Discovery of a Covalent Kinase Inhibitor from a DNA-Encoded Small-Molecule Library × Protein Library Selection

Article abstract of DOI:10.1021/jacs.7b04880

We previously reported interaction determination using unpurified proteins (IDUP), a method to selectively amplify DNA sequences encoding ligand:target pairs from a mixture of DNA-linked small molecules and unpurified protein targets in cell lysates. In this study, we applied IDUP to libraries of DNA-encoded bioactive compounds and DNA-tagged human kinases to identify ligand:protein binding partners out of 32 096 possible combinations in a single solution-phase library × library experiment. The results recapitulated known small molecule:protein interactions and also revealed that ethacrynic acid is a novel ligand and inhibitor of MAP2K6 kinase. Ethacrynic acid inhibits MAP2K6 in part through alkylation of a nonconserved cysteine residue. This work validates the ability of IDUP to discover ligands for proteins of biomedical relevance.

Full text of DOI:10.1021/jacs.7b04880

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Communication
Discovery of a covalent kinase inhibitor from a DNA-
encoded small-molecule library X protein library selection
Alix I. Chan, Lynn M. McGregor, Tara Jain, and David R Liu
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b04880 • Publication Date (Web): 09 Jul 2017
Downloaded from http://pubs.acs.org on July 10, 2017
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Discovery of a covalent kinase inhibitor from a DNA-encoded small-
molecule library protein library selection
´
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†
‡
Alix I. Chan, Lynn M. McGregor , Tara Jain , and David R. Liu*
The Broad Institute of Harvard and MIT, Howard Hughes Medical Institute, and the Department of Chemistry and Chemical Biolo-
gy, Harvard University, 75 Ames St, Cambridge, Massachusetts 02142, USA.
Supporting Information Placeholder
library of DNA-
linked ligands
library of DNA-linked protein
targets in cell lysate
ABSTRACT: We previously reported interaction determina-
tion using unpurified proteins (IDUP), a method to selec-
tively amplify DNA sequences encoding ligand:target pairs
from a mixture of DNA-linked small molecules and unpuri-
fied protein targets in cell lysates. In this study we applied
IDUP to libraries of DNA-encoded bioactive compounds
and DNA-tagged human kinases to identify ligand:protein
binding partners out of 32,096 possible combinations in a
single solution-phase library × library experiment. The re-
sults recapitulated known small molecule:protein interac-
tions and also revealed that ethacrynic acid is a novel ligand
and inhibitor of MAP2K6 kinase. Ethacrynic acid inhibits
MAP2K6 in part through alkylation of a non-conserved cys-
teine residue. This work validates the ability of IDUP to dis-
cover ligands for proteins of biomedical relevance.
ligand:target
binding induces
hairpin formation
+
complementary
regions
ligand:target binding pairs decoded
using high-throughput DNA
sequencing
all interactions evaluated
simultaneously in library X
library experiment
Figure 1. Overview of IDUP. DNA-barcoded small molecules
and proteins are combined in cell lysate. Primer extension, PCR
and DNA sequencing reveal the identity of protein:ligand pairs.
To address some of these drawbacks, our group devel-
oped interaction determination using unpurified proteins
(
IDUP), a solution-phase method for in vitro identification
Discovering small molecules that specifically modulate
the activity of proteins of biomedical interest remains a cru-
cial activity in the life sciences. DNA-encoded chemical li-
braries have emerged as a rich source of such small molecules
as biological probes and leads for therapeutics development,
and are typically evaluated for binding to individual protein
of protein-binding ligands from combinations of ligands and
unpurified proteins in a single experiment. ꢀIn IDUP, binding
6
of a DNA-tagged protein and a DNA-encoded ligand stabi-
lizes the hybridization of short (6- to 8-nt) complementary
regions at the 3’ ends of their associated DNA barcodes (Fig-
ure 1). The resulting short DNA duplex undergoes primer
extension by a DNA polymerase, encoding both the small
molecule and the protein it binds on a single oligonucleotide.
