Synthesis and Structure-Activity Relationships of 3,5-Disubstituted-pyrrolo[2,3- b]pyridines as Inhibitors of Adaptor-Associated Kinase 1 with Antiviral Activity

Article abstract of DOI:10.1021/acs.jmedchem.9b00136

There are currently no approved drugs for the treatment of emerging viral infections, such as dengue and Ebola. Adaptor-associated kinase 1 (AAK1) is a cellular serine-threonine protein kinase that functions as a key regulator of the clathrin-associated host adaptor proteins and regulates the intracellular trafficking of multiple unrelated RNA viruses. Moreover, AAK1 is overexpressed specifically in dengue virus-infected but not bystander cells. Because AAK1 is a promising antiviral drug target, we have embarked on an optimization campaign of a previously identified 7-azaindole analogue, yielding novel pyrrolo[2,3-b]pyridines with high AAK1 affinity. The optimized compounds demonstrate improved activity against dengue virus both in vitro and in human primary dendritic cells and the unrelated Ebola virus. These findings demonstrate that targeting cellular AAK1 may represent a promising broad-spectrum antiviral strategy.

Full text of DOI:10.1021/acs.jmedchem.9b00136

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Article
Synthesis and structure-activity relationships of 3,5-
disubstituted-pyrrolo[2,3-b]pyridines as inhibitors of
adaptor associated kinase 1 (AAK1) with antiviral activity
Sven Verdonck, Szu-Yuan Pu, Fiona J. Sorrell, Jonathan M Elkins, Matheus Froeyen,
Ling-Jie Gao, Laura I. Prugar, Danielle E. Dorosky, Jennifer M. Brannan, Rina Barouch-
Bentov, Stefan Knapp, John M. Dye, Piet Herdewijn, Shirit Einav, and Steven De Jonghe
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00136 • Publication Date (Web): 28 May 2019
Downloaded from http://pubs.acs.org on May 29, 2019
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Synthesis and structure-activity relationships of 3,5-disubstituted-pyrrolo[2,3-
b]pyridines as inhibitors of adaptor associated kinase 1 (AAK1) with antiviral activity
§
,†
#,†
ǁ
ǁ,φ
§
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Sven Verdonck, Szu-Yuan Pu, Fiona J. Sorrell, Jon M. Elkins, Mathy Froeyen, Ling-Jie
§
◊
◊
◊
#
Gao, Laura I. Prugar, Danielle E. Dorosky, Jennifer M. Brannan, Rina Barouch-Bentov,
Stefan Knapp, John M. Dye, Piet Herdewijn, Shirit Einav Steven De Jonghe§,*
ǁ,‡
◊
§
#,*
§
Medicinal Chemistry, Rega Institute for Medical Research, KU Leuven, Herestraat 49 – bus
041, 3000 Leuven, Belgium.
1
#
Department of Medicine, Division of Infectious Diseases and Geographic Medicine, and
Department of Microbiology and Immunology, Stanford University School of Medicine,
Stanford, California 94305, USA.
ǁ
Nuffield Department of Clinical Medicine, Target Discovery Institute (TDI) and Structural
Genomics Consortium (SGC), University of Oxford, Old Road Campus, Roosevelt Drive,
Oxford OX3 7DQ, United Kingdom.
φ
Structural Genomics Consortium, Universidade Estadual de Campinas, Cidade Universitária
Zeferino Vaz, Av. Dr. André Tosello, 550, Barão Geraldo, Campinas / SP 13083-886, Brazil.
‡
Institute for Pharmaceutical Chemistry, Buchmann Institute for Life Sciences Campus
Riedbeerg, Goethe-University Frankfurt, 60438 Frankfurt am Main, Germany.
◊
US Army Medical Research Institute of Infectious Diseases, Viral Immunology Branch, Fort
Detrick, Maryland 21702, USA.
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Abstract
There are currently no approved drugs for the treatment of emerging viral infections, such as
dengue and Ebola. Adaptor associated kinase 1 (AAK1) is a cellular serine/threonine protein
kinase that functions as a key regulator of the clathrin-associated host adaptor proteins and
regulates the intracellular trafficking of multiple unrelated RNA viruses. Moreover, AAK1 is
overexpressed specifically in dengue virus-infected but not bystander cells. Since AAK1 is a
promising antiviral drug target, we have embarked on an optimization campaign of a previously
identified 7-azaindole analogue, yielding novel pyrrolo[2,3-b]pyridines with high AAK1
affinity. The optimized compounds demonstrate improved activity against dengue virus both in
vitro and in human primary dendritic cells and the unrelated Ebola virus. These findings
demonstrate that targeting cellular AAK1 may represent a promising broad-spectrum antiviral
strategy.
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Introduction
Dengue virus (DENV) is an enveloped, positive-sense, single-stranded RNA virus belonging
to the Flaviviridae family. DENV is transmitted by the mosquitoes Aedes aegypti and Aedes
albopictus, which mainly reside in (sub)tropical climates. Hence, dengue outbreaks are mainly
confined to equatorial areas, where more than 100 countries have been declared dengue-
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endemic. The World Health Organization (WHO) estimates that up to 3.9 billion people are at
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risk for dengue infection at any given time. In 2013, the WHO reported 3.2 million cases of
3
severe dengue and more than 9,000 dengue-related deaths worldwide. Up to 80% of DENV-
infected patients remain asymptomatic. Symptomatic patients usually experience an acute
1
febrile illness, characterized by high fever, muscle and joint pain, , and sometimes rash. The
likelihood of progression to severe dengue, manifesting by shock, hemorrhage and organ
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failure, is greater upon secondary infection with a heterologous dengue serotype (of four that
circulate) due to antibody-dependent enhancement.4
Ebola virus (EBOV) is a member of the Filoviridae family. Four of the five known EBOV
species have been responsible for over twenty outbreaks and over 10,000 deaths since their
identification in 1976.5
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Current efforts in search for drugs active against DENV focus primarily on viral targets, such
as the NS3 helicase, NS2B-NS3 protease, NS4B, NS5 methyltransferase, NS5 polymerase and
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the viral envelope. In search for anti-EBOV drugs, the RNA-dependent RNA polymerase L,
the viral surface glycoprotein GP, and viral proteins VP24 and VP35 have been explored as
6
candidate targets. However, targeting viral functions is often associated with the rapid
emergence of drug resistance and usually provides a ‘one drug, one bug’ approach. DENV and
EBOV rely extensively on host factors for their replication and survival. These cellular factors
represent attractive candidate targets for antiviral agents, potentially with a higher barrier for
resistance. In addition, such host-targeted antivirals are more likely to exhibit broad-spectrum
antiviral activity when targeting a host function required for the replication of several unrelated
viruses.7,8
Intracellular membrane trafficking is an example of a cellular process that is hijacked by various
9
viruses . Intracellular membrane trafficking depends on the function of tyrosine and dileucine
based signals in host cargo proteins, which are recognized by µ1-5 subunits of the clathrin
adaptor protein (AP) complexes AP1-5. Adaptor complexes mediate the sorting of cargo
proteins to specific membrane compartments within the cell. While AP2 sorts in the endocytic
pathway, AP1 and AP4 sort in the secretory pathway.10
The activity of AP2M1 and AP1M1, the µ subunits of AP2 and AP1, respectively, is regulated
by two host cell kinases, adaptor-associated kinase 1 (AAK1) and cyclin G associated kinase
(GAK). Phosphorylation of specific threonine residues in AP2M1 and AP1M1 by these kinases
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is known to stimulate their binding to tyrosine signals in cargo protein and enhance vesicle
assembly and internalization. Both AAK1 and GAK regulate clathrin-mediated endocytosis by
recruiting clathrin and AP2 to the plasma membrane. AAK1 also regulates clathrin-mediated
endocytosis of cellular receptors via alternative sorting adaptors that collaborate with AP-2, e.g.
