SGC-AAK1-1: A Chemical Probe Targeting AAK1 and BMP2K

Article abstract of DOI:10.1021/acsmedchemlett.9b00399

Inhibitors based on a 3-acylaminoindazole scaffold were synthesized to yield potent dual AAK1/BMP2K inhibitors. Optimization furnished a small molecule chemical probe (SGC-AAK1-1, 25) that is potent and selective for AAK1/BMP2K over other NAK family members, demonstrates narrow activity in a kinome-wide screen, and is functionally active in cells. This inhibitor represents one of the best available small molecule tools to study the functions of AAK1 and BMP2K.

Full text of DOI:10.1021/acsmedchemlett.9b00399

Letter
pubs.acs.org/acsmedchemlett
Cite This: ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX
SGC-AAK1-1: A Chemical Probe Targeting AAK1 and BMP2K
Carrow Wells,†,‡ Rafael M. Counago,§,∥ Juanita C. Limas,# Tuanny L. Almeida,§,∥
̃
Jeanette Gowen Cook,⊥ David H. Drewry,†,‡ Jonathan M. Elkins,§,∇ Opher Gileadi,§,∇
Nirav R. Kapadia,†,‡ Alvaro Lorente-Macias,○ Julie E. Pickett,†,‡ Alexander Riemen,□
Roberta R. Ruela-de-Sousa,§,∥ Timothy M. Willson,†,‡ Cunyu Zhang,● William J. Zuercher,†,‡,◆
Reena Zutshi,□ and Alison D. Axtman*,†,‡
†Structural Genomics Consortium (SGC), UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill
(UNC−CH), Chapel Hill, North Carolina 27599, United States
‡Division of Chemical Biology and Medicinal Chemistry, UNC Eshelman School of Pharmacy, UNC−CH, Chapel Hill, North
Carolina 27599, United States
§
́
SGC, Departamento de Genetica e Evoluca
SP 13083-886, Brazil
̧ o, Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP), Campinas,
̃
∥
́
Centro de Química Medicinal, Centro de Biologia Molecular e Engenharia Genetica, UNICAMP, Campinas, SP 13083-875, Brazil
#Department of Pharmacology, UNC−CH, Chapel Hill, North Carolina 27599, United States
⊥Department of Biochemistry and Biophysics, UNC−CH, Chapel Hill, North Carolina 27599, United States
∇SGC, Nuffield Department of Clinical Medicine, University of Oxford, Old Road Campus Research Building, Oxford, OX3 7DQ,
U.K.
○
́
́
Departamento de Química Farmaceutica y Organica, University of Granada, Granada, 18071, Spain
◆Lineberger Comprehensive Cancer Center (LCCC), UNC−CH, Chapel Hill, North Carolina 27599, United States
□Luceome Biotechnologies, LLC, Tucson, Arizona 85719, United States
●Platform Technology Sciences, GlaxoSmithKline, Collegeville, Pennsylvania 19426, United States
S
* Supporting Information
ABSTRACT: Inhibitors based on a 3-acylaminoindazole
scaffold were synthesized to yield potent dual AAK1/
BMP2K inhibitors. Optimization furnished a small molecule
chemical probe (SGC-AAK1-1, 25) that is potent and
selective for AAK1/BMP2K over other NAK family members,
demonstrates narrow activity in a kinome-wide screen, and is
functionally active in cells. This inhibitor represents one of the
best available small molecule tools to study the functions of
AAK1 and BMP2K.
KEYWORDS: AAK1, BMP2K, acylaminoindazole, NAK family, endocytosis, protein kinase
stimulates binding to cargo proteins.3 AAK1 also modulates
uman protein Ser/Thr kinases Adaptor protein 2-
HAssociated Kinase 1 (AAK1) and BMP-2-Inducible
the Notch pathway, partially through its phosphorylation of
Numb.4,5 BMP2K plays a role in osteoblast differentiation, is a
Kinase (BMP2K/BIKE) play critical roles in mediating
endocytosis and other key signaling pathways. Both are
broadly expressed members of the NAK family of human
kinases, which also includes Cyclin G-Associated Kinase
(GAK) and Myristoylated and Palmitoylated Serine/Threo-
nine Kinase 1 (MPSK1/STK16). The family shares little
homology outside of their kinase domains (KDs).1 AAK1 and
BMP2K are the most closely related, with overall sequence
identity of 50% and KD sequence identity of 74%.2 A key
function of AAK1 is regulation of receptor-mediated
endocytosis via binding directly to clathrin and phosphorylat-
ing the medium subunit of AP2 (adaptor protein 2), which
clathrin-coated vesicle-associated protein, and, like AAK1, also
associates with Numb.6,7
Due to their many functions, AAK1 and BMP2K have been
implicated as potential drug targets for diverse conditions.