Only these extended oligonucleotides with primer sequences
from both libraries can undergo PCR amplification. Subse-
quent high-throughput DNA sequencing reveals the identi-
ties of all ligand:protein partner pairs. IDUP enables simul-
taneous evaluation of small molecule and protein libraries in
a single experiment in cell lysate and leverages the efficien-
cy of DNA-encoded libraries and high-throughput DNA
sequencing. We previously validated IDUP’s ability to enrich
DNA sequences encoding known binding pairs from an ex-
cess of mock barcodes not conjugated to small molecules or
1
targets by affinity enrichment using immobilized, purified
2
,3
protein targets. The effectiveness of these methods is lim-
ited by artefactual enrichment of library members that bind
the solid support or non-physiologically relevant forms of
target proteins, incomplete knowledge of the biological con-
text necessary for the target to adopt its relevant form, and
the inability to simultaneously explore interactions with mul-
tiple proteins of interest. Few methods of screening DNA-
encoded libraries, such as selections on cell-surface displayed
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proteins, parallel selections under varied conditions, or the
use of photocrosslinking probes to perform selections on
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unmodified proteins, have begun to address these limita-
tions.
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target proteins. In this study, we conducted a discovery-
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oriented IDUP experiment using libraries of DNA-barcoded (calculated cLogP of -9.8 to 7.4, mean = 1.8), and number of
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proteins and small molecules to identify novel binding pairs.
H-bond donors (1 to 40, mean = 4.7) (Figure S1).
The majority of our library of protein targets consisted
of human kinases, many of which are of biomedical interest.
The ability of IDUP to assess the selectivity of small mole-
cules could, in principle, distinguish promiscuous and selec-
tive kinase ligands. To assemble this protein library, we iden-
tified a set of 289 cytosolic and soluble human kinase ORFs
included in pDONR221 vectors for Gateway cloning (Har-
vard PlasmID Repository). The ORFs were subcloned into
an N-terminal SNAP-tag fusion protein plasmid by Gateway
cloning to enable DNA barcoding. The resulting pDEST-
SNAP-kinase vectors were transiently transfected into
HEK293T cells. The corresponding cell lysates were indi-
vidually treated with 31-nt benzylguanine-linked oligonucle-
otides (DNA-BG) that each contained a unique 6-nt barcode
and the common 3’ 8-nt hybridization region required for
IDUP. DNA-BG barcodes were validated computationally
and in a mock IDUP experiment to remove sequences that
were subject to positive or negative PCR bias. Unlabeled
SNAP protein was quenched using SNAP-Cell Block (New
England Biolabs) and the lysates were pooled to obtain 236
SNAP-tagged, DNA-barcoded target proteins. In parallel, an
aliquot of pooled lysates was quenched with SNAP-Cell
Block, then combined with pooled DNA-BG, to generate a
non-DNA-tagged negative control sample.
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Figure 2. Results of seven replicate IDUP experiments of com-
bined protein and small molecule libraries. Eight DNA barcodes
corresponding to bona fide interactions (Bak:Bcl-xL,
PKI:PRKX, JQ1:BRD2, JQ1:BRD3, bisX:GSK3a, EA:
We constructed a library of DNA-linked compounds
with annotated bioactivity, hypothesizing that those com-
pounds may have more favorable solubility, stability, or pro-
tein-binding properties. We identified a candidate set of 500
carboxylic acid-containing compounds and 250 aliphatic
primary amines within the databases of the Broad Institute
and Harvard’s Department of Chemistry and Chemical Biol-
ogy. By inspection, we removed compounds containing func-
tional groups that would interfere with DNA conjugation
and compounds from overrepresented structural classes (e.g.,
quinolones, cephalosporins, or penicillins), arriving at a set
of 177 carboxylate- and 87 amine-containing compounds.
MAP2K6) enriched (red) out of 32,096 possibilities. K and
D
IC50 values are for compounds and proteins not linked to DNA.
Nonspecific amplification across some protein barcodes may
arise from poor expression of those targets (see Fig. S2).
We combined 2 pmol of the DNA-linked small-molecule
library with the DNA-tagged protein library and performed
IDUP primer extension (see Supplemental Information p.
