2-Aminothiazole Derivatives as Selective Allosteric Modulators of the Protein Kinase CK2. 1. Identification of an Allosteric Binding Site

Article abstract of DOI:10.1021/acs.jmedchem.8b01766

CK2 is a ubiquitous Ser/Thr protein kinase involved in the control of various signaling pathways and is known to be constitutively active. In the present study, we identified aryl 2-aminothiazoles as a novel class of CK2 inhibitors, which displayed a non-ATP-competitive mode of action and stabilized an inactive conformation of CK2 in solution. Enzyme kinetics studies, STD NMR, circular dichroism spectroscopy, and native mass spectrometry experiments demonstrated that the compounds bind in an allosteric pocket outside the ATP-binding site. Our data, combined with molecular docking studies, strongly suggested that this new binding site was located at the interface between the αC helix and the flexible glycine-rich loop. A first hit optimization led to compound 7, exhibiting an IC₅₀ of 3.4 μM against purified CK2α in combination with a favorable selectivity profile. Thus, we identified a novel class of CK2 inhibitors targeting an allosteric pocket, offering great potential for further optimization into anticancer drugs.

Full text of DOI:10.1021/acs.jmedchem.8b01766

Subscriber access provided by University of Winnipeg Library
Article
2-Aminothiazole derivatives as selective allosteric modulators of the
protein kinase CK2. Part 1: Identification of an allosteric binding site
Benoît BESTGEN, Isabelle Krimm, Irina Kufareva, Ahmed Kamal, WEI GUANG SEETOH, Chris Abell, Rolf W.
Hartmann, Ruben Abagyan, Claude COCHET, Marc Le Borgne, Matthias Engel, and Thierry LOMBERGET
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01766 • Publication Date (Web): 28 Jan 2019
Downloaded from http://pubs.acs.org on January 29, 2019
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 47
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
2-Aminothiazole derivatives as selective allosteric
modulators of the protein kinase CK2.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Part 1: Identification of an allosteric binding site
Benoît Bestgen†‡§∥⊥, Isabelle Krimm#, Irina Kufareva∇, Ahmed Ashraf Moustafa Kamal○, Wei-
◆
◆
§
∥⊥
Guang Seetoh , Chris Abell , Rolf W. Hartmann○, Ruben Abagyan∇, Claude Cochet , Marc
Le Borgne‡, Matthias Engel†, Thierry Lomberget*,‡
†Pharmaceutical and Medicinal Chemistry, Saarland University, Campus C2.3, 66123,
Saarbrücken, Germany
‡Universite de Lyon, Universite Lyon 1, Faculte de Pharmacie – ISPB, EA 4446 Bioactive
́
́
́
Molecules and Medicinal Chemistry, SFR Sante Lyon-Est CNRS UMS3453 - INSERM US7, F-
́
69373, Lyon cedex 08, France
§Institut National de la Santé et de la Recherche Médicale, U1036, 38000 Grenoble, France
∥Institute of Life Sciences Research and Technologies, Biology of Cancer and Infection,
Commissariat à l’Energie Atomique, 38000 Grenoble, France
⊥Unité Mixte de Recherche-S1036, University of Grenoble Alpes, 38000 Grenoble, France
#
Institut des Sciences Analytiques, UMR 5280, Université de Lyon, CNRS, Université Lyon 1,
ENS Lyon - 5, rue de la Doua, 69100 Villeurbanne, France
ACS Paragon Plus Environment
1

Journal of Medicinal Chemistry
Page 2 of 47
1
2
3
4
5
6
∇Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego,
La Jolla, CA 92093, USA
7
8
9
○Pharmaceutical and Medicinal Chemistry, Saarland University & Department of Drug Design
and Optimization, Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Campus
C2.3, 66123 Saarbrücken, Germany
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
◆Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, CB2 1EW,
United Kingdom
ABSTRACT
CK2 is a ubiquitous Ser/Thr protein kinase involved in the control of various signaling pathways
and is known to be constitutively active. In the present study, we identified aryl 2-aminothiazoles
as a novel class of CK2 inhibitors, which displayed a non ATP-competitive mode of action and
stabilized an inactive conformation of CK2 in solution. Enzyme kinetics studies, STD-NMR,
circular dichroism spectroscopy and native mass spectrometry experiments demonstrated that the
compounds bind in an allosteric pocket outside the ATP-binding site. Our data, combined with
molecular docking studies, strongly suggested that this new binding site was located at the
interface between the αC helix and the flexible glycine-rich loop. A first hit optimization led to
compound 7, exhibiting an IC50 of 3.4 µM against purified CK2α in combination with a favorable
selectivity profile. Thus, we identified a novel class of CK2 inhibitors targeting an allosteric
pocket, offering great potential for further optimization into anti-cancer drugs.
Keywords
Kinase inhibitors, protein kinase CK2, enzymatic studies, allosteric modulators, protein NMR
ACS Paragon Plus Environment
2

Page 3 of 47
Journal of Medicinal Chemistry
1
2
3
4
5
6
INTRODUCTION
7
8
9
A large fraction of the cellular phosphoproteome relies on the activity of protein kinase CK2,1 a
heterotetrameric serine/threonine kinase, formed of α catalytic and β regulatory subunits
(Supplementary Figure S1) and involved in the activation of several pro-oncogenic pathways that
are critical for cell proliferation, differentiation and survival.2,3 As CK2 is implicated in cancerous
transformation, its inhibition proved to be an effective method to induce tumor regression, and the
ATP-competitive compound CX-49454 (5-(3-chlorophenylamino)benzo[c][2,6]naphthyridine-8-
carboxylic acid, silmitasertib, IC50 = 14 nM) is currently in phase II clinical trials for the treatment
of cholangiocarcinoma.5 However, CX-4945 was recently reported to be more potent against
several additional kinases such as Clk (Cdc-like kinase) 2 (IC50 = 4 nM),6 as well as Dyrk1A and
Dyrk1B (IC50s = 6.8 and 6.4 nM, respectively),7 kinases involved in the regulation of alternative
splicing (Clk2 and Dyrk1A), NFAT signaling and neuronal functions (Dyrk1A), and myogenesis
(Dyrk1B). This underlines the fact that potent and selective CK2α inhibitors are difficult to obtain
through targeting the ATP binding pocket. Therefore, research efforts currently strive to identify
inhibitors that exploit other regions of the protein to suppress the catalytic activity of the kinase.8
Only few CK2α inhibitors with a non-ATP competitive mode of action were hitherto reported;
among them were azonaphthalene dye–derived compounds; however, the binding site and
potential mechanism of inhibition remained elusive.9 Besides, inorganic PolyOxoMetalate
complexes (POMs), e.g. [P2Mo18O62]6-, were described as potent allosteric inhibitors of CK2α,
exhibiting nanomolar IC50s and great selectivity in a panel of 29 kinases.10 However, POMs lack
cellular activity and there is little perspective to develop them further as in vivo active agents.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ACS Paragon Plus Environment
3

Journal of Medicinal Chemistry
Page 4 of 47
1
2
3
4
5
6
7
8
9
A small alternative binding pocket, termed αD pocket, was recently identified by Spring and co-
workers, located below the ATP binding pocket on the large lobe of the catalytic subunit.11 In their
follow-up study, an IC50 of 7 µM against CK2α was reached with the most optimized ligand
CAM4712, however, the selectivity over other kinases had dropped during hit optimization.12
In the present study, we describe the identification of a novel active class of non ATP-competitive
CK2 inhibitors. Using different methodologies, we provide evidence that the compounds target an
alternative binding pocket, distinct from the ATP binding site and from the αD pocket.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
RESULTS AND DISCUSSION
Hit identification and analysis of the inhibition mode. A Virtual Ligand Screening (VLS)
campaign13 targeting the α/β interface of CK2 (Supplementary Figure S1) was performed with
2·106 compounds (in-house library). The first hundred, highest-ranked molecules were evaluated
for their inhibitory activity against CK2α using a standard radioactive assay.10,14 Originally
designed as a potential protein–protein interaction inhibitor, compound 1 (Chart 1), possessing a
2-aminothiazole scaffold, was identified as the most active hit exhibiting an IC50 value of 27.7 µM
for CKα.
To define the biochemical mechanism of action of compound 1, we examined the effects of
increasing concentrations of ATP or peptide substrate, and the presence of CK2β on the inhibitory
activity of the compound. We found that CK2 inhibition by 1 was independent of the
concentrations of both ATP (Figure 1A) and peptide substrate (Figure 1B). More surprisingly, both
CK2α and the CK2α2β2 holoenzyme were inhibited to the same extent by 1 (Figure 1C), indicating
that the inhibition pattern was also independent of the presence of CK2This last result clearly
indicated that the binding site of hit compound 1 was not located in the CK2 α/β subunit interface.
ACS Paragon Plus Environment
4

Page 5 of 47
Journal of Medicinal Chemistry
1
2
3
4
5
6
We then searched for commercially available analogs of 1 and purchased the derivative 2 (Chart
1), having a para acid substitution; it showed an improved IC50 value of 7.0 µM for CK2α.
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Chart 1. Chemical structures of CX-4945 (silmitasertib) and hit compounds 1,2
COOH
HN
Cl
COOH
N
O2N
O2N
N
S
N
S
N
NH
NH
COOH
1
2
CX-4945
Lineweaver-Burk inhibition plots indicated that in the presence of a saturating peptide substrate
concentration (600 μM), compound 2 could bind to either the CK2–peptide substrate complex or
the CK2–ATP–peptide complex showing a mixed-type mechanism of inhibition with respect to
ATP (Figure 2A).15 This indicated that compound 2 was not an ATP site–directed inhibitor. Again,
the inhibitory potency of compound 2 was similar toward the monomeric CK2α or the CK2α2β
holoenzyme (Figure 2B), and was barely affected by increasing concentrations of CK2β (Figure
2C). Collectively, the absence of competition by ATP or the CK2β subunit strongly suggested that
2 bound outside the CK2α/CK2β interface and did not compete with ATP, at least not directly;
both a type II-like inhibition mode or binding outside the ATP-binding pocket were in agreement
with the data obtained this far.
ACS Paragon Plus Environment
5

Journal of Medicinal Chemistry
Page 6 of 47
1
2
3
4
5
6
7
8
9
A
B
C
100
75
50
25
0
100
75
50
25
0
5
4
3
2
1
0
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
0
50
100
150
200
0
50
100
150
0
50
100
[ATP] (μM)
[peptide substrate] (μM)
[compound 1] (μM)
Figure 1. Analysis of the inhibition mode of compound 1. A. Effects of increasing ATP
concentrations on the inhibition of CK2α activity by compound 1. CK2α (20 ng) was
incubated with or without 15 M of 1 after which its activity was assayed with 200 M
of CK2β–independent peptide substrate in the presence of increasing ATP
concentrations. B. Effects of increasing peptide substrate concentrations on the
inhibition of CK2 activity by 1. CK2 (20 ng) was incubated with (o open circles) or
without ( filled circles) 15 M of 1 after which its activity was assayed with 100 M
ATP in the presence of increasing concentrations of CK2β–independent peptide
substrate. C. Effects of the presence of CK2β on the enzymatic activity. CK2α (20 ng)
( filled circles) or CK2α2β2 (40 ng) (o open circles) were incubated in the presence of
increasing concentrations of compound 1 after which CK2 activity was assayed with a
CK2–independent peptide substrate
ACS Paragon Plus Environment
6

Page 7 of 47
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ACS Paragon Plus Environment
7

Journal of Medicinal Chemistry
Page 8 of 47
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 2. Analysis of the inhibition mode of compound 2. A. Lineweaver-Burk inhibition plot of
human recombinant CK2α by compound 2 at various ATP concentrations. The Ki was determined
by plotting the slopes at varying inhibitor concentrations. B. CK2α and CK2α2β2 activity in the
presence of increasing concentrations of compound 2. C. Effect of increasing CK2β concentrations
on the inhibition of a fixed amounts of CK2α (150 nM) by compound 2 (20 µM).
Hit compound 2 is highly selective. To further evaluate compound 2, a selectivity profiling
against 44 human kinases was performed (Table 1). At 50 µM of 2, which led to a full inhibition
of CK2α, only three other kinases from the panel showed a reduction of activity by more than 50
%: EGFR (74 % inhibition), EphA4 (55 %) and pim-1 (54 %). This remarkable selectivity of the
non-optimized hit compound argued for an interaction with a non-conserved binding site.
ACS Paragon Plus Environment
8

Page 9 of 47
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Table 1. Residual kinase activity in the presence of compound 2 at 50 µM.a
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Residual
activity
(%)
0
Residual
activity
(%)
83
Residual
activity
(%)
93
Residual
activity
(%)
Protein kinase
Protein kinase
Protein kinase
Protein kinase
CK2α
EGFR
HIPK2
MAPK1
LKB1
IR, activated
CDK6/cyclinD3
cSRC
CaMK1
CDK2/cyclinE
Met
101
26
84
95
101
EphA4
45
85
97
102
pim-1
46
PKBα
85
FGFR1
97
PAK2
102
MUSK
PDGFRα
AMPKα1
Aurora-B
Dyrk2
62
CDK2/cyclinA
PKCα
86
Fyn
97
IKKα
103
65
86
HIPK1
97
MEK1
103
71
cKit
87
HIPK3
98
Abl
104
74
PKA
87
CDK9/cyclinT1
Rsk1
99
CDK7/cyclinH
JAK2
104
78
CDK1/cyclinB
CHK2
89
99
104
Aurora-A
GSK3β
79
89
ROCK-1
Yes
100
100
Lyn
104
79
c-RAF
92
mTOR
104
a Results were obtained with ATP concentration adjusted to the respective Km of each kinase. Duplicate values did
not differ by more than 15% in all assays.
Scheme 1. Synthesis of aminothiazole derivatives 3-9.a
ACS Paragon Plus Environment
9

Journal of Medicinal Chemistry
Page 10 of 47
1
2
3
COOH
4
5
6
7
8
9
OEt
R2
R2
Cl
a,b
S
N
S
Cl
NH
c
NH2
N
H
NH2
5
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
O
c
Br
R1
R2
R1
N
d
4
NH
S
3,6-9
aReagents and conditions: (a) CS2, Et3N in THF/H2O 1/1, RT, 24h, then I2 in THF, 0°C to RT,
3h. (b) NH4OH 30%, RT, 6h. (c) EtOH, reflux, 3-12h. (d) Cat. H2SO4, MeOH, reflux, 2h (R2 =
COOH and R1 = m-nitrophenyl).
Preliminary hit optimization. Derivatives of compound 2 were synthesized (Scheme 1) to
establish preliminary structure–activity–relationships (SAR) (Table 2). The presence of a
carboxylic acid function turned out to be important, as it could not be removed (compound 3) or
replaced by the methyl ester (4) without significant loss of activity, suggesting that the binding
could involve ionic interactions with a basic residue of the protein. The presence of an aryl group
at position 4 of the aminothiazole core was found to be crucial, as indicated by the complete lack
of activity of the truncated analog 5. While modulation of the electron density of this aromatic ring
(compounds 2 and 8) did not substantially change the inhibitory activity compared with that of the
unsubstituted congener 6, increasing the lipophilicity by a bromo-substituent significantly
enhanced the affinity to reach the sub-micromolar range (Ki = 0.7 µM, compound 7, Table 2).
ACS Paragon Plus Environment
10

Page 11 of 47
Journal of Medicinal Chemistry
1
2
3
4
Thus, the preliminary SAR obtained with the 4-phenyl substitutions suggested that binding of this
5
6
moiety to CK2α was driven by hydrophobic interaction with a lipophilic pocket.
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 2. Chemical structures and CK2 inhibitory activities of compounds 1-9.
Compound
CK2α inhibitory activitya
COOH
3
N
4
O2N
IC50 = 27.7 µM
2
NH
5
S1
1
COOH
IC50 = 7.0 µM
Kib = 1.6 µM
O2N
N
S
NH
2
O2N
N
27 (±10) % inh. at 10 µM
9 (±7) % inh. at 10 µM
NH
S
3
ACS Paragon Plus Environment
11

Journal of Medicinal Chemistry
Page 12 of 47
1
2
3
4
CO2Me
5
6
7
8
9
O2N
N
S
NH
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
4
COOH
0 (±6) % inh. at 10 µM
N
S
NH
5
COOH
IC50 = 9.0 µM
Kib = 1.1 µM
N
NH
S
6
COOH
IC50 = 3.4 µM
Kib = 0.7 µM
Br
N
NH
S
7
COOH
IC50 = 12.0 µM
MeO
N
S
NH
ACS Paragon Plus Environment
12

Page 13 of 47
Journal of Medicinal Chemistry
1
2
3
4
8
5
6
7
8
COOH
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
IC50 = 14.0 µM
N
S
N
NH
9
a IC50 values are the mean of ≥2 experiments (SD < 20 %). b Ki values were determined from graphical plotting of the
Lineweaver-Burk plot slopes.
Selectivity and non-ATP competitive behavior of the optimized inhibitor 7. The selectivity of
the most potent compound 7, which retained a mixed-type mechanism of inhibition toward ATP
(Figure 3), was evaluated against a panel of 32 selected kinases, specifically chosen from closely
related kinases of the CMGC family but also including kinases from other branches of the kinome
(Table 3). Compound 7 was screened at a concentration of 50 µM, which is approx. 15 times the
IC50 value for CK2α. Under these conditions, six other kinases were inhibited by more than 60%:
EphA4 (73%), GSK3β (71%), CK1γ1 (69%), ACVR1 (66%), Clk4 (61%) and MLCK (61%).
Notably, Clk2 and Dyrk1A, the main off-targets of the ATP-competitive compound CX-4945,
were only weakly inhibited (by approximately 30%).
ACS Paragon Plus Environment
13

Journal of Medicinal Chemistry
Page 14 of 47
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 3. Lineweaver-Burk inhibition plot of human recombinant CK2α by compound 7 at various
concentrations (µM). Ki was determined by plotting the slopes at varying inhibitor concentrations.
ACS Paragon Plus Environment
14

Page 15 of 47
Journal of Medicinal Chemistry
1
2
3
4
Table 3. Residual kinase activity in the presence of compound 7 at 50 µM.a
5
6
7
8
Residual
activity
(%)
Residual
activity
(%)
Residual
activity
(%)
Protein kinase
Protein kinase
Protein kinase
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
EphA4
GSK3β
CK1γ1
27
Clk2
Abl
67
PKCα
MEK1
92
29
69
95
31
Chk2
72
JAK2
98
MKNK1
(MNK1)
PDGFRα
ACVR1 (ALK2)
34
39
39
TBK1
CDK1/cyclinB
Dyrk1A
75
78
79
101
102
104
Clk4
MLCK
DAPK3
EGFR
(MLCK2)
STK17A
(DRAK1)
CK2α´
48
Clk1
80
106
48
55
57
57
HIPK3
Clk3
82
86
86
90
CDK2/cyclinE
ROCK1
107
107
116
GSG2 (Haspin)
pim1
RIPK2
MuSK
cKit
HIPK2
aResults were obtained with ATP concentration adjusted to the respective Km of each kinase. Duplicate values did
not differ by more than 17% in all assays.
The novel inhibitors decrease the thermal stability of CK2α. To further investigate the non-
ATP competitive binding mechanism, the thermal stability of CK2 in the presence of our
compounds was compared to its thermal stability in the presence of CX-4945 using circular
dichroism (CD) spectroscopy, which allows monitoring the transition to the unfolded state. For
these experiments, compounds 6 and 9 were chosen owing to their superior water solubility. An
enhancement of the thermal stability was consistently reported for ATP-competitive ligands.16,17
In the presence of compounds 6 or 9, however, we observed a clear reduction of the melting
ACS Paragon Plus Environment
15

Journal of Medicinal Chemistry
Page 16 of 47
1
2
3
4
5
6
7
8
9
temperature Tm (Table 4, entries 2 and 3), compared with the sole enzyme (Table 4, entry 1). In
contrast, the ATP-competitive reference compound CX-4945 stabilized the protein structure,
resulting in an increased Tm (Table 4, entry 4). These results added to the evidence that our 2-
aminothiazole derivatives did not target the ATP-binding pocket.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ACS Paragon Plus Environment
16

Page 17 of 47
Journal of Medicinal Chemistry
1
2
3
4
Table 4. Unfolding temperatures of CK2α in the presence of compound 6, 9 or CX-4945, as
5
6
monitored by thermal denaturation of protein by circular dichroism (TdCD).
7
8
9
Entry
Sample
CK2α
Tm (°C)
1
2
3
4
53.2 (± 0.3)
50.3 (± 0.3)
52.0 (± 0.2)
55.4 (± 0.2)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
CK2α + 6 (50 µM)
CK2α + 9 (6.25 µMa)
CK2α + CX-4945 (6.25 µMa)
The midpoint unfolding temperatures (Tm) are averages of three independent experiments (± SD). CK2α
concentration was 5 µM. aConcentrations were limited by the compound solubility in methanol.
Experimental evidence that the novel compounds bind outside the ATP binding pocket. To
elucidate the binding region of this series, NMR Saturation Transfer Difference18 (STD)
competition experiments were performed between our 2-aminothiazole ligands and the ATP-
competitive ligands CX-4945 or AMP-PNP using CK2α protein. NMR-STD experiments are
typically used to characterize the interaction of compounds with macromolecular targets. While
the NMR signals of all the compounds are observed in the 1D spectrum, the STD spectrum display
signals only for the compound(s) that bind(s) the protein target. Again, compounds 6 and 9 were
chosen because of their relatively good solubility in water. Figure 4 shows the STD spectrum of
compound 6 bound to CK2α. Upon addition of CX-4945, simultaneous binding of both compounds
6 and CX-4945 was observed (Figure 4A). The absence of competition between these compounds
indicated that compound 6 did not bind to the same site as CX-4945. By comparison, the
competition between CX-4945 and AMP-PNP was observed in STD experiments that were
recorded under similar experimental conditions (Supplementary Figure S2). AMP-PNP occupies
a larger space than CX-4945, due its more extended molecular structure. While the binding of
AMP-PNP to CK2α was observed with the apo enzyme (Figure 4B), AMP-PNP was not able to
ACS Paragon Plus Environment
17

Journal of Medicinal Chemistry
Page 18 of 47
1
2
3
4
5
6
7
8
9
bind to CK2α in the presence of 2-aminothiazole 6 (Figure 4B). Identical results were observed
for compound 9, as illustrated by Figure 5A. Altogether, our results proved that compounds 6 and
9 did not bind in the region of the ATP binding pocket of CK2 where CX-4945 was shown to
interact with in cocrystal structures (PDB entries 3PE119 and 3NGA20). The fact that an apparent
competition was observed between the binding of 6 or 9 and AMP-PNP can be interpreted in two
ways: either a direct steric clash occurs between the compound and AMP-PNP, or the compound
triggers an allosteric change which leads to a partial occlusion of the ATP binding site, e. g. where
the terminal phosphate binds, while sparing all areas engaged in CX-4945 binding (these
possibilities are investigated further below, cf. section “Binding mode prediction by molecular
docking”).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
STD experiments can also discriminate parts of the ligand that are buried in the protein structure
upon binding from parts of the compound exposed to the solvent, through the so-called STD–based
epitope mapping. STD factors were calculated for the protons of compound 6 (Figure 4C). These
data indicate that the benzoic acid moiety of 6 was exposed to the solvent, while the proton of the
thiazole ring and the para protons of the phenyl ring were buried within the protein (Figure 4D).
ACS Paragon Plus Environment
18

Page 19 of 47
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
6
1D
A
8
9
STD
1D
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
6 + CX-4945 (x)
x
x
x
x x x
STD
AMPPNP
B
1D
STD
AMPPNP (*) + 6
1D
*
*
*
*
STD
9
C
10
8
buried
6
D
Buried
1, 2, 3, 4
5
67%
7
S
N
8, 9
exposed
64%
6, 7
exposed
45%
buried
100 %
5
Exposed
4
2
buried
100%
10
HN
1
OH
3
O
Figure 4. STD–based evidence for the simultaneous binding of compound 6 and CX-4945 to
CK2α, epitope mapping for compound 6. A. 1D and STD NMR experiments of compound 6 (500
µM) recorded alone and in the presence of the kinase inhibitor CX-4945 (100 µM) with 5 µM
CK2α; NMR peaks for CX-4945 are labeled by crosses (x). B. 1D and STD NMR experiments of
the ATP-binding site inhibitor AMP-PNP (500 µM) recorded alone and in the presence of
ACS Paragon Plus Environment
19

Journal of Medicinal Chemistry
Page 20 of 47
1
2
3
4
5
6
7
8
9
compound 6 (500 µM) with 5 µM CK2α. The 1D and STD NMR spectra are shown in black and
red, respectively. C. STD factors measured for compound 6 upon binding to CK2α. D.
Representation of the STD factors with a color scale on the chemical structure of 6.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
AMP-PNP (◊) + 9
1D
◊
◊
◊
◊
STD
Figure 5. STD experiments for compound 9 bound to CK2α. 1D and STD NMR experiments of the
ATP-binding site inhibitor AMP-PNP (500 µM) recorded alone and in the presence of compound 9 (500
µM) with 5 µM CK2α.
Mapping of the binding site by alanine scan mutational analysis. Our cocrystallization attempts
aiming at elucidating the non-ATP competitive binding mode of 2-aminothiazole derivatives with
CK2α were not successful. Some crystallization issues were already noticed for non-ATP
competitive CK2 inhibitors.9 Therefore, we employed alternative techniques to complement the
results from the NMR competition experiments.
In the literature, crystallographic studies21,22,23 and long timescale molecular dynamics
simulations24 suggested that the glycine-rich loop is a highly flexible part of CK2. Indeed, in
most CK2α crystal structures, the glycine-rich loop conformation is maintained by two hydrogen
bonds linking the Tyr50 of this loop and two lysine residues (Lys74, Lys77) of the basic cluster.
Nonetheless, two crystal structures (PDB entries: 3JUH25 and 3FWQ26) were identified, in which
the key hydrogen bonds fixing Tyr50 are lacking. Further examination of these structures revealed
ACS Paragon Plus Environment
20

Page 21 of 47
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
a hydrophobic pocket between the glycine-rich loop and the αC-helix, delimited by residues Tyr50,
Lys71, Val73, Lys74 and Lys77 (Figure 6). We hypothesized that this hydrophobic pocket, in
combination with the adjacent lysine residues, could be an allosteric binding site for our
aminothiazole inhibitors. Our findings clearly proved that the pocket targeted by our compounds
was distinct from the so-called αD pocket, which was located near the αD-helix and delimited by
Val162, Asn119, Pro159, Met221, Met137, Ile133, Ile128, Ile124 residues.11
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Interestingly, the basic lysine cluster was addressed by small molecules before, being the target of
the acidic part (aspartic or glutamic tri/tetrapeptides) of TBI-derived bisubstrate inhibitors, which
bound to both the ATP site and this basic environment.27,28
Figure 6. A. Superposition of four CK2α structures (3PE1, magenta; 3FWQ, blue; 3JUH, cyan;
3Q9Z29; green). The ATP-binding site is occupied by CX-4945 in 3PE1 and by AMP-PNP in 3JUH
ACS Paragon Plus Environment
21

Journal of Medicinal Chemistry
Page 22 of 47
1
2
3
4
5
6
7
8
9
and 3Q9Z (not shown for clarity). B. A separated view from front or top of each structure is
depicted. The pockets were identified using ICM pocket finder and represented with a mesh
surface. The putative allosteric pocket (colored in yellow) is partially available in 3PE1, fully
accessible in 3FWQ, 3JUH and 3Q9Z.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
To investigate more deeply this protein region, nine GST-CK2α single-point alanine mutants were
tested in enzymatic assays in the presence of compound 7 (Figure 7 and Supplementary Table S1).
Three mutants significantly less sensitive to inhibition by 7 (Lys74Ala, Lys77Ala and His160Ala)
were further tested in dose–response experiments. Compared with wild type CK2α, the IC50 values
of compound 7 were increased for the Lys74Ala (1.8-fold) and Lys77Ala (3.2-fold) mutants, even
considering that the remaining lysine residue 76 (located nearby) might compensate in part for the
loss of electrostatic interactions, thus attenuating the drop in binding affinity. Rather unexpectedly,
the His160Ala mutant also showed an increased IC50 (2.2 fold) (Figure 7).
ACS Paragon Plus Environment
22

Page 23 of 47
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Figure 7. Percentage of inhibition of GST-CK2α wild-type and single alanine mutants in the
presence of compound 7 (4 µM). IC50s were determined for GST-CK2α wild-type and mutants
with reduced sensitivity toward inhibition (S.D. < 20%). The fold increase in the respective IC50
over that of the wild-type is indicated at the bottom. nd: not determined.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Although His160 is not in close proximity to the putative allosteric pocket, it displayed a direct
interaction with the P-loop tip residueTyr50 in both the crystallized inactive states of CK2α, 3FWQ
and 4UB7.23 This might indicate that at least in some inactive conformational states, His160 helps
to stabilize the position of Tyr50 and hence of the entire glycine-rich loop, thus opening the gate
to the allosteric pocket. In contrast, in the active conformation of CK2α, the position of Tyr50 is
rather stabilized by H-bonding or cation–π interactions with lysines from the basic cluster, as can
be observed e. g. in the crystal structures of PBD entries 3PE1, 3OWJ30 and 3Q9Z. In light of these
considerations, the mutation of His160 might diminish the population of inactive conformational
states in the equilibrium that can be recognized by our allosteric ligand.
Confirmation of the alternative binding site by competition experiments followed by native
mass spectrometry-based detection. To complete the binding site mapping based on the single
alanine mutants scan, we were looking for probe compounds known to bind in the region of the
assumed allosteric pocket. To this end, we selected inositol hexakisphosphate (also named IP6),
which was reported as a CK2α ligand binding to the named basic cluster, as demonstrated by X-
ray studies (Figure 8A).31 Native nano-electrospray ionization-mass spectrometry (nanoESI-MS)
was employed as a fast, label-free, and highly accurate biophysical technique to characterize the
binding properties of 6 to CK2α. In the gas phase of the mass spectrometer, proteins and protein-
ligand complexes have been known to be preserved in near-native states, thus closely reflecting
ACS Paragon Plus Environment
23

Journal of Medicinal Chemistry
Page 24 of 47
1
2
3
4
5
6
7
8
9
solution-phase conditions.32,33 In native MS, the ions of proteins and their complexes are resolved
based on their mass-to-charge ratio (m/z), which allows precise molecular mass determination.
Therefore, parameters such as ligand binding, binding stoichiometry, and binding site information
could be obtained from native MS experiments.34,35 Native mass spectra of CK2α1–335 (deletion
mutant construct used in native MS experiments; henceforth referred to as CK2α) were acquired
using 10 μM protein in 0.50 M ammonium acetate pH 8.5 and 5% (v/v) DMSO under non-
denaturing conditions by nano-electrospray ionization-mass spectrometry (nESI-MS) that preserve
non-covalent interactions. CK2α itself produced a well-resolved charge state series, with ions
possessing low charges, signifying the presence of native-like folded structures (Figure 8B). CK2α
produced three charge states spanning 9+ to 11+ in the m/z range of 3,600-4,500, with the main
charge state being 10+. The observed mass (39,879 ± 3 Da) and calculated mass (39,871 Da) from
the protein sequence of the CK2α construct are in good agreement, and establish the oligomeric
state of CK2α as a monomer, consistent with published structural data (Figure 8B).31 The binding
of IP6 to CK2α was evaluated by incubating 10 μM CK2α with 100 μM IP6. Native mass
spectrometry showed that CK2α binds IP6 with 1:1 and 1:2 stoichiometry, with 1:1 binding being
the predominant stoichiometry, and the 1:2 stoichiometry probably arising from non-specific
binding (Figure 8C).36 CK2α binds to 6 with various stoichiometries, possibly reflecting the
presence of multiple ligand-binding sites, or non-specific binding (Supplementary Figure S3).28b,36
In the presence of CX-4945, the observation of the CK2α-CX-4945-6 ternary complex peak
indicated that 6 binds to CK2α at a site apart from the ATP site (Supplementary Figure S3), thus
corroborating the results obtained with NMR STD experiments. To verify our hypothesis that a
part of 6 binds to the same basic pocket as IP6, we performed binding competition experiments
between CK2α inhibitor 6 and IP6 using native mass spectrometry for detection. In the presence
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ACS Paragon Plus Environment
24

Page 25 of 47
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
of 6, we observed the dissociation of the CK2α-IP6 complex, while the CK2α-6 complex was
formed, as indicated by the disappearance and appearance of the complex peaks, respectively
(Figure 8C). In addition, no CK2α-IP6-6 ternary complex peak was found, altogether suggesting
that 6 is a competitive ligand of IP6 and binds to the same basic pocket region also including Lys74
and Lys77 (Figure 8A).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 8. MS–based evidence for competitive binding between IP6 and compound 6 in CK2α. A.
Crystal structure of CK2α in complex with IP6 (PDB file 3W8L31), with an inset showing
ACS Paragon Plus Environment
25

Journal of Medicinal Chemistry
Page 26 of 47
1
2
3
4
5
6
7
8
9
interactions with amino acid residues Lys74, Lys77, Asn189 and Arg191. Hydrogen bonding
interactions are indicated with blue dashed lines. B. Native mass spectra of monomeric CK2α (10
µM), showing three charge states, in the presence of 5% (v/v) DMSO. C. Competitive effect of 6
(10 µM) on the CK2α-IP6 interaction (10 µM CK2α, 100 µM IP6), with an inset showing a
magnification of the 10+ charge state. Addition of 6 caused the dissociation of the CK2α-IP6
complex to yield only CK2α-6 complexes. All charge states are colored and indicated with
symbols. The charge states of each species are indicated in the spectra.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Binding mode prediction by molecular docking. Guided by the above experimental data as a
whole, we performed molecular modeling studies to gain insight in a potential binding mode. On
the basis of the results from the STD NMR competition experiments, compound 6 was docked
simultaneously with either CX-4945 or AMP-PNP into the CK2α structures where the presumed
allosteric pocket was most accessible for a small ligand, in PDB entries 3JUH (chain A) and 3FWQ
(chain B) (Figure 9). With both PDB coordinates, similar binding poses were predicted for 6, which
bound in an allosteric pocket adjacent to the ATP binding site, located between the αC-helix and
the glycine–rich loop (cf. Figure 9B). In all cases, basic residues from the C-helix (Lys74, Lys77
and Arg80) and the activation segment (Arg191) were found to interact with the carboxyl–
substituted phenyl ring of 6 via a network of favorable hydrogen bonds and salt bridges or cation–
π interactions. In addition, a cation-π interaction between His160 and Arg47 of the glycine rich
loop was observed in the inactive structure 3FWQ (which was retained after docking), but not in
the active conformations of CK2α (e.g. in 3PE1), in which Arg47 was turned to the opposite
direction by almost 180°. Thus, it could be speculated that the interaction triad
Arg47/His160/Tyr50 may contribute to the stabilization of the allosteric pocket, facilitating
ACS Paragon Plus Environment
26

Page 27 of 47
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
binding of our ligands. Altogether, the depicted binding modes in Figure 9 were fully consistent
with the SAR, the pattern of exposed/buried protons as determined by STD-NMR experiments (cf.
Figure 4C), the alanine scan mutational analysis (Figure 7), and the competition experiments
between IP6 and 6 (Figure 8C). In further agreement with the STD-NMR experiments, the docking
simulations indicated that 6 and CX4945 can bind simultaneously to both CK2α structures without
mutual interference (Figures 9A, C). In PDB entry 3JUH, CK2α was cocrystallized with AMP-
PNP; using this cocrystal structure, 6 was docked in close proximity to the ATP analogue in some
poses, but not in all. Figure 9B depicts the binding pose with the closest distance between the two
ligands. As indicated by the van der Waals volumes (cyan and orange surfaces of AMP-PNP and
6, respectively, Figure 9B), no significant steric clash with the ATP analogue occurred. The closest
distance between the terminal phosphate of AMP-PNP and the benzene ring was 3.57 Å, which is
above the sum of the van der Waals radii, hence this binding model rather suggested that
simultaneous binding would be possible, conflicting with the rigorous displacement of the AMP-
PNP by 6 and 9 that was observed in the STD-NMR experiments (Figures 4B and 5). In addition,
it is hardly conceivable that binding of a small ligand to an active CK2α conformation such as in
3JUH would induce a full inhibition of the catalytic activity, given that the ligand does neither
compete with ATP binding nor with substrate binding (cf. Figure 1). Contrastingly, in the inactive
CK2α structure in 3FWQ, Arg47 and Tyr50 from the glycine–rich loop collapse in the space
normally accommodating the α- and γ-phosphate moieties of ATP26 (cf. Figure 9D). Hence, the
remaining space is sufficient for the binding of CX-4945 (Figure 9C) but not for AMP-PNP or
ATP (Figure 9D). Although is unlikely that one of the binding poses obtained with the 3FWQ or
3JUH coordinates reflects the exact binding mode of our inhibitors, the binding model involving
3FWQ provided a better explanation for the STD-NMR competition experiments of 6 and 9 with
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ACS Paragon Plus Environment
27

Journal of Medicinal Chemistry
Page 28 of 47
1
2
3
4
AMP-PNP (cf. Figures 4B and 5), as the stabilization of such a CK2α conformation upon binding
5
6
of an effector would strongly diminish the affinity of ATP for its binding pocket.
7
8
9
A
C
αC-helix
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
V73
V73
K77
K68
K77
K68
K74
R80
K74
R80
Y50
R47
R47
Y50
Glycine
rich loop
R191
CX-4945
R191
Glycine
rich loop
CX-4945
H160
V73
B
D
Glycine
rich loop
K74
V73
K77
K68
K77
R80
K74
R47
Y50
R191
Y50
Glycine
rich loop
R47
R191
AMP-PNP
AMP-PNP
H160
PBD: 3JUH
PBD: 3FWQ
Figure 9. Prediction of the binding mode of 6 in CK2α. To derive a potential binding mode
consistent with the STD-NMR experimental results (cf. Figures 4 and 5), molecular docking of 6
was performed in the presence of either CX-4945 or AMP-PNP using MOE. Two different CK2α
structures were employed, which showed an accessible alternative binding pocket near the region
identified through site directed mutagenesis (Figure 7) and binding competition experiments with
IP6 (Figure 8). PDB entry 3JUH (chain A) was used in A and B, and 3FWQ (chain B) in C and D.
Relevant residues and structural elements are labeled. In addition, transparent van der Waals
surfaces are shown in A and B for AMP-PNP (cyan, in the ATP binding pocket) and for 6 (orange,
in the allosteric pocket). In C, the partially occluded ATP binding pocket (with bound CX-4945)
and the allosteric pocket (with bound 6) are outlined by a gold surface. In D, the van der Waals
ACS Paragon Plus Environment
28

Page 29 of 47
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
surface of AMP-PNP is indicated by a mesh; the molecule was placed in ATP pocket region of
3FWQ following to a superimposition with the CK2α/AMP-PNP cocrystal of 3JUH. The steric
clashes with the glycine-rich loop residues Arg47 and Tyr50 are indicated by red circles. In A–D,
H-bonds are indicated by blue dashed lines, and CH–π interactions by brown dashed lines,
respectively. The grey tubular element denotes the smoothened backbone trace of the glycine–rich
loop and adjacent parts of beta sheet β3.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Conclusions
To date, few studies described alternative strategies for the pharmacological inhibition of CK2
through exploiting binding sites outside the ATP pocket8-11,27 and even fewer presented drug-like
small molecules.12 Originally designed as protein–protein interaction inhibitors, the aminothiazole
derivatives described in this paper exhibited an original allosteric mode of inhibition toward
protein kinase CK2. Using a comprehensive set of experiments, including enzyme kinetics studies,
STD NMR, circular dichroism studies, alanine scan analysis and competition experiments
monitored by native mass spectrometry, we could prove that the novel compounds target an
alternative binding pocket outside the ATP site; this novel binding site could be mapped to a region
between the glycine-rich loop and the αC-helix of CK2α, which was – to the best of our knowledge
– not reported previously to be targeted by small molecule inhibitors.
In light of the favorable selectivity profile – even prior to any optimization – and the still small
molecule size, we anticipate that the compounds presented herein can be developed further into
selective and drug-like leads.
ACS Paragon Plus Environment
29