AAK1 has been linked to diseases affecting the brain such as
Special Issue: Women in Medicinal Chemistry
Received: August 29, 2019
Accepted: October 23, 2019
Published: October 23, 2019
© XXXX American Chemical Society
A
DOI: 10.1021/acsmedchemlett.9b00399
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ACS Medicinal Chemistry Letters
Letter
schizophrenia, Parkinson’s disease, and amyotrophic lateral
sclerosis as well as implicated as a potential antiviral target for
treatment of hepatitis C.8,9 BMP2K has been associated with
myopia and evaluated as a potential treatment for HIV.10,11
Figure S1 shows the current inhibitor landscape and how their
pharmacophores bind to AAK1/BMP2K. Patented AAK1
inhibitors were recently reviewed.12
X-ray crystal structures for the KDs of all NAK family
members have been solved and reported.2,13 Published and
novel high-resolution crystal structures of AAK1 and BMP2K
reveal target-specific structural features that enabled our design
of specific chemical probes and allowed further interrogation of
the roles that these kinases play.2 Screening a library of kinase
inhibitors (Published Kinase Inhibitor Set, PKIS) via differ-
ential scanning fluorimetry (DSF) revealed a chemical starting
point showing strong AAK1 binding.14 Structure−activity
relationships (SAR) were initially established by examining
the 3-acylaminoindazole analogs profiled within PKIS (Figures
1 and S2). Next, more than 200 analogs were prepared via the
size, electronics, and hydrogen-bonding capacity (Table 1). A
sulfonamide was found to be optimal for AAK1 inhibition but
only when appended at the 3-position of the aryl ring (1 versus
2). Sulfonamides with small saturated rings (<6 atoms) and
short alkyl chains or alkyl amines were the most potent (1, 11,
13, 14). This part of the binding pocket was not tolerant to
incorporation of large groups. 3,5-Disubstitution and 3,6-
disubstition were disfavored, supporting a small binding pocket
for this aryl ring that is sensitive to even minor changes, such as
incorporation of a fluorine in place of a hydrogen (data not
shown). Finally, activity of 1 and 8 suggests the presence of a
hydrogen-bonding network within the ATP-binding pocket
that can be accessed by 3-position groups capable of donating
a hydrogen. Interestingly, methylation of the aryl 3-nitrogen
(11) or insertion of a methylene spacer between the
sulfonamide and aryl ring (12) increased potency for AAK1.
These results motivated design of a range of sulfonamides to
further explore the SAR of this part of the molecule.
The portion of the ATP-binding pocket accessed by the 3-
position of the indazole was explored through substitution with
a variety of groups (Table 2). Compounds in Table 2 were
prepared using the route in Scheme S1, but the step order was
reversed. The indazole 3-position substituent appears to form a
polar interaction with the kinase hinge region and is sensitive
to structural modifications. The alkyl group was varied from
cyclopropyl to methyl, isopropyl, cyclobutyl, phenyl, and
Table 2. SAR of 3-Acylaminoindazole Carboxamide
Figure 1. Structures of PKIS hit (1) and key analog 14 with parts of
the molecule modified during SAR exploration boxed.
route shown in Scheme S1. A summary of key analogs
prepared to arrive at 14 is included in Table 1. Compounds
were profiled using the AAK1 split luciferase assay developed
by Luceome.15 Potent inhibition of BMP2K was not observed
in this assay format.