S30). Extended products, encoding protein:ligand pairs,
were selectively amplified by PCR and analyzed by high-
throughput DNA sequencing. The abundance of each bar-
code out of the 32,096 possible ligand:protein combinations
was compared to its frequency in the control IDUP experi-
ment to define an enrichment value for each possible combi-
nation (Figure 2). Across seven technical replicates, the most
significantly enriched sequence corresponded to Bcl-xL:Bak
binding (205-fold average enrichment), the only interaction
tested that we previously validated in an IDUP experiment.6
In addition, we observed high enrichment (89.5-fold) of the
barcodes corresponding to PKI peptide (a cAMP-dependent
Each small molecule’s 43-nt DNA barcode included an
internal 7-nt barcode and a constant 3’ 8-nt hybridization
region complementary to that of the protein library. Carbox-
ylic acids were coupled to a 3’-amine-linked DNA oligonu-
cleotide using DMT-MM or EDC and purified by HPLC,
resulting in 97 DNA-linked compounds. Amine-containing
small molecules were coupled to 3’-thiol-functionalized
DNA using a heterobifunctional crosslinker containing both
a maleimide and an NHS ester (Thermo Scientific Pierce),
yielding an additional 39 DNA-linked compounds. We in-
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kinase inhibitor ) and PRKX (a cAMP-dependent kinase ).
Two different barcodes corresponding to variants of the BET
inhibitor JQ1 enriched for binding to BET family proteins
cluded the Bak peptide as a positive control, as we previously
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BRD2 (6.8- or 6.9-fold, K = 128 nM ) and BRD3 (15.2- or
9.4-fold, K = 60 nM ). Although DNA-encoded library
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detected its binding to Bcl-xL protein (K
D
= 480 nM) in the
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D
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IDUP format. The final library contained an equimolar mix-
ture of each of the 136 DNA-linked compounds. The mole-
cules span a range of chemical properties, including molecu-
lar weight (123 to 2,222 Da, mean = 357 Da), lipophilicity
selections can suffer from interference between the DNA and
binding of a library member to a protein, this possibility did
not preclude enrichment of these ligand:protein partners. We
did not observe a strong correlation between DNA-free bind-
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that covalent modification of MAP2K6 by EA at Cys38 is
ing affinity and IDUP enrichment, potentially due to factors
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such as the DNA tag affecting IDUP enrichment positively
or negatively.
partially, but not solely, responsible for kinase inhibition.
Next, we evaluated if protein:small-molecule combina-
tions encoded by other enriched amplicons corresponded to
bona fide protein:ligand pairs. We tested 11 interactions en-
coded by enriched barcodes in either kinase activity or bind-
ing assays using the corresponding non-DNA tagged ligands
(
Table S3). Using Z’-LYTE assays (Invitrogen) we measured
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the inhibition of PRKX by PKI (IC50 = 52 nM) and GSK3a
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by bisindolylmaleimide X (bisX) (4.7-fold IDUP enrich-
ment, IC50 = 115 nM). Finally, we discovered that ethacrynic
acid (EA) inhibits MAP2K6 (4.7-fold IDUP enrichment, Z’-
LYTE IC50 = 4.5 µM).
Figure 4. Kinases were incubated with 100 µM EA (+EA) or
DMSO (-EA), then dialyzed to remove free EA. Activity was
assayed as a function of kinase concentration. Ethacrynic acid
inhibits MAP2K6 to a greater degree when Cys38 is present
(
left) than when this residue is mutated to an alanine (right).
A member of the MAP2K family, MAP2K6 activates
p38 MAP kinase in response to environmental stresses.15
Previous cheminformatic and proteome-wide studies impli-
cated Cys128 (in the Gatekeeper region) or Cys196 (adja-
cent to the DFG motif) as more accessible or reactive to-
wards small-molecule electrophiles.16 In contrast, Cys38 is
located within a non-active site region with poorly under-
Figure 3. Inhibition of MAP2K6 by EA is dependent on the
Michael acceptor. The non-electrophilic analogs shown are >30-
fold less potent.
17
stood function and is not conserved among other MAP2Ks
(
Figure S5). We confirmed that EA has higher affinity for
EA is an FDA-approved loop diuretic that inhibits the
MAP2K6 than other MAP2Ks (Figure 5). These trends are
consistent with the results of the IDUP library ´ library ex-
periment, suggesting that IDUP can illuminate a compound’s
selectivity even within a protein family.
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NKCC symporter and has not been previously reported to
inhibit any kinases. EA contains a Michael acceptor that re-
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acts with glutathione and EA derivatives have been previ-
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4
ously used as covalent bromodomain inhibitors. We inves-
tigated whether it inhibits MAP2K6 by forming a covalent
adduct with the protein. Non-electrophilic analogs of EA
(
dihydro-ethacrynic acid and tienilic acid) exhibited >30-
fold weaker inhibition of MAP2K6 (Figure 3). Incubating
MAP2K6 with EA yielded a +303 adduct in the intact pro-
tein mass spectrum, consistent with covalent modification by
EA (Figure S3B). Sequential treatment of MAP2K6 with EA
and then iodoacetamide (IAA), a cysteine alkylating agent,
resulted in modification by IAA at only five of MAP2K6’s six
cysteines (Figure S3D), suggesting that EA modifies
MAP2K6 at a cysteine residue. Weꢀ analyzed EA-treated
MAP2K6 by tryptic digest and MALDI-TOF and observed
only one peptide (residues 37-49) with a modification con-
sistent with EA adduct formation (Figure S4A). This peptide
contains a single cysteine residue, and fragmentation of this
peptide by tandem mass spectrometry confirmed that Cys38
was the site of covalent modification (Figure S4C).
Figure 5. EA has higher affinity for MAP2K6 than the related
MAP2K family members, as measured in both LanthaScreen Eu
assays and our initial IDUP assay. n.d. = not determined.
The one-pot experiment described here enriched bar-
codes encoding seven different binding interactions out of
32,096 possible combinations. Only one (Bak:Bcl-xL) had
been previously validated in the IDUP format, showing that
IDUP is a generalizable method for detecting binding inter-
actions in complex mixtures. The inhibition of MAP2K6 by
EA by targeting Cys38 was also unexpected. Such selective
probes could be used to investigate the role of MAP2K6 in
To better assess the mechanism of EA inhibition, we
incubated with EA a constitutively active MAP2K6 mutant
containing phospho-mimetic S207E and T211E mutations
(MAP2K6EE), dialyzed the protein into EA-free buffer, and
observed 9-fold apparent loss of kinase activity in the Z’-
LYTE assay. Preincubation of EA with a C38A point mutant
of MAP2K6EE resulted in a smaller loss in inhibition poten-
cy of ~3.3-fold (Figure 4). Together, these results suggest
1
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redox sensing, development, and cancer. EA’s inhibition of
MAP2K6, a cellular target unrelated to current uses of EA to
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treat edema or as a probe for GST function or Wnt signal-
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ing , suggests that further studies of EA and related com-
pounds as biological probes might be fruitful. The discovery
of this novel ligand interaction site in MAP2K6 through
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(
Corresponding Author
B. A.; Rocco, T. P., Goodman & Gilman's: The Pharmacological Basis of
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drliu@fas.harvard.edu
Present Addresses
†
Department of Cellular and Molecular Pharmacology, Univer-
6
057–6061.
sity of California, San Francisco, CA 94158, United States.
(15)Raman, M.; Chen, W.; Cobb, M. H. Oncogene 2007, 3100-12.
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H.; Gray, N. S. Chem. Biol. 2013, 146-59. (b) Weerapana, E.; Wang, C.;
Simon, G. M.; Richter, F.; Khare, S.; Dillon, M. B.; Bachovchin, D. A.;
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‡
Harvard Medical School, Boston, MA 02115, United States.
Funding Sources
This work was supported by NIH R01 EB022376 (formerly
R01 GM065400), NIH R35 GM118062, DARPA N66001-14-
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2
-4053, and the Howard Hughes Medical Institute.
Notes
The authors declare the following competing financial interest:
D.R.L. is a founder of Ensemble Therapeutics, a company that
uses DNA-templated synthesis for drug discovery.
ACKNOWLEDGMENT
JQ1-CO
2
H and JQ1-NH were gifts from Jay Bradner. We thank
2
Dr. Sunia Trauger for assistance with mass spectrometry.
REFERENCES
(
21)Zambaldo, C.; Daguer, J. P.; Saarbach, J.; Barluenga, S.; Winssinger,
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González-Páez, G. E.; Chatterjee, S.; Lanning, B. R.; Teijaro, J. R.; Olsen,
A. J.; Wolan, D. W.; Cravatt, B. F. Nature 2016, 570-574.
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2,096 interactions
evaluated simultaneously
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36 DNA-encoded
compounds
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proteins in cell lysate
Cl
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MAP2K6
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ligand:target binding pairs
decoded using high-
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discovery of a selective,
covalent inhibitor
!
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