebitz, P. Drueckes, J.
Trappe, D. Fabbro, D. Seebach, Eur. J. Med. Chem. 2012, 57, 1 –
9.
[15] S. E. Thomas, J. S. Brugge, Annu. Rev. Cell Dev. Biol. 1997, 13,
513 – 609.
[16] G. S. Martin, Nat. Rev. Mol. Cell Biol. 2001, 2, 467 – 475.
[17] H. Zegzouti, M. Zdanovskaia, K. Hsiao, S. Goueli, Assay Drug
Dev. Technol. 2009, 7, 560 – 572.
Figure 1. Synergy studies of combinations of substrate-competitive
inhibitor 12 with ATP-competitive inhibitors PP2 or PP5. IC35 concen-
trations were dosed individually and in combination. The dotted line
denotes predicted additivity [(eA+eB)À(eA*eB)] of 12+PP2 (or
PP5).[25] A higher level of inhibition than the predicted additivity
indicates synergism.
[18] B. L. Martin, D. Wu, D. Jakes, D. J. Graves, J. Biol. Chem. 1990,
265, 7108 – 7111.
[19] J. A. Wells, C. L. McClendon, Nature 2007, 450, 1001 – 1009.
[20] K. R. Brandvold, M. E. Steffey, C. C. Fox, M. B. Soellner, ACS
Chem. Biol. 2012, 7, 1393 – 1398.
provide synergistic inhibition of the target kinase. Our
methodology is the only screening technique to selectively
identify substrate-competitive kinase inhibitors and should be
applicable to any tyrosine kinase of interest.
[21] J. H. Hanke, J. P. Gardner, P. S. Changelian, W. H. Brissette, E. J.
Weringer, D. A. Pollock, P. A. Connelly, J. Biol. Chem. 1996, 271,
695 – 701.
[22] M. I. Davis, J. P. Hunt, S. Herrgard, P. CIceri, L. M. Wodicka, G.
Pallares, M. Hocker, D. K. Treiber, P. P. Zarrinkar, Nat. Biotech-
nol. 2011, 29, 1046 – 1051.
[23] W. Sherman, T. Day, M. P. Jacobson, R. A. Friesner, R. Farid, J.
Med. Chem. 2006, 49, 534 – 553.
[24] X. Zheng, R. J. Resnick, D. Shalloway, Int. J. Cancer 2008, 122,
1999 – 2007.
Received: December 20, 2013
Revised: March 6, 2014
Published online: May 2, 2014
Keywords: drug discovery · inhibitors · kinases ·
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medicinal chemistry · substrate activity screening
[1] A. Levitzki, A. Gazit, Science 1995, 267, 1782 – 1788.
[2] P. Cohen, Nat. Rev. Drug Discovery 2002, 1, 309 – 315.
[3] J. M. Zhang, P. L. Yang, N. S. Gray, Nat. Rev. Cancer 2009, 9, 28 –
39.
[25] B. J. Park, Z. L. Whichard, S. J. Corey, Cancer Lett. 2012, 320,
104 – 110.
[26] M. Fallahi-Sichani, S. Honarnejad, L. M. Heiser, J. W. Gray, P. K.
Sorger, Nat. Chem. Biol. 2013, 9, 708 – 714.
[4] Z. A. Knight, K. M. Shokat, Chem. Biol. 2005, 12, 621 – 637.
[5] K. J. Cox, C. D. Shomin, I. Ghosh, Future Sci. 2011, 3, 29 – 43.
[6] G. Ye, R. Tiwari, K. Parang, Curr. Opin. Invest. Drugs 2008, 9,
605 – 613.
[27] G. Scapin, Curr. Drug Targets 2006, 7, 1443 – 1454.
[28] A. C. Dar, M. S. Lopez, K. M. Shokat, Chem. Biol. 2008, 15,
1015 – 1022.
[29] M. C. Berenbaum, Adv. Cancer Res. 1981, 35, 269 – 335.
[7] D. Lavogina, E. Enkvist, A. Uri, ChemMedChem 2010, 5, 23 – 34.
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