The effects of aryl substitution on the distal aryl ring were
explored, including 3- and 4-position substituents of variable
Table 1. SAR of 3-Acylaminoindazole Aryl Ring
Cmpd
R
AAK1 IC50 (nM)22
1
2
3
4
5
6
7
8
3-NHSO2Me
4-NHSO2Me
3-CONH2
4-CONH2
3-NHAc
3-CO2H
4-CO2H
3-OH
4-OH
220
1200
3800
910
1100
18000
2800
350
280
800
120
71
9
10
11
12
13
14
3-NH2
3-N(CH3)SO2Me
3-CH2NHSO2Me
3-NHSO2(isopropyl)
3-NHSO2(cyclopropyl)
54
31
B
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Table 3. NAK Family Binding-Displacement Assay Selectivity Analysis
Cmpd
R
AAK1 Ki (nM)23
BMP2K Ki (nM)23
GAK Ki (nM)23
STK16 Ki (nM)23
14
23
24
3-NHSO2(cyclopropyl)
3-NHSO2N(CH3)2
3-NHSO2CH2(cyclopropyl)
8.3
10
6.3
13
19
17
1600
2600
870
330
350
91
cyclopropylmethyl to explore the steric bulk tolerated by the
pocket. Alternatively, a urea or sulfonamide was incorporated
in place of the acylated amine (20, 21, 22) but resulted in loss
of AAK1 affinity. The cyclopropanecarboxamide was found to
be optimal (14). Minor changes, such as ring opening of the
cyclopropane (16) or expansion to a cyclobutane (17), were
found to result in loss of AAK1 affinity.
Based on the initial SAR, two additional 3-acylaminoinda-
zoles (23 and 24) were prepared (Table 3) incorporating the
aryl 3-cyclopropanecarboxamide that was found to yield potent
inhibitors of AAK1. The analogs were designed to probe
whether an alkyl amine or longer chain hydrocarbon could be
tolerated at the indazole 3-position. Like lead compound 14,
these analogs demonstrated promising inhibition of AAK1 in
the split luciferase assay (93%I and 94%I of AAK1 at 1 μM for
23 and 24, respectively). TR-FRET binding-displacement
assays were employed to further interrogate the biochemical
selectivity of these promising 3-aminoacylindazoles across the
NAK family (Table 3).21 Compounds 23 and 24 were found to
be dual AAK1/BMP2K inhibitors with more than 5-fold and
50-fold selectivity over STK16 and GAK, respectively.
To understand the selectivity of 23 and 24 across NAK
family kinases, cocrystal structures were solved with the
BMP2K KD (Figure 2, Table S1). Attempts to obtain
cocrystals of acylaminoindazoles bound to the AAK1 KD
were not successful. The BMP2K cocrystal structures were
solved to ∼2.4 Å resolution and showed that the core indazole
formed the critical interaction with the hinge region of the KD.
Given the high structural and sequence conservation
between the ATP-binding sites of the BMP2K and AAK1
KDs (Figures 2 and S3), it is likely that both kinases bind to
the indazole inhibitors using common interactions. Thus it is
unlikely, using this chemotype, that we can design inhibitors
that target only AAK1 or BMP2K. The pendant aryl ring is
accommodated by the hydrophobic region adjacent to the
gatekeeper residue (residue Met130/126, BMP2K/AAK1
numbering) within the kinase ATP-binding site. These
cocrystal structures suggest that large groups attached to the
4-position of the aryl ring (2 and 4) are likely not tolerated due
to steric hindrance. Smaller groups at the 3- and 4-positions
that can act as hydrogen-bond donors (8 and 9) can make
favorable interactions with the DFG motif aspartate residue
(Asp198/194), whereas small acidic groups at these positions
(6 and 7) are not favored due to charge repulsion. The
structures also reveal that a sulfonamide at the 3-position can
form hydrogen-bonds with multiple polar residues within or
adjacent to the protein ATP-binding pocket (Gln137/133,
Asn185/181, and Asp198/194). Groups attached to this
sulfonamide are accommodated by a small cavity within the
protein P-loop. When considering substituents attached to the
Figure 2. Cocrystal structures of 23 (A) and 24 (B) bound to the
BMP2K-KD. Main-chain atoms for residues (131−137) within the
BMP2K-KD hinge region, side-chain atoms for BMP2K-KD gate-
keeper residue (Met130), and those taking part in polar interactions
with the ligands are shown as sticks. Black dashed lines depict possible
hydrogen-bonds between protein and ligand atoms. Red spheres
denote the position of crystallographic water molecules. The 2Fo−Fc
electron density maps (contoured at 1.0σ) are shown below.
indazole ring, poor binding is observed for compounds bearing
sulfonamide groups in place of an amide (21 and 22). This
finding is due to steric clash and electronic effects caused by
the proximity of the sulfonamide oxygen atoms to nearby
protein side chains. Compound 20 is proposed to adopt a
planar conformation that introduces a steric clash not observed
with smaller groups and/or those that are not flat (14 and 17).
Cocrystal structures also reveal that groups attached to the 3-
position amide are solvent exposed, an observation that helps
rationalize the moderate tolerance of AAK1 for larger and
bulkier substituents at this position (15, 17, and 19).