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by phosphorylation of NUMB. Additionally, AAK1 has been implicated in the regulation of
EGFR internalization and recycling to the plasma membrane via its effects on and interactions
with alternate endocytic adaptors. We have demonstrated that AAK1 and GAK regulate
hepatitis C (HCV) entry and assembly by modulating AP2 activity.10,11 and viral release and
cell-to-cell spread via regulation of AP1.7,12 AAK1 and GAK are also required in the life cycles
of DENV and EBOV. 7
We have reported that the approved anticancer drugs sunitinib and erlotinib that potently inhibit
AAK1 and GAK, respectively, demonstrate broad-spectrum in vitro antiviral activity against
different members of the Flaviviridae family (HCV, DENV, Zika virus, West Nile virus), as
7
well as against various unrelated families of RNA viruses. We have also demonstrated that the
combination of these two drugs effectively reduces viral load, morbidity and mortality in mice
infected with DENV and EBOV.7,13 These data provide a proof-of-concept that small molecule
inhibition of AAK1 and GAK can yield broad-spectrum antiviral agents.7,13 Moreover, using
single-cell transcriptomic analysis, AAK1 has been validated as a particularly attractive target
since it is overexpressed specifically in DENV-infected and not bystander cells (uninfected
cells from the same cell culture), and its expression level increases with cellular virus
abundance.13
AAK1 has been studied primarily as a drug target for the treatment of neurological disorders,
1
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such as schizophrenia, Parkinson’s disease, neuropathic pain , bipolar disorders and
Alzheimer’s disease.15,16 Consequently, very potent AAK1 inhibitors based on different
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chemotypes have been disclosed in the patent literature. Figure 1 shows representative
examples of these AAK1 inhibitors and their enzymatic inhibition data.
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O
O
N
N
O
N
N
N
CN
O
O
N
H
N
CH2F
O
H
O
H3C NH2
NH2
H3C NH2
IC50 = 0.2 nM
WO2017/059080
IC50 = 0.3 nM
WO2017/059085
IC50 = 0.62 nM
WO2016/164295
OH
HN
N
O
CN
N
2
H N
N
N
N
N
HN
N
N
N
N
N
O
N
N
N
H
NH2
NH2
N
IC50 = 6 nM
WO2016/053794
IC50= 4 nM
WO2016/022312
IC50 = 3.3 nM
WO2015/054358
Figure 1. Known AAK1 inhibitors
Despite the fact that AAK1 emerged as a promising antiviral target, none of these compound
classes have been evaluated and/or optimized for antiviral activity. The only exception being a
series of imidazo[1,2-b]pyridazines, which were originally developed by Lexicon
1
4
Pharmaceuticals (Figure 2). We have previously resynthesized these molecules, confirmed
their potent AAK1 affinity and demonstrated their antiviral activity against HCV and DENV.7
N
O
N
R
N
N
O
N
OCH3
N
R = tert-butyl or isopropyl
Figure 2. AAK1 inhibitors with documented antiviral activity
Here, rather than starting from a potent AAK1 inhibitor, we started from a structurally simple
compound with reasonable AAK1 activity from which easy structural variation could be
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introduced. A screening campaign of 577 structurally diverse compounds (representing kinase
inhibitor chemical space) across a panel of 203 protein kinases using the DiscoverX binding
assay format previously identified a pyrrolo[2,3-b]pyridine or 7-aza-indole derivative
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(
compound 1, Figure 3) as a potent AAK1 inhibitor (K = 53 nM). In this manuscript, we
D
describe our efforts to optimize the AAK1 affinity and anti-DENV activity of compound 1.
O
NC
HN
N
N
N
H
1
Figure 3. A pyrrolo[2,3-b]pyridine (7-aza-indole) based AAK1 inhibitor
Results and Discussion
Chemistry
Synthesis of 3-substituted-5-aryl pyrrolo[2,3-b]pyridines
A regioselective Sonogashira coupling of trimethylsilylacetylene with commercially available
18
5
-bromo-3-iodo-2-aminopyridine 2 afforded the alkynyl derivative 3 (Scheme 1). Compound
was then reductively ring closed with a strong base yielding pyrrolo[2,3-b]pyridine 4.
3
1
8
19
Initially, NaH was used as base , but later on potassium tert-butoxide was applied as this
gave a cleaner reaction outcome and an improved yield. Initial attempts to nitrate position 3 of
2
0
the 7-azaindole scaffold employed a mixture of a 65% HNO solution and sulfuric acid .
3
However, the desired product was difficult to isolate from this reaction mixture and therefore,
2
1
the nitration was performed by treatment of compound 4 with fuming nitric acid. The 3-nitro
derivative 5 precipitated from the reaction mixture and was conveniently isolated by filtration.
Suzuki coupling of compound 5 with a number of arylboronic acids yielded the 3-nitro-5-aryl-
2
2
pyrrolo[2,3-b]pyridines 6a-h in yields ranging from 65 to 85%. Catalytic hydrogenation of
the nitro moiety yielded the corresponding amino derivatives 7a-h. Because of the instability
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of the 3-amino-pyrrolo[2,3-b]pyridines, these were not purified and used as such for further
2
3
reaction. Coupling with an acid chloride in a mixture of pyridine and dichloromethane or
alternatively, reaction with carboxylic acid using (benzotriazol-1-
a
yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP) as coupling reagent24
yielded a small library of pyrrolo[2,3-b]pyridines 8a-o. A sulfonamide derivative 8p was
prepared via reaction of 7g with phenylsulfonyl chloride in pyridine.