Guided by these cocrystal structures, analogs were designed
focusing on modifications to the sulfonamide portion of the
molecule. Sulfonamides with pendant alkyl chains, alkyl
amines, fluorinated alkyl chains, small hydrocarbon-based
rings, and fluorinated aryl rings were synthesized. Table 4
includes some of our most promising analogs, the design of
which was informed by more than 200 compounds prepared
before them, some of which are included herein. Our years of
effort on this scaffold enabled us to produce many highly
potent compounds. Compounds built on Scaffold B were
prepared via Scheme S1. Those built on Scaffold A were
prepared using a slightly modified route (Scheme S2). Analogs
25−39 were evaluated for potency and selectivity across the
C
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Table 4. In Vitro and Cellular SAR of 3-Acylaminoindazole Probe Candidates
AAK1 (nM)
BMP2K(nM)
GAK (nM)
Ki23
NB IC50
STK16 (nM)
24
24
25
25
Cmpd
Scaffold
R
Ki23
NB IC50
Ki23
NB IC50
Ki23
NB IC50
S10(1 μM)26
14
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
A
A
A
A
A
A
A
A
A
B
B
B
B
B
B
B
cyclopropyl
8.3
9.1
8.2
6.2
12
59
22
24
8.5
32
44
39
30
33
22
20
641
770
375
490
655
1020
528
379
192
2710
3400
2350
1530
1470
1260
1790
13
17
20
17
30
120
51
53
17
27
42
33
59
74
37
36
1420
2800
1890
1490
2180
2950
1620
1190
707
1890
3030
1620
6550
3830
2740
2960
1600
1700
1700
1300
1100
7000
4600
4600
2900
510
780
580
3900
3200
3200
360
>10000
>10000
>10000
>10000
9250
330
270
310
78
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
0.005
0.02
0.017
0.002
0.01
0.007
0.022
0.017
0.007
0.022
N(CH2CH3)2
N(CH2CH3)(CH3)
CH2CH(CH3)2
CH2CH2CH3
4-F-benzyl
2-F-benzyl
3-F-benzyl
cyclobutyl
CH2CH2CF3
N(CH2CF3)(CH3)
CH2CF3
CH2CH(CH3)2
N(CH2CH3)2
CH2(cyclopropyl)
N(CH2CH3)(CH3)
110
430
470
510
240
180
290
180
450
220
150
86
9240
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
0.005
0.002
0.01
NAK family TR-FRET binding assays. Furthermore, cellular
target engagement was determined via NanoBRET (NB)
assays, where the respective kinases were fused to 19-kDa
luciferase (NLuc) and transiently expressed in HEK293 cells,
and then incubated with a cell-permeable fluorescent energy
transfer probe (tracer).16 Using increasing concentrations of
test compounds, dose-dependent displacement of the tracer
was observed, allowing calculation of the IC50 values in Table
4. These NB assays enabled measurement of potency and
selectivity of the compounds across the NAK family in living
cells (Figure S4).
All analogs (25−39) were found to be dual AAK1/BMP2K
inhibitors. Insertion of the methylene spacer between the aryl
ring and sulfonamide (Scaffold B) reduced potency for AAK1/
BMP2K. Within the Scaffold B group of molecules,
incorporation of an alkyl chain capped with fluorines (33,
34, and 35) increased the in vitro biochemical affinity for GAK,
but not sufficiently to demonstrate GAK activity in cells (Table
S3). In previous work, we had also observed that only low
nanomolar inhibitors of GAK possessed cellular activity.17
Nearly all of the analogs were also devoid of activity in the
STK16 NB assays. Several single-digit nanomolar biochemical
inhibitors of AAK1 were prepared based upon Scaffold A that
also demonstrated target engagement in cells with IC50 values
between 190−1020 nM. Incorporation of fluorinated aryl rings
was tolerated by AAK1 and BMP2K but not by GAK or
STK16 (29, 30, and 31).
the cell-based activity, 14, 26, 27, and 30−32 were the best
performers in both AAK1 and BMP2K NB assays, while 25
and 28 exhibited a slight drop in potency when transitioning
from the biochemical to cell-based assays. Confirmation of cell
penetrance and target engagement enables the facile translation
of these compounds to in vivo studies. Furthermore, LipE was
calculated for all compounds in Table 4 to estimate their
quality as drug candidates (Figure S5).