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Scheme 1. Synthesis of 3-substituted-5-aryl pyrrolo[2,3-b]pyridines 8a-p
SiMe3
NO2
d
Br
I
Br
b
Br
c
Br
a
N
H
N
N
N
NH2
N
NH2
N
H
2
3
4
5
8a: R1 = Ph; R2 = 3-pyridyl
8
8
b: R1 = 4-F-Ph; R2 = 3-pyridyl
c: R1 = 4-CF -Ph; R = 3-pyridyl
O
3
2
HN
8d: R1 = 4-OCH3-Ph; R2 = 3-pyridyl
8e: R1 = 4-CH -Ph; R = 3-pyridyl
R2
f or g
R1
3
2
8
f: R1 = 3-CH -Ph; R = 3-pyridyl
3 2
8g: R1 = 3,4-di-OCH -Ph; R = 3-pyridyl
N
H
3
2
N
8h: R1 = 3-thienyl; R2 = 3-pyridyl
i: R1 = 3,4-di-OCH -Ph; R = Ph
8
3
2
NH2
NO2
8j: R1 = 3,4-di-OCH -Ph; R = 4-F-Ph
3
2
R1
R1
e
8k: R1 = 3,4-di-OCH -Ph; R = 4-OCH -Ph
3 2 3
8
l: R1 = 3,4-di-OCH -Ph; R = 4-pyridyl
3 2
N
N
N
8m: R1 = 3,4-di-OCH -Ph; R = benzyl
N
3 2
H
H
8n: R1 = 3,4-di-OCH -Ph; R = cyclopentyl
3 2
8
o: R1 = 3,4-di-OCH -Ph; R = cyclohexyl
6
6
a: R1 = Ph
b: R1 = 4-F-Ph
3
2
7a: R1 = Ph
7b: R1 = 4-F-Ph
7c: R1 = 4-CF -Ph
7d: R1 = 4-OCH -Ph
7e: R1 = 4-CH -Ph
7f: R1 = 3-CH -Ph
7g: R1 = 3,4-di-OCH -Ph
7h: R1 = 3-thienyl
O
6c: R1 = 4-CF -Ph
3
S
3
HN
6
d: R1 = 4-OCH -Ph
3
h
3
R1
O
6
e: R1 = 4-CH -Ph
3
3
6f: R1 = 3-CH -Ph
3
3
N
H
6g: R1 = 3,4-di-OCH -Ph
N
3
3
6
h: R1 = 3-thienyl
8p: R1 = 3,4-di-OCH -Ph
3
Reagents and conditions. a) TMSA, Pd(PPh ) Cl , CuI, Et N, THF, rt ; b) KOtBu, NMP, 80°C ; c)
3
2
2
3
HNO , 0°C to rt ; d) Pd(PPh ) , K CO , ArB(OH) , H O, dioxane, 105°C ; e) H , Pd/C, THF, rt ; f)
3
3 4
2
3
2
2
2
RCOCl, pyridine, THF, 1M NaOH, rt ; g) RCOOH, BOP, Et N, DMF, rt ; h) PhSO Cl, pyridine, rt.
3
2
Synthesis of 3-benzoyl-5-(3,4-dimethoxyphenyl)-pyrrolo[2,3-b]pyridine
Formylation of compound 4 by a Duff reaction25 yielded 3-formyl-5-bromo-azaindole 9
Scheme 2). The pyrrole nitrogen was protected26 using NaH and tosylchloride yielding
(
compound 10. Suzuki coupling reaction with 3,4-dimethoxyphenylboronic acid furnished
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compound 11. Nucleophilic addition of phenylmagnesium bromide to the aldehyde furnished
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the secondary alcohol 12. Oxidation of the benzylic alcohol using MnO afforded ketone 13.
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Finally, alkaline deprotection of the tosyl group yielded the desired compound 14.
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Scheme 2. Synthesis of 3-benzoyl-5-(3,4-dimethoxyphenyl)-pyrrolo[2,3-b]pyridine 14
O
O
CHO
CHO
CHO
Br
c
Br
Br
b
a
N
N
N
N
N
N
N
H
H
Ts
N
Ts
4
9
10
11
O
O
O
O
HO
O
O
O
O
f
e
d
N
N
N
N
N
N
H
Ts
Ts
12
13
14
Reagents and conditions. a) hexamine, H O, CH COOH, 120°C ; b) NaH, TsCl, 0°C to rt ; c) 3,4-
2
3
dimethoxyphenylboronic acid, Pd(PPh ) , 2M K CO , toluene, EtOH, 105°C ; d) 3M PhMgBr, THF, rt;
3 4
2
3
e) MnO , THF, rt ; f) KOH, EtOH, 80°C.
2
Synthesis of 3-phenyl-5-(3,4-dimethoxyphenyl)-pyrrolo[2,3-b]pyridine
Iodination of compound 4 with N-iodosuccinimide30 afforded compound 15 (Scheme 3).
Reaction of compound 15 with phenylboronic acid led only to recovery of unreacted starting
material. Therefore, the pyrrole nitrogen of the 7-azaindole scaffold was protected as a tosyl
group,26 affording compound 16. A regioselective Suzuki coupling reaction using
phenylboronic acid furnished the 3-phenyl-pyrrolo[2,3-b]pyridine analogue 17. A subsequent
Suzuki reaction22 with 3,4-dimethoxyphenylboronic acid yielded compound 18. Finally,
alkaline cleavage of the tosyl protecting group afforded the desired target compound 19.29
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Scheme 3. Synthesis of 3-phenyl-5-(3,4-dimethoxyphenyl)-pyrrolo[2,3-b]pyridine 19
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
I
I
Br
Br
c
Br
b
a
Br
N
N
N
N
N
N
N
N
H
Ts
Ts
H
4
15
16
17
O
O
O
O
d
e
N
N
N
N
H
Ts
18
19
Reagents and conditions. a) NIS, acetone, rt ; b) NaH, TsCl, THF, 0°C to rt ; c) PhB(OH) , Pd(PPh ) ,
2
3 4
K CO , toluene, EtOH, H O, 90°C ; d) 3,4-dimethoxyphenylboronic acid, Pd(PPh ) , K CO , toluene,
2
3
2
3 4
2
3
EtOH, H O, 105°C ; e) KOH, EtOH, 80°C.
2
Synthesis of 3-alkynyl-5-(3,4-dimethoxyphenyl)-pyrrolo[2,3-b]pyridines
Sonogashira reaction of compound 15 with a number of (hetero)arylacetylenes yielded
1
8
regioselectively compounds 20a-f in yields varying from 20-70% (Scheme 4). In contrast to
Suzuki couplings (Scheme 3), protection of the pyrrole nitrogen was not necessary. Subsequent
2
2
Suzuki coupling with 3,4-dimethoxyphenylboronic acid gave access to final compounds 21a-
f.