All compounds from Table 4 built upon Scaffold A and a few
representative compounds from Scaffold B (25−33, 35, 36,
and 38) were evaluated for selectivity by KINOMEscan
(DiscoverX) against 403 wildtype human protein kinases at 1
μM (Table 4).18 The compounds demonstrated remarkable
selectivity for AAK1/BMP2K across the 403 human kinases.
All proved to be narrow spectrum inhibitors of AAK1/BMP2K
with >90% inhibition of only 1−9 kinases (S10(1 μM) =
0.005−0.022, Figure S6).
To explore the phenotypic effect of AAK1/BMP2K
inhibition, lead compound 14 and inactive analog 16 were
tested in several cell lines. Surprisingly, we observed that 14
had cytostatic effects on cells after 24 h of continuous
treatment. Further study in U2OS human osteosarcoma cells
showed that 14 elicited cell cycle arrest in G2/M phase at 5−
10 μM, which was not captured at the 5 μM dose (Figure S7).
Although the molecular mechanism of the cell cycle arrest
proved to be elusive, we employed U2OS cells as a screen to
remove analogs that displayed this phenotype for consideration
as chemical probes. A total of 12 analogs were screened for
their effect on G2/M arrest (Figure 3). Nocodazole treatment
was used as a positive control for a strong arrest in mitosis.
Only analogs 27, 28, and 32 demonstrated the collateral G2 or
M phase arrest at 5 μM, while the remaining analogs were
devoid of this phenotype (Figures 3 and S8). Potent inhibition
of AAK1/BMP2K by arrestors and nonarrestors alike indicated
that G2 or M phase arrest was likely due to inhibition of an
unidentified off-target kinase.
3-Fluoroaryl analog (31) was one of the most potent
compounds in the NB assays for AAK1 and BMP2K, while the
4-fluoro analog (29) was 3-fold less potent on both kinases.
Compounds bearing small hydrocarbon chains and rings or
alkyl amines on the sulfonamide proved to be the best dual
AAK1/BMP2K inhibitors biochemically (14, 25, 26, 27, 28,
and 32) and demonstrated at least 35-fold selectivity for
BMP2K over GAK. The biochemical selectivity for BMP2K
over STK16 was more variable (4−16-fold). When considering
D
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Accession Codes
PDB codes: compound 23, 5I3O; compound 24, 5I3R.
AUTHOR INFORMATION
Corresponding Author
*Tel: 919-962-5349. E-mail: alison.axtman@unc.edu.
■
ORCID
Carrow Wells: 0000-0003-4799-6792
David H. Drewry: 0000-0001-5973-5798
Nirav R. Kapadia: 0000-0002-2826-1689
Alvaro Lorente-Macias: 0000-0001-9510-714X
Julie E. Pickett: 0000-0002-9535-8528
Timothy M. Willson: 0000-0003-4181-8223
William J. Zuercher: 0000-0002-9836-0068
Alison D. Axtman: 0000-0003-4779-9932
Figure 3. Cell cycle distributions. Cell cycle phases were determined
by analytical flow cytometry of DNA content and DNA synthesis in
U2OS cells after 24 h treatment with 5 μM of the indicated
compounds (n = 3).
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Funding
Compounds 25 and 26 emerged as probe candidates based
on their potency on AAK1 and BMP2K, kinome-wide
selectivity, lack of a G2/M arresting phenotype, and LipE
values. Following KINOMEscan of compounds 25 (SGC-
AAK-1) and 26, the binding affinities (KD) of all kinases
inhibited >80% at 1 μM plus AAK1 and a few kinases inhibited
by structurally related analogs were determined (Table S4).
Only three kinases were bound by 25 within 30-fold of the
potency of 25 for AAK1: RIOK1 (KD = 72), RIOK3 (KD =
290), and PIP5K1C (KD = 260).19 Compound 26 bound eight
kinases within 30-fold of the potency of AAK1 (Table S4).
Most compounds in Table 4 demonstrate potent affinity for
RIOK1 and RIOK3 in the corresponding DiscoverX binding
assays. Given that RIOK1 and RIOK3 are ATPases, an assay
that has ATP present would be much more relevant but has
not yet been developed.20 In accordance with our previously
described probe criteria,21 SGC-AAK1-1 (25) is a high quality
dual AAK1/BMP2K chemical probe. Compound 16 (SGC-
AAK1-1N) is the complementary negative control analog. The
probe set has been made commercially available, making SGC-
AAK1-1 the only AAK1-targeting chemical probe that is widely
available alongside its negative control.