Scheme 4. Synthesis of 3-alkynyl-5-(3,4-dimethoxyphenyl)-pyrrolo[2,3-b]pyridines 21a-f
R
R
O
O
I
Br
Br
a
b
N
N
N
N
N
N
H
H
H
15
21a: R = phenyl
21b: R = 3-pyridyl
21c: R = 3-OCH -phenyl
2
2
2
2
2
0a: R = phenyl
0b: R = 3-pyridyl
0c: R = 3-OCH -phenyl
3
3
0d: R = 4-CH -phenyl
21d: R = 4-CH -phenyl
3
3
0e: R = 4-F-phenyl
21e: R = 4-F-phenyl
21f: R = 3-CN-phenyl
20f: R = 3-CN-phenyl
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Reagents and conditions. a) RC≡CH, Pd(PPh ) Cl , CuI, THF, Et N, rt ; b) 3,4-dimethoxyphenylboronic
3
2
2
3
acid, Pd(PPh ) , K CO , H O, dioxane, 105 °C.
3
4
2
3
2
Synthesis of 1-methyl-1H-pyrrolo[2,3-b]pyridine
Methylation31 of compound 4 using NaH and MeI furnished compound 22 (Scheme 5).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
21
22
Nitration, followed by Suzuki coupling gave access to compound 24. Finally, catalytic
2
3
reduction of the nitro group, followed by condensation of the exocyclic amino group of
compound 25 with nicotinoyl chloride yielded target compound 26.
Scheme 5. Synthesis of 1-methyl-1H-pyrrolo[2,3-b]pyridine 26.
O
O
NO2
NO2
Br
Br
Br
b
c
a
N
N
N
N
N
N
H
N
N
4
22
23
24
O
O
O
O
NH2
O
d
e
HN
N
N
N
N
N
25
26
Reagents and conditions. a) NaH, MeI, THF, 0°C to rt ; b) HNO , 0°C to rt ; c) 3,4-
3
dimethoxyphenylboronic acid, Pd(PPh ) , K CO , H O, dioxane, 105°C ; d) H , THF, rt ; e) nicotinoyl
3
4
2
3
2
2
chloride, pyridine, THF, 1M NaOH, rt.
Synthesis of pyrazolo[3,4-b]pyridine
2
2
Suzuki coupling between commercially available 5-bromo-2-chloronicotinonitrile 27 and 3,4-
dimethoxyphenylboronic acid yielded regioselectively compound 28. Nucleophilic
displacement of the chlorine by hydrazine, with a concomitant nucleophilic addition at the
3
2
cyano group allowed to construct the pyrazole moiety, yielding compound 29. Finally, amide
3
3
formation using nicotinoyl chloride yielded the final compound 30.
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Scheme 6. Synthesis of pyrazolo[3,4-b]pyridine 30
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
O
O
O
O
O
O
a
b
NH2
N
HN
c
Br
CN
Cl
CN
Cl
N
N
N
N
N
N
N
N
H
H
27
28
29
30
Reagents and conditions. a) 3,4-dimethoxyphenylboronic acid, Pd(PPh ) , K CO , H O, dioxane, 105°C
3
4
2
3
2
;
b) 35% hydrazine hydrate, EtOH, 80°C ; c) nicotinoyl chloride, pyridine, rt.
Synthesis of pyrrolo[2,3-b]pyrazine
Treatment of 2-aminopyrazine 31 with N-bromosuccinimide yielded the 3,5-dibromopyrazine
intermediate 32 (Scheme 7).34
A
regioselective Sonogashira coupling with
trimethylsilylacetylene, followed by a reductive ring closure with potassium tert-butoxide
furnished pyrrolo[2,3-b]pyrazine 34.35 Iodination36 with N-iodosuccinimide yielded the
1
8
dihalogenated intermediate 35 that was subsequently treated with 3-ethynylpyridine and 3,4-
2
2
dimethoxyphenylboronic acid leading to the desired pyrrolo[2,3-b]pyrazine 37.
Scheme 7. Synthesis of pyrrolo[2,3-b]pyrazine 37
SiMe3
Br
N
N
Br
N
Br
Br
N
b
c
a
N
N
N
NH2
N
NH2
N
NH2
H
31
32
33
34
N
N
O
O
I
Br
N
N
Br
N
N
N
N
d
e
f
N
N
H
N
H
H
35
36
37
Reagents and conditions. a) NBS, DMSO, rt ; b) Me SiC≡CH, Pd(PPh ) Cl , CuI, THF, Et N, rt ; c)
3
3 2
2
3
KOtBu, NMP, 100°C ; d) NIS, acetone, rt ; e) 3-ethynylpyridine, Pd(PPh ) Cl , CuI, THF, Et N, rt ; f)
3
2
2
3
3,4-dimethoxyphenylboronic acid, Pd(PPh ) , K CO , H O, dioxane, 105 °C.
3
4
2
3
2
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Kinase profiling and X-ray crystallography of compound 1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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8
9
0
1
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5
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9
0
1
2
3
4
5
6
7
8
9
0
AAK1 is a serine-threonine kinase that belongs to the NUMB-associated family of protein
kinases (NAKs). Other members of this kinase family include BIKE/BMP2K (BMP-2 inducible
kinase), GAK (cyclin G associated kinase) and MPSK1 (myristoylated and palmitoylated
serine-threonine kinase 1, also known as STK16). As part of an early profiling of hit compound
1
, we assessed its selectivity by a binding-displacement assay against each of the four NAK
family kinases (Figure 4). Conversion of the experimentally determined IC values to K values
5
0
i
to allow estimation of the selectivity showed that compound 1 was 3-fold more selective for
AAK1 over GAK, and 8-fold and 22-fold more selective for AAK1 over BMP2K and STK16,
respectively (Table 1).
Figure 4. Binding displacement assay of compound 1 against the NAK family members.
Table 1. Selectivity of compound 1 for AAK1 against the NAK family kinases
Binding Displacement Assay
NAK
AAK1
BMP2K
GAK
IC50 (µM)
1.17
K
i
(µM)
K
i
/ K
i
(AAK1)
1.0
0.541
4.40
1.75
10.1
3.25
8.13
3.23
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STK16
20.0
11.9
22.0
To analyse the binding mode of compound 1, the crystal structure of compound 1 bound to
AAK1 to 2.0 Å resolution was determined (Figure 5). Data collection and refinement statistics
are provided in Supplemental Table 1 (see Supporting Information) and PDB coordinates are
deposited under PDB ID 5L4Q. Compound 1 binds in the ATP-binding site of AAK1 in a
relatively planar manner (Figure 5), with the pyrrolo[2,3-b]pyridine moiety bound directly
between the side-chains of two highly conserved residues: Ala72 from β2 in the kinase N-lobe
and Leu183 of the C-lobe, the location where the adenine ring of ATP would bind. The nitrogen
atoms of the pyrrolo[2,3-b]pyridine moiety form two hydrogen bonds to the peptide backbone
of residues Asp127 and Cys129 at the kinase hinge region (Figure 5). The 4-cyanophenyl
moiety is oriented towards the solvent with the phenyl ring directly sandwiched between the
backbone of Gly132 of the hinge and Leu52 of β1 in the N-lobe, and the nitrogen of the cyano
moiety forming a polar interaction with the side-chain of Asn136. The nicotinamide moiety is
bound against the “gatekeeper” residue Met126, and forms a hydrogen bond to the side chain
of Lys74, another highly conserved residue that normally links the phosphate of ATP to the αC-
helix and required for correct positioning of the N-lobe for efficient catalysis. Compound 1 also
interacts with two water molecules, one situated at the back of the ATP pocket that bridges
between the oxygen of the amide moiety and the backbone nitrogen of Asp194, and a second
at the front of the adenosine binding site directly below Val60 in the β1 strand that interacts
with the amide nitrogen and surrounding solvent. The DFG motif (Asp194) is in the
conformation expected of active AAK1 (Figure 5), with the activation loop of AAK1 in the
same conformation as seen in previous AAK1 and BMP2K crystal structures, including the
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
activation-segment C-terminal helix (ASCH), a structural element that is rare amongst other
protein kinases, but conserved across the NAK family.37
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 5. Crystal structure of AAK1 (grey) in complex with compound 1 (purple), PDB ID
5
L4Q. A: Overview of the crystal structure of AAK1. Highlighted are areas that are important for kinase
function. Compound 1 bound at the hinge is shown. ASCH = Activation Segment C-terminal Helix, a
feature unique to kinases of the NAK family. B: 2F -F Electron density map contoured at 1σ around
o
c
compound 1, showing the fit of the model to the map. C and D: Detailed views of the interactions of
compound 1 in the ATP-binding site from two orientations. Black dotted lines indicate polar
interactions, red spheres indicate water molecules. Note that residues 48-63 from β1 and β2 are removed
from C for clarity.
Structure-activity relationship (SAR) study
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All compounds synthesized in this study were evaluated for AAK1 affinity using two different,
commercially available AAK1 binding assays. In the early stage of the program, the proprietary
KINOMEscan screening platform of DiscoverX was used. In this assay, compounds that bind
the kinase active site prevent kinase binding to an immobilized ligand and reduce the amount
of kinase captured on the solid support. Hits are then identified by measuring the amount of
kinase captured in test versus control samples via qPCR that detects an associated DNA label.38
Later on in the project, compounds were evaluated by the LanthaScreen™ Eu Kinase Binding
Assay (ThermoFisher Scientific), in which binding of an Alexa Fluor™ conjugate or “tracer”
to a kinase is detected by addition of a Eu-labeled anti-tag antibody. Binding of the tracer and
antibody to a kinase results in a high degree of fluorescence resonance energy transfer (FRET),
whereas displacement of the tracer with a kinase inhibitor results in loss of FRET.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
The compounds were also assessed for antiviral activity in human hepatoma (Huh7) cells
infected with DENV2. Their effect on overall infection was measured at 48 hours postinfection
with DENV2 via luciferase assays and the half-maximal effective concentration and the 90%
effective concentrations (EC and EC values, respectively) were calculated. In parallel, the
5
0
90
cytotoxicity of the compounds (expressed as the half-maximal cytotoxic concentration or CC50
value) was measured via an AlamarBlue assay in the DENV-infected Huh7 cells.
In all these enzymatic and antiviral assays, sunitinib was included as a positive control (Tables
2
, 3, 4, 5 and 6). Sunitinib has potent AAK1 affinity as measured by the KinomeScan format
(K = 11 nM) and LanthaScreen assay (IC = 47 nM) and displayed potent activity against
D
50
DENV with an EC value of 1.35 µM.
5
0
SAR at position 5 of the 7-aza-indole scaffold
We confirmed the AAK1 binding affinity of hit compound 1, yet in our hands a K value of
D
1
7
1
20 nM was measured (vs. the reported K = 53 nM). This hit demonstrated antiviral activity,
D
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
although rather weak (EC = 8.37 µM). Substitution of the 5-(4-cyanophenyl) moiety of
5
0
compound 1 by phenyl (compound 8a), thienyl (compound 8h) and substituted phenyl rings
with electron-withdrawing groups (compound 8c), electron-donating substituents (compounds
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
8
d-g) and a halogen (compound 8b) gave rise to a series of analogues with structural variety
that were at least equipotent to compound 1, suggesting that structural modification at this
position is tolerated for AAK1 binding (Table 2). The synthesis of this limited number of
analogues allowed us to quickly identify the 5-(3,4-dimethoxyphenyl) congener (compound 8g)
with low nM AAK1 binding affinity in both the KinomeScan and LanthaScreen assay,
indicating an excellent correlation between the two assays. As compound 8g showed stronger
AAK1 affinity than the positive control sunitinib, no additional efforts were done to further
explore the SAR at this position of the 7-aza-indole scaffold.
All analogues within this series had an improved antiviral activity against DENV, relative to
the original hit 1. The compound with the highest binding affinity for AAK1 (compound 8g)
also demonstrated the most effective anti-DENV activity, with EC and EC values of 1.64
5
0
90
µM and 7.46 µM, respectively (Figure 6).
Table 2. SAR at position 5 of the 7-aza-indole scaffold
O
HN
R5
N
N
N
H
AAK1 enzymatic data
DENV antiviral
activity
Cytotoxicity
CC50 (µM)
Cmpd#
1
R
5
AAK1 K
µM)
D
AAK1 IC50
(µM)
EC50
EC90
(
(µM)
(µM)
(
DiscoverX)
LanthaScreen
a
4-cyanophenyl
phenyl
4-F-phenyl
0.120
ND
ND
ND
8.37
5.29
6.43
2.94
4.39
2.60
4.86
46.8
87.1
25.2
12.0
25.8
33.6
10.8
>50
a
b
8
a
0.0822
0.141
NE
NE
a
b
8
b
a
8
c
4-CF
3
-phenyl
-phenyl
ND
0.23
57.0
a
b
8
d
4-OCH
3
0.0194
0.0532
ND
ND
NE
a
8
e
4-Me-phenyl
3-Me-phenyl
>100
16.0
a
8
f
ND
0.0186
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8g
3,4-di-OCH
phenyl
3-thienyl
3
-
0
.00673
0.00432
1.64
7.46
39.7
8h
Sunitinib
0.0161
0.0110
NDa
0.0474
3.01
1.35
66.8
2.71
NEb
246
-
a
b
ND : not determined ; NE : No effect (no apparent effect on cellular viability up to a concentration of
0 µM).
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
SAR of the N-acyl moiety
Given the improved AAK1 affinity and antiviral activity of compound 8g, the SAR of this
compound was further examined through the replacement of the 3-pyridyl group by a number
of (hetero)aromatics and cycloaliphatic groups, while the 3,4-dimethoxyphenyl residue was
kept fixed (Table 3). Our findings indicate that the 3-pyridyl moiety is critical for AAK1
binding as all analogues showed a 100-fold decreased AAK1 affinity, when compared to
compound 8g, giving rise to AAK1 IC values in the 0.1-0.6 µM range. Only the congener
5
0
with a 4-methoxyphenyl residue (compound 8k) was endowed with an enhanced AAK1 affinity
with an IC value of 0.072 µM. In correlation with their weaker affinity for AAK1, these
5
0
compounds had a diminished antiviral activity relative to compound 8g.
Table 3. SAR of the N-acyl moiety
O
O
O
HN
R
N
N
H
AAK1 enzymatic
data
AAK1 IC50 (µM)
LanthaScreen
0.00432
0.108
DENV antiviral activity
Cytotoxicity
CC50 (µM)
Cmpd#
R
EC50 (µM)
EC90 (µM)
8
g
3-pyridyl
phenyl
4-F-phenyl
1.64
4.07
8.34
NE
12.6
142
7.46
50.5
30.2
39.7
17.3
15.9
23.6
8
i
8
j
0.161
0.0721
0.381
0.612
0.398
0.319
a
a
8
k
4-OCH
3
-phenyl
NE
26.8
a
8
l
4-pyridyl
benzyl
cyclopentyl
cyclohexyl
NE
NE
a
a
8
m
NE
8n
8o
3.42
4.27
8.34
10.8
15.2
14.7
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
aNE : No effect (no apparent antiviral effect or effect on cellular viability up to a concentration of 10
µM).
SAR of the linker moiety at position 3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
To evaluate the importance of the amide linker, a number of surrogates were prepared (Table
4
). For synthetic feasibility reasons, compound 8i, having a phenyl residue instead of a 3-
pyridyl ring, and endowed with quite potent AAK1 affinity and moderate antiviral activity, was
selected as a reference compound. Removing the amide linker furnished the 3-phenyl
substituted analogue 19, which was 4-fold more potent as AAK1 ligand than compound 8i, and
showed a slightly improved antiviral activity against DENV (EC and EC values of 3.02 µM
5
0
90
and 8.34 µM, respectively). When the amide linker of compound 8i was replaced by a ketone
(compound 14) or alkyne (compound 21a) functionality, AAK1 affinity was retained.
Compound 14 exhibited an improved antiviral activity in comparison with compound 8i.
Finally, replacement of the amide moiety by a sulfonamide linker was not well-tolerated, as
compound 8p showed close to 200-fold drop in AAK1 affinity.
Table 4. SAR of the linker moiety at position 3
O
O
R
N
N
H
AAK1 enzymatic
data
AAK1 IC50 (µM)
LanthaScreen
DENV antiviral activity
Cytotoxicity
CC50 (µM)
Cmpd#
R
EC50 (µM)
EC90 (µM)
H
N
8
i
0.108
0.0236
0.184
4.07
3.02
1.07
50.5
8.34
3.73
17.3
990
O
1
1
9
4
O
35.3
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8
9
21a
8p
0.209
4.44
5.64
8.14
10.58
106
20.1
NEa
H O
N
S
O
aNE : No effect (no apparent effect on cellular viability up to a concentration of 10 µM).
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
SAR of phenylacetylene
The data in Table 4 suggest that the amide moiety is not essential for AAK1 binding and can
be replaced. Although the 3-phenyl derivative 19 displayed potent AAK1 affinity, 3,5-diaryl
pyrrolo[2,3-b]pyridines are well known in the literature as kinase inhibitors. In contrast, 3-
alkynyl-7-aza-indoles are studied to a much less extent. We, therefore, selected the acetylene
derivate 21a to decipher if it was possible to improve AAK1 affinity and antiviral activity by
further modifying the substitution pattern. A number of substituted phenylacetylene derivatives
was prepared (compounds 21b-f). The SAR in this series was quite flat, as all compounds
displayed very similar AAK1 affinity with IC values in the 0.1-0.4 µM range (Table 5). In
5
0
contrast, the introduction of a 3-pyridylacetylene (compound 21b) led to a substantial
improvement in AAK1 affinity (IC50 = 0.0042 µM) with a concomitant improvement in
antiviral activity (EC = 0.72 µM) and only a moderate cytotoxic effect (CC = 17 µM, Figure
5
0
50
6
).
Table 5. SAR of phenylacetylene moiety
R
O
O
N
N
H
AAK1
enzymatic data
DENV antiviral activity
Cytotoxicity
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2
2
2
2
2
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3
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3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Cmpd#
R
AAK1 IC50
(µM)
LanthaScreen
EC50 (µM)
EC90 (µM)
CC50 (µM)
21a
21b
21c
21d
21e
phenyl
3-pyridyl
0.209
0.00402
0.149
0.420
0.387
5.64
0.72
10.58
4.16
20.1
17.0
19.6
a
a
3-OCH
4-CH
3
-phenyl
-phenyl
NE
NE
a
3
7.45
4.21
10.2
20.5
8.57
41.2
NE
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4-F-phenyl
3-CN-phenyl
19.9
a
2
1f
0.381
NE
a
NE : No effect (no apparent antiviral effect or effect on cellular viability up to a concentration of 10
µM).
Scaffold modifications
Lastly, our SAR exploration focused on the 7-aza-indole scaffold itself (Table 6). Methylation
of the pyrrole nitrogen afforded compound 26, displaying a greatly decreased AAK1 affinity
(
>800-fold loss in activity relative to compound 8g) and antiviral activity (EC > 10 µM).
50
Insertion of an additional nitrogen atom in the pyrrole moiety yielded the pyrazolo[3,4-
b]pyridine analogue 30 that is 100-fold less active as an AAK1 ligand relative to the parent
compound 8g. When the pyridine moiety of compound 21b was replaced by a pyrazine ring,
the pyrrolo[2,3-b]pyrazine analogue 37 was obtained. This compound was endowed with very
potent AAK1 affinity (IC = 0.00927 µM), comparable to its 7-aza-indole counterpart 21b.
5
0
Unfortunately, compound 37 demonstrated greater cytotoxicity than compound 21b in Huh7
cells with CC ’s of 4.81 µM vs. 17.0 µM, respectively.
5
0
Table 6. Scaffold modifications
AAK1
enzymatic data
AAK1 IC50
(µM)
DENV antiviral activity
Cytotoxicity
CC50 (µM)
Cmpd#
Structure
EC50 (µM)
EC90 (µM)
LanthaScreen
O
O
O
HN
2
6
3.39
NEa
NEa
NEa
N
N
N
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O
O
O
HN
3
3
0
7
0.462
2.09
3.28
19.0
7.68
16.0
4.81
N
N
N
N
H
N
O
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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3
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4
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4
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4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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7
8
9
0
1
2
3
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8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
0.00927
N
N
N
H
aNE : No effect (no apparent antiviral effect or effect on cellular viability up to a concentration of 10
µM).
Figure 6. Compounds 8g and 21b suppress DENV infection more effectively than
compound 1. Dose response of DENV infection (blue) and cell viability (black) to compounds 1, 8g,
and 21b measured by luciferase and alamarBlue assays, respectively, 48 hours after infection. Data are
plotted relative to vehicle control. Shown are representative experiments from at least two conducted,
each with 5 biological replicates; shown are means ± SD.
Broad-spectrum antiviral activity
AAK1 has been demonstrated to be important for the regulation of intracellular viral trafficking
7
of multiple unrelated viruses. Sunitinib, an approved anticancer drug with potent anti-AAK1
7
activity, displayed antiviral activity against RNA viruses from six different families. To
evaluate for potential broad-spectrum antiviral coverage of our molecules beyond DENV
infection, hit compound 1 and the optimized congeners (compounds 8g and 21b) were tested
for their activity against the unrelated EBOV. Huh7 cells were infected with EBOV and treated
for 48 hours with each compound (Figure 7). Whereas compound 1 did not show effective
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activity against EBOV, the more potent AAK1 inhibitors 8g and 21b displayed anti-EBOV
activity with EC values in the low µM range and CC > 10-20 µM .
5
0
50
0
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1
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8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 7. Compounds 1, 8g and 21b suppress EBOV infection. Dose response of EBOV
infection (blue) and cell viability (black) to compounds 1, 8g and 21b measured by plaque assay (for
compound 1) or immunofluorescence assays (for compounds 8g and 21b) and CellTiter-Glo
luminescent cell viability assay in Huh7 cells 48 hours after infection. Data are plotted relative to vehicle
control. Shown are representative experiments from at least two conducted, each with 3 biological
replicates; shown are means ± SD.
Correlation of the antiviral effect of compounds 1, 8g and 21b with functional AAK1
inhibition
To confirm that the observed antiviral activity is correlated with functional inhibition of AAK1
activity, we measured levels of the phosphorylated form of the µ subunit of the AP2 complex,
AP2M1, upon treatment with compounds 1, 21b and 8g. Since AP2M1 phosphorylation is
2
7
transient (due to phosphatase PP2A activity), to allow capturing of the phosphorylated state,
Huh7 cells were incubated for 30 minutes in the presence of the PP2A inhibitor calyculin A
prior to lysis. Treatment with compounds 1, 21b and 8g reduced AP2M1 phosphorylation
(Figure 8), indicating modulation of AP2M1 phosphorylation via AAK1 inhibition.
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1
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8
9
0
1
2
3
4
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6
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8
9
0
Figure 8. Antiviral effect of compounds 1, 8g and 21b correlate with functional inhibition
of AAK1. Dose response of AP2M1 phosphorylation to treatment with 1 (A), 8g (B), and 21b
(C) by Western analysis in lysates derived from Huh7 cells. Representative membranes (from two
independent experiments) blotted with anti-phospho-AP2M1 (pAP2M1), anti-AP2M1 (AP2M1), and
anti-Actin (Actin) antibodies and quantified data of pAP2M1/actin protein ratio normalized to DMSO
controls are shown. ** p < 0.01, *** p < 0.001 by 2-tailed unpaired t test.
Inhibition of DENV infection in human primary monocyte-derived dendritic cells
(MDDCs)
To determine the therapeutic potential of the AAK1 inhibitors, their antiviral activity was
studied in human primary dendritic cells. Primary cells are a physiologically more relevant
model for DENV infection than immortalized cell lines, and are considered an ex vivo model
39
for DENV infection. Compounds 21b and 8g showed a dose-dependent inhibition of DENV
infection with EC and EC values of 0.0428 µM and 1.49 µM and 0.739 µM and 3.32 µM,
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respectively (Figure 9). This very potent activity in MDDCs associated with minimal
cytotoxicity (CC > 20 µM), particularly of compound 21b demonstrates the potential of
5
0
AAK1 inhibitors as antiviral agents.
0
1
2
3
4
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1
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8
9
0
1
2
3
4
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6
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8
9
0
Figure 9. Ex vivo antiviral activity of 21b and 8g in human primary dendritic cells. Dose
response of DENV infection (blue) and cell viability (black) to compounds 21b (A) and 8g (B) measured
by plaque assays and alamarBlue assays, respectively, 72 hours after infection of primary human
monocyte-derived dendritic cells (MDDCs). Shown is a representative experiment with cells from a
single donor, out of 2 independent experiments conducted with cells derived from 2 donors, each with
6
biological replicates; shown are means ± SD.
Kinase selectivity
To assess the kinase selectivity of the optimized AAK1 inhibitors, compound 21b was screened
against 468 kinases via the KINOMEScan assay (DiscoverX) at a single concentration of 10
µM. As can be derived from the kinase interaction map (Figure 10), compound 21b cannot be
considered as a selective AAK1 inhibitor. Beyond AAK1, compound 21b targets multiple other
kinases (including the other members of the NAK family), which might contribute to its
antiviral effect.
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8
9
0
Figure 10. Kinome tree of compound 21b. Kinases that bind compound 21b are marked with red
circles. The larger the circle, the stronger the binding affinity.
To quantitatively characterize the selectivity of compound 21b, selectivity scores (S-scores)
4
0
were calculated (Table 7). The S-score is calculated by dividing the number of kinases that
compounds bind to by the total number of distinct kinases tested, excluding mutant variants.
Up to now, the AAK1 inhibitor that often has been used for antiviral activity studies is sunitinib.
The S(3 µM)-score of sunitinib, which is the number of kinases found to bind with a
dissociation constant less than 3 µM by the total number of non-mutant kinases tested, is 0.57.40
The S (3 µM) score is comparable with the S(10) score and therefore it can be deduced that the
7
-aza-indole based AAK1 inhibitor 21b, although still not very selective, has an improved
kinase selectivity profile, relative to that of sunitinib, and it may therefore represent an
improved pharmacological tool to probe the role of AAK1 in viral infection.
Table 7. Selectivity scores (S scores) for compound 21b at 10 µM
S-score type
Number of Hits
Number of Non-Mutant
Selectivity Score
Kinases
403
403
403
S(35)a
212
138
49
0.526
0.342
0.122
b
S(10)
c
S(1)
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aS(35) = (number of non-mutant kinases with %Ctrl <35)/(number of non-mutant kinases tested)
b
S(10) = (number of non-mutant kinases with %Ctrl <10)/(number of non-mutant kinases tested)
S(1) = (number of non-mutant kinases with %Ctrl <1)/(number of non-mutant kinases tested)
c
Molecular modeling
0
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2
3
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6
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8
9
0
To rationalize the improved AAK1 affinity of compounds 8g and 21b, when compared to the
4
1
original hit 1, a docking study was performed using Autodock Vina. A control docking
experiment with the original inhibitor lkb (compound 1), present in the 5L4Q PDB file, allowed
to reproduce the original X-ray position very well. The best Vina docking scores are reported
in Table 8. Despite the higher AAK1 affinity of compounds 8g and 21b, their Vina docking
scores are worse than reference compound 1. This might be due to the fact that a rigid enzyme
in the docking process is used that blocks induced-fit effects.
Table 8. Vina docking and MM/PBSA results calculated on last 4 ns.
a
b
a
c
Compound
1
Vina score
MM /GBSA in
MM /PBSA in
d
d
(kcal/mol)
kcal/mol (SD )
kcal/mol (SD )
-10.7
-10.3
-31.5 (1.6)
-38.6 (2.2)
-40.6 (2.2)
-0.7 (2.3)
-3.0 (2.9)
-3.1 (2.8)
8
g
2
1b
-10.1
a
b
c
MM : molecular mechanics ; GB: Generalized Born Surface Area; PBSA: Poisson Boltzmann Surface
d
Area; SD: standard deviation.
Therefore, for the three docked systems with the best Vina docking score, a molecular dynamics
4
2
(MD) simulation using the Amber 18 software was performed. Figure 11 shows an overlap of
representative structures of compounds 1, 8g and 21b, as extracted from the MD simulations.
The three inhibitors form a hydrogen bond between the NH of the pyrrole ring and the backbone
carbonyl group of D127. The nitrogen at position 3 of the pyridine ring seems to be essential
for strong AAK1 affinity. In the X-ray of AAK1 with compound 1, this nitrogen of the
nicotinamide moiety makes a hydrogen bond with K74 side chain. However, this
hydrogen/ionic bond is not maintained in the molecular dynamics simulations of AAK1 with
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compound 1, and neither with compounds 8g and 21b. On the other hand, for the three
compounds, frequent van der Waals contacts between this nitrogen and the charged nitrogen in
the sidechain of K74 (distances < 4 Å) are observed.
1
1
1
1
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For the last 4 ns of the trajectories, MM/GBSA and MM/PBSA (molecular mechanics energies
with a generalized Born surface area continuum solvation model and molecular mechanics
energies combined with the Poisson–Boltzmann surface area continuum solvation model,
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respectively) binding affinity calculations were conducted. No entropy contributions were
calculated (Table 8). From the data in Table 8, it is clear (more from the General Born model
than from the Poisson Boltzmann model) that compounds 8g and 21b are stronger AAK1
binders than reference compound 1. Both methoxy groups of compounds 8g and 21b contribute
to the binding energy via van der Waals interactions with specific amino acids of AAK1.
Residues L52, L62, F128, C129, R130, G131, G132, Q133, V135 and N136 are involved in
binding to compound 21b. The same residues plus E50 make contact with compound 8g. On
the other hand, the nitrile function of compound 1 shows clearly less van der Waals contacts
with surrounding residues (L52, G132, Q133 and N136, Figure 12).
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Figure 11. AAK1 snapshot structures with 3 ligands. Compound 1 (green, lightgreen), compound 8g
(magenta, rose) and compound 21b (cyan; lightblue), taken extracted from the last 4 ns of the MD
trajectory as representative structure from the biggest cluster using the average linkage clustering
method available in cpptraj. The AAK1 and lkb X-ray structures are also shown (orange, yellow). Image
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made by the Chimera software.
Figure 12. AAK1 enzyme with compound 21b (same snapshot as in Figure 11). Amino acids making
contact with the two methoxy groups (heavy atom distances < 4 Å) are coloured in yellow and magenta.
The magenta colour represents also the residues having Van der Waals contacts with the nitrile group
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of compound 1. Image made by the Chimera software.
Conclusions
AAK1 is a promising host target for the development of broad-spectrum antiviral agents. In this
manuscript, the first systematic SAR study of AAK1 inhibitors as antiviral agents is presented.
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Journal of Medicinal Chemistry
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Starting from a known ligand with moderate AAK1 binding affinity and anti-DENV activity, a
systematic SAR study was carried out. It led to the discovery of AAK1 ligands with low nM
AAK1 binding affinity that display improved antiviral activity against DENV. Moreover, the
optimized AAK1 inhibitors exhibit very potent activity in DENV-infected primary dendritic
cells and anti-EBOV activity, supporting the potential to develop broad-spectrum antiviral
agents based on AAK1 inhibition. Kinase profiling revealed that these compounds have an
improved selectivity profile relative to sunitinib, yet further research is necessary to improve
their kinase selectivity.
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Experimental section
All commercial reagents were obtained via Acros Organics, Sigma-Aldrich, AK Scientific and
Fluorochem at at least 99% purity unless indicated otherwise. Furthermore, all dry solvents
were obtained via Acros Organics with an AcroSeal system and regular solvents were obtained
via Fisher Scientific at technical grade. Thin layer chromatography (TLC) of the reactions was
performed on silica gel on aluminum foils (60 Å pore diameter) obtained from Sigma-Aldrich
and visualized using ultraviolet light. Recording of the NMR spectra was performed using a
Bruker 300 MHz, 500 MHz or 600 MHz spectrometer. The chemical shifts are reported in ppm
with Me Si as the reference and coupling constants (J) are reported in hertz. Mass spectra were
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acquired on a quadrupole orthogonal acceleration time-of-flight mass spectrometer (Synapt G2
HDMS, Waters, Milford, MA). Samples were infused at 3 µL/min and spectra were obtained
in positive or negative ionization mode with a resolution of 15000 (FWHM) using leucine
enkephalin as lock mass. Purity of the compounds was analyzed on a Waters 600 HPLC system
equipped with a Waters 2487 Dual λ absorbance detector set at 256 nm using a 5µm 4.6x150
mm XBridge Reversed Phase (C ) column. The mobile phase was a gradient over 30 minutes
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starting from 95% A and 5% B and finishing at 5% A and 95% B with a flow rate of 1 ml per
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