We have described the design, synthesis, and biological
evaluation of a series of acylaminoindazoles as selective dual
inhibitors of AAK1/BMP2K. Compound 25 emerged as our
best probe candidate. X-ray cocrystal structures of selected
acylaminoindazoles bound to BMP2K revealed key inter-
actions within the ATP binding pocket that explain the high
affinity of this series for AAK1 and BMP2K. The effect of 25
on WNT signaling was recently reported.19 Further inves-
tigations into effects of selective pharmacological inhibition of
AAK1 are ongoing.
SGC is a registered charity (number 1097737) that receives
funds from AbbVie, Bayer Pharma AG, Boehringer Ingelheim,
Canada Foundation for Innovation, Eshelman Institute for
Innovation, Genome Canada, Innovative Medicines Initiative
(EU/EFPIA) [ULTRA-DD 115766], Janssen, Merck KGaA
Darmstadt Germany, MSD, Novartis Pharma AG, Ontario
Ministry of Economic Development and Innovation, Pfizer,
Sao Paulo Research Foundation-FAPESP [2013/50724-5,
̃
2014/50897-0, 2016/17469-0], Takeda, and Wellcome
[106169/ZZ14/Z]. This work was also supported by the
Brazilian agency CNPq [465651/2014-3]. J.G.C. is supported
by grants from the NIH/NIGMS (GM083024, and
R25GM089569); J.C.L. is supported by an HHMI Gilliam
Fellowship for Advanced Study (GT10886) and a T32 award
(T32GM007040). The UNC Flow Cytometry Core Facility is
supported in part by P30 CA016086 to the UNC LCCC. NC
Biotech Center Institutional Support Grants 2017-IDG-1025
and 2018-IDG-1030 and NIH 1UM2AI30836-01 enabled this
work. A.L.M. acknowledges support from the Spanish MECD
(FPU 14/00818). D.H.D. and R.N. acknowledge
1R44TR001916 for support.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
R.M.C. thanks Diamond Light Source for access to beamline
I03 (proposal number MX14664) that contributed to the
results presented here. Constructs for NanoBRET measure-
ments of AAK1, BMP2K, GAK, and STK16 were kindly
provided by Promega. Dr. Ehrmann from the UNC Mass
Spectrometry Core Laboratory provided HRMS support.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsmedchem-
lett.9b00399.
ABBREVIATIONS
■
■
S
AAK1, AP2-associated protein kinase 1; BMP2K/BIKE,
BMP2-inducible kinase; STK16, serine/threonine kinase 16;
GAK, cyclin-G-associated kinase; NAK, numb-associated
kinase; AP2, adaptor protein 2; TR-FRET, time-resolved
fluorescence resonance energy transfer; PKIS, published kinase
inhibitor set; DSF, differential scanning fluorimetry; SAR,
structure−activity relationships; HEK, human embryonic
kidney; NB, NanoBRET; U2OS, U2 osteosarcoma; KD,
Protein crystallization conditions, NB and cell cycle
assay details, experimental methods, and synthesis and
characterization of compounds 25, 26, 27, and 28
(PDF)
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DOI: 10.1021/acsmedchemlett.9b00399
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ACS Medicinal Chemistry Letters
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Meisenheimer, P. L.; Fan, F.; Wood, K. V.; Robers, M. B.
Quantitative, Wide-Spectrum Kinase Profiling in Live Cells for
Assessing the Effect of Cellular ATP on Target Engagement. Cell
Chem. Biol. 2018, 25 (2), 206−214.
(17) Asquith, C. R. M.; Laitinen, T.; Bennett, J. M.; Godoi, P. H.;
East, M. P.; Tizzard, G. J.; Graves, L. M.; Johnson, G. L.; Dornsife, R.
E.; Wells, C. I.; Elkins, J. M.; Willson, T. M.; Zuercher, W. J.
Identification and Optimization of 4-Anilinoquinolines as Inhibitors
of Cyclin G Associated Kinase. ChemMedChem 2018, 13 (1), 48−66.
(18) Davis, M. I.; Hunt, J. P.; Herrgard, S.; Ciceri, P.; Wodicka, L.
M.; Pallares, G.; Hocker, M.; Treiber, D. K.; Zarrinkar, P. P.
Comprehensive analysis of kinase inhibitor selectivity. Nat. Biotechnol.
2011, 29 (11), 1046−51.
kinase domain; NLuc, nanoluciferase; tx, treatment; LipE,
lipophilic efficiency
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(26) Calculation of S10 discussed in Figure S6.
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F
DOI: 10.1021/acsmedchemlett.9b00399
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX