Structure-Based Design of Selective, Covalent G Protein-Coupled Receptor Kinase 5 Inhibitors

Article abstract of DOI:10.1021/acsmedchemlett.9b00365

The ability of G protein-coupled receptor (GPCR) kinases (GRKs) to regulate desensitization of GPCRs has made GRK2 and GRK5 attractive targets for treating heart failure and other diseases such as cancer. Although advances have been made toward developing inhibitors that are selective for GRK2, there have been far fewer reports of GRK5 selective compounds. Herein, we describe the development of GRK5 subfamily selective inhibitors, 5 and 16d that covalently interact with a nonconserved cysteine (Cys474) unique to this subfamily. Compounds 5 and 16d feature a highly amenable pyrrolopyrimidine scaffold that affords high nanomolar to low micromolar activity that can be easily modified with Michael acceptors with various reactivities and geometries. Our work thereby establishes a new pathway toward further development of subfamily selective GRK inhibitors and establishes Cys474 as a new and useful covalent handle in GRK5 drug discovery.

Full text of DOI:10.1021/acsmedchemlett.9b00365

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Letter
Structure-Based Design of Selective, Covalent
G Protein-Coupled Receptor Kinase 5 Inhibitors
Rachel A. Rowlands, M. Claire Cato, Helen V. Waldschmidt, Renee A. Bouley, Qiuyan
Chen, Larisa Avramova, Scott D Larsen, John J. G. Tesmer, and Andrew D. White
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.9b00365 • Publication Date (Web): 12 Nov 2019
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ACS Medicinal Chemistry Letters
Structure-Based Design of Selective, Covalent G Protein-Coupled Receptor Kinase 5 Inhibitors
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Rachel A. Rowlandsa , M. Claire Catob , Helen V. Waldschmidta,†, Renee A. Bouleyb, Qiuyan Chenc, Larisa Avramovac, Scott D.
Larsena, John J. G. Tesmer c, and Andrew D. Whitea*
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a University of Michigan, Vahlteich Medicinal Chemistry Core, College of Pharmacy, 428 Church St, Ann Arbor, MI 48109, USA
b University of Michigan, Life Sciences Institute, Departments of Pharmacology and Biological Chemistry, 210 Washtenaw Ave,
Ann Arbor MI 48109, USA
c Purdue University, Departments of Biological Sciences and Medicinal Chemistry and Molecular Pharmacology, 915 W State St,
West Lafayette, IN 47907
Keywords: covalent inhibitor, kinase inhibitor, GPCR kinase, heart failure
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ABSTRACT: The ability of G protein-coupled receptor (GPCR) kinases (GRKs) to regulate desensitization of GPCRs has made
GRK2 and GRK5 attractive targets for treating heart failure and other diseases such as cancer. Although advances have been made
towards developing inhibitors that are selective for GRK2, there have been far fewer reports of GRK5 selective compounds. Herein,
we describe the development of GRK5 sub-family selective inhibitors, 5 and 16d that covalently interact with a non-conserved
cysteine (Cys474) unique to this sub-family. Compounds 5 and 16d feature a highly amenable pyrrolopyrimidine scaffold which
affords high nanomolar to low micromolar activity that can be easily modified with Michael acceptors with various reactivities and
geometries. Our work thereby establishes a new pathway towards further development of sub-family selective GRK inhibitors and
establishes Cys474 as a new and useful covalent handle in GRK5 drug discovery.
Many cellular events are modulated in response to extracel-
lular signals by G protein-coupled receptors (GPCRs)1. GPCR
kinases (GRKs) selectively recognize and phosphorylate acti-
vated GPCRs, leading to their desensitization and internaliza-
tion, which is critical for a normal return to cellular homeosta-
sis2. Based on their phylogeny, the seven mammalian GRKs are
divided into the GRK1 (GRK1 and 7), GRK2 (GRK2 and 3),
There is growing evidence that GRK2 and GRK5 have dis-
tinct pathological roles within the failing heart21–25. Increased
GRK2 levels are thought to mediate the decrease in cell-surface
βARs and the prolonged sympathetic nervous system activa-
tion, leading to decreased contractility22. GRK5 is unique
among GRKs in that it undergoes Ca2+·calmodulin-dependent
nuclear localization that allows GRK5 to translocate into the
nucleus where it phosphorylates histone deacetylase 5, turning
on the transcription of hypertrophic genes24. Indeed, cardiac-
specific GRK2 knockout mice have improved contractility and
increased cell-surface βARs post-myocardial infarction16, and
GRK5 knockout mice are protected from cardiac hypertrophy
following controlled cardiac stress. The extent of the functional
differences in GRK2 and GRK5 within cardiomyocytes re-
mains to be elucidated, but selective inhibition of each of these
kinases would offer the opportunity to further understand their
distinct roles in the progression of heart failure. In addition, the
selective inhibition of GRK2 or GRK5 presents the possibility
of treating different aspects of heart failure without affecting
the entire cardiac regulatory system.
3
and GRK4 (GRK4, 5, and 6) subfamilies . GRK1 and 7 are
expressed primarily in the retina and GRK4 in the testes,
whereas GRK2, 3, 5, and 6 are more ubiquitously expressed.4
Among the GRKs, the β-adrenergic receptor (βAR) kinases
(GRK2 and GRK3) are the most widely studied, due to their
role in various disease states such as cardiovascular disease,
cancer and inflammation.5,6 Although GRK5 has been studied
for its role in multiple disease states including cancer, neuro-
degeneration, type 2 diabetes and cardiovascular disease, few
examples of GRK5 selective inhibitors are found in the litera-
ture.7,8 Given its cross-functionality, a selective and potent
probe is needed to further investigate the role of GRK5 in the
disease states it is implicated in.
Despite high structural similarity in the active site among
GRKs, we have had success in developing potent and selective
small molecule inhibitors for the GRK2 subfamily that improve
contractility in isolated adult mouse cardiomyocytes, in part be-
cause GRK2, and presumably GRK3, adopts a distinct inactive
pose from other GRKs and other protein kinases26–29. Compari-
son of GRK2 and GRK5 crystal structures revealed a more spa-
cious ATP-binding pocket in GRK2/3 that was able to accom-
modate bulkier chemical substituents, which allowed us to build
out GRK5 binding26. We have also developed pan GRK-
selective compounds (CCG-215022 and CCG-258748) with na-
nomolar potency for both GRK2 and GRK526,27,29, but thus far
we have not been successful in developing GRK5 selective (or
GRK4 subfamily selective) inhibitors using the canonical re-
versible binding model. Others have reported GRK4 subfamily
selective compounds, but they are also potent inhibitors of other
kinase families8 and have not been independently confirmed.
Cardiac function is controlled in part by βARs. Under physi-
ological conditions, βARs at the cardiomyocyte cell surface are
activated in response to increased circulating levels of the fight-
or-flight hormones, epinephrine and norepinephrine, leading to
an increase in cardiac output9. GRK2 and GRK5, the predomi-
nant GRKs expressed in the heart, then regulate signal termina-
tion through phosphorylation leading to subsequent internaliza-
tion of the βARs10. In the failing heart, epinephrine and norepi-
nephrine levels remain high in an attempt to compensate for de-
creased cardiac output11. Although initially beneficial for in-
creasing heart contractility, prolonged exposure to catechola-
mines exacerbates the problem as evidenced by increased
GRK2 and GRK5 levels, a decreased number of βARs at the
cell surface, and initiation of a pathological hypertrophic stress
response12. βAR antagonists (β-blockers) are used to treat heart
failure, but there is growing evidence that inhibition of GRK2,
GRK5, or both could improve the currently available heart fail-
ure therapies13–20
.
Examination of the crystal structure of GRK6 in what is be-
lieved to be an active conformation (PDB 3NYN30) revealed
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that the thiol group of Cys474, located within the flexible active
Page 2 of 7
1
site tether (AST) loop of the kinase domain, is positioned adja-
cent to the ATP-binding site, at least when the kinase adopts a
more closed conformation (Figure 1A). Because this cysteine
is unique to GRK4 subfamily members it could be exploited as
a handle for covalent inhibition to gain selectivity for GRK5
over GRK2. In recent years, the popularity of covalent war-
heads has risen because they offer the possibility of more po-
tency and selectivity than reversible inhibitors31. In particular,
specifically targeting non-conserved cysteines in the ATP-
binding pocket of kinases has demonstrated utility31–33. The
most successful irreversible modifiers have come from design-
ing a reversibly-binding compound with low or sub-µM affinity
to also contain a covalent warhead that is within reach and in
the proper orientation to interact with the free thiol of a nearby
cysteine. The most widely used reaction to achieve irreversible
covalent attachment onto a cysteine is a Michael addition using
electrophilic warheads such as acrylamides, vinyl sulfones, and
alkynes33. Recent advances have also been made with the use
of an N,N-dimethyl-butenoic amide, which improves solubility
and contains an internal base that can deprotonate and activate
the thiol group.33–35
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Figure 1. GSK2163632A and related compounds suggest a route to selective
inhibition of GRK5 via covalent modification of Cys474. (A) GSK2163632A
(green) bound to GRK1 (yellow, PDB entry 4PNI) superimposed with the active
conformation of GRK6 (blue, PDB entry 3NYN) (B) Previously identified pyr-
rolopyrimidine based GRK inhibitors and covalent inhibitor design rationale.
Green ovals highlight the base of the scaffold that is generally conserved, and
orange circles represent a basic nitrogen that was removed except in the case of
analogs where it is replicated potentially by a N,N-dimethyl-butenoic amide.
In a previous screen, GSK2163632A was identified as a mod-
estly potent GRK5 inhibitor (IC50 = 3.2 µM) with high potency
for GRK1 (IC50 = 130 nM) and lower potency for PKA (IC50
>
500 µM). Two related compounds, GSK1713088A and
GSK1326255A (Figure 1B), were shown to have similar po-
tency for GRK5 (IC50 = 3.2 and 2.5 µM respectively), but also
modest selectivity over both GRK1 and GRK2.36 All three GSK
compounds share a common pyrrolopyrimidine core, which
binds within the ATP pocket with the nitrogens from the core
forming hydrogen bonds with the hinge of the kinase domain in
a donor-acceptor-donor motif, as observed for GSK2163632A
in complex with GRK1 (PDB entry 4PNI, Figure 1A). We
therefore hypothesized that we could append covalent warheads
to the methoxyphenyl of GSK1713088A or GSK1326255A that
could react with Cys474 (Figure 1B). To avoid a highly substi-
tuted ring, we replaced the tertiary amine appendages with our
covalent warheads in hopes that the potency gained by the co-
valent bond would overcome the loss of a hydrogen bond ac-
cepted by the tertiary amine. The N,N-dimethyl-butenoic amide
warhead would, however, place a basic tertiary amine in a sim-
ilar position.
respectively. Tosyl protection of commercially available 4, 6 –
dichloropyrrolopyrimidine S1 followed by base catalyzed aro-
matic substitution with 2- aminobenzamide gave S3.37 Acid
promoted lactam cyclization activated the 2-chloro for substi-
tution with either aniline S8 or S12 to give lactams S4 and S9.
Hydrolysis using ammonium hydroxide afforded amides S5 and
S10. Tosyl deprotection followed by amide coupling provided
analogs 4-9.
The para and meta ethylamide controls, 4 and 7 respectively,
showed substantially different biochemical results indicating a
possible structure-activity-relationship (SAR) cliff. The para
substituted ethyl amide 7 showed no activity up to 100 µM
against all GRKs tested, whereas 4 showed low µM inhibition
for GRK5 with an IC50 of 5.9 µM. There was no consistent time
dependent change in inhibition of GRK5 by 4 or 7 as a function
of time, as expected. The para substituted acrylamide, 5, exhib-
ited an IC50 of 6.2 µM for GRK5 after one hour of preincubation
on ice, with no potency against GRK1 or GRK2. After 4 h, 5
showed a convincing increase in potency as a function of time
consistent with covalent inhibition, with an IC50 of 0.2 µm (Ta-
ble 1, Figure 2). The >16-fold difference in GRK5 potency for
5 relative to its non-covalent control 4 at 1 h also suggests a
covalent mechanism of action. When 5 was also tested using
light activated rod outer segments (ROS) as a substrate instead
of tubulin, the inhibitory potency was comparable across all in-
cubation times (Figure S5). This result is consistent with our
prior GRK inhibitor studies.27,38 The compounds also exhibited
>450 selectivity over GRK2. The meta acrylamide analog 8 had
similar potency for GRK5 at all incubation times with respect
to its non-covalent control 7 suggesting that it is not acting co-
valently. It also showed no inhibition of GRK1 but modest
GRK2 inhibition (IC50 = 39 µM).
We first rationally designed 6 different variants of the GSK
inhibitor series with short amide linkers. We overlaid a GRK5
crystal structure (PDB entry 4WNK) with that of the active con-
formation of GRK6 (PDB entry 3NYN) (Figure 1A). Building
a covalent warhead meta to the aniline (ring C) likely retains
the hydrogen bond of the amide carbonyl that GSK2163632A
forms with the backbone nitrogen of GRK1-Asp271 (GRK5-
Asp270) but this may position the Michael acceptor too distant
from Cys474. Alternatively, building the warhead para to the
aniline would likely hinder the hydrogen bond with Asp271, but
may put the Michael acceptor within 1.5 Å of the Cys474 thiol
group. However, it is not possible to accurately model these in-
teractions because the degree of kinase domain closure and the
conformations of the flexible AST and P loops cannot be pre-
dicted a priori. Therefore, both para and meta substituents were
synthesized, including unreactive saturated ethyl amide analogs
as negative controls (Table 1).
Both 6 and 9, featuring the N,N-dimethyl-butenoic amide in
para and meta positions, respectively, had increased potency
for all three GRKs relative to the acrylamide inhibitors 5 and 8,
indicating that addition of the basic nitrogen augments potency
Synthesis of para analogs 4, 5, and 6 (Table 1) and meta sub-
stituted analogs 7, 8, and 9 are described in Schemes S1 and S2,
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ACS Medicinal Chemistry Letters
Table 1: IC50 Values for Pyrrolopyrimidine Compounds (µM ± SD)
1
2
3
4
5
6
7
8
9
GRK5
C474S
(4 hr)
GRK5
GRK5
GRK5
GRK5
(4 hr)
GRK1
(4 hr)
GRK2
(4 hr)
GRK6
(4 hr)
GRK2/
GRK5§
Compound
R1
R2
R3
(0 min) (30 min) (60 min)
‡GSK
2163632A
‡GSK
-
-
-
3.2
3.2
0.13
13
20
6.3
-
-
-
6.2
2
1
2
3
4
5
6
7
8
9
NA
NA
NA
NA
NA
NA
H
H
H
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
-
-
1713088A
‡GSK
-
-
2.5
> 100
> 100
> 100
7.9
-
-
3
H
1326255A
CCG
262604
CCG
258903
CCG
263045
CCG
262606
CCG
258904
> 100
59 ± 90
0.57±0.5
18 ±10
20 ± 10
> 100
11.3 ± 5
0.30±0.1
5.4 ±8
6.3 ±4
> 100
6.2 ±3
-
> 100
> 100
-
-
NA
>450
2
H
0.22±0.1
-
0.41±0.2
-
>100
H
H
0.35±0.1
5.9 ±4
0.76±0.2 0.68±0.03
-
H
H
>100
-
> 100
> 100
39 ±3
2.7±0.2
>100
>100
-
>100
>17
7
H
H
H
H
5.5 ±4
>100
-
H
CCG
0.22±0.03 0.26±0.03 0.27±0.03
-
2.1±0.6
-
-
10
H
263115
CCG
264561
CCG
264099
CCG
264629
CCG
265328
CCG
265327
-
-
-
-
-
-
-
-
-
-
-
-
>100
78±20
17±10
1.1±0.4
19±20
>100
22±5
4.8±2
>100
7.1±3
-
-
-
-
-
-
-
-
-
-
>100
>100
>100
1.8±2
7.7±6
>100
18±8
2.6±2
>100
2.5±3
>100
NA
>1.3
>6
16a
16b
16c
16d
16e
16f
H
H
H
H
H
CH3
CH3
CH3
CH3
CH3
CH3
-
-
>100
42±40
-
-
>100
-
>100
28±20†
>100
0.7±1
>90
>5
-
-
-
-
-
-
-
-
>100
-
-
-
-
-
-
CCG
265041
CCG
2.9±0.6
3.2±0.8
2.1±2
>100
<0.01
0.1
0.4
NA
>14
H
Figure 2. Time-dependent inhibition of GRK5 by 5 and 16d bu-t not CCG-
16g
H
CH3
265042
215022. Compounds were pre-incubated for times of 0 min (black), 100 min
CCG
(magenta), and 240 min (green). (A) CCG-215022, which has no covalent mod-
-
16h
H
CH3
265044
ifier, does not show a significant change in IC50 (380 nM) over time. (B) 5 and
CCG
265268
(C) 16d exhibit the expected leftward shift in IC50 for covalent inhibitors as pre-
-
16i
H
CH3
incubation times increase, as indicated by the blue arrows. Each curve is the
CCG
average of either 3 (215022 and 16d) or 4 (5) experiments. Error bars represent
-
>100
16j
standard deviation.
H
CH3
265267
Experimental values derived from this report were run three times in duplicate with the exception of assays involving 5 against GRK5, GRK6, and GRK5-C474S with
4 hr incubations, which were performed two times in duplicate. Inhibitor incubation times are given in parentheses.
‡From Homan K. et al.38. Note that these compounds were not assayed after 4 hr incubations and are listed here for comparison purposes only.
§Selectivity for GRK5 over GRK2 based on IC50 ratio. The IC50 value for the longest incubation with GRK5 was used for the calculation.
† Measured at 100 min.
against all three kinases. The para substituted analog, 6, was
predicted to be closer to the AST loop, and thus more likely to
form a covalent bond with Cys474. In our time dependent inhi-
bition of GRK5, it shows some improvement from 0 min to 1 h
(GRK5 IC50 from 0.57 to 0.35 µM, respectively) but exhibits no
selectivity over GRK1 or GRK2 (IC50 = 0.76 and 0.68 µM, re-
spectively). The meta substituted analog, 9, did not show time
dependent GRK5 inhibition (IC50 range = 0.22 – 0.27 µM), alt-
hough it showed ~10-fold selectivity for GRK5 over GRK1 and
GRK2 (IC50 = 2.1 and 2.7 µM, respectively). This difference in
GRK selectivity between the meta and para substituted analogs
6 and 9 indicates that the position of the amide linked append-
age from the methoxyphenyl ring is one route for gaining
GRK5 selectivity. Despite their GRK5 potency 6 and 9 were
not further pursued due to their lack of selectivity against GRK1
and 2.
We next tested whether homologation of the covalent war-
head linkers to the methoxyphenyl ring would allow them to
better engage Cys474 by adding in an additional rotatable bond.
For each series of para and meta-substituted analogs we ex-
panded our search to test five unique covalent warheads of var-
ying softness (acrylamides through vinyl sulfones; Table 1). In
Scheme S3, the benzamide on Ring A was methylated to avoid
an acid catalyzed intramolecular ring closure.37 Synthesis of
16a-j starts with SEM protection of 4,6-dicholopyrrolopyrimi-
dine (10) to give starting material 11. Compound 11 underwent
a base catalyzed aromatic substitution to give 12. Using a tradi-
tional Buchwald-Hartwig cross coupling with 2-methoxy-4-cy-
anoaniline, compound 13 was achieved. From 13, the nitrile
was reduced using Raney Nickel in methanolic ammonia to
give precursor 14. Using standard amide coupling conditions
compounds 15a-e were achieved in moderate yields (50-80%).
Final compounds 16a-e were then produced by acid catalyzed
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Daltons
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Figure 3. Evidence from intact mass spectrometry for covalent modifica-
tion of GRK5 by 5, 16b, and 16d. (A) GRK5 only (black trace), GRK5·5
(purple). (B) MS traces for GRK5 (black trace), GRK5·16d (ocean blue),
and GRK5·16b (pink). The latter trace indicates that 16b only partially
labeled GRK5.
amide of 16c (IC50 = 17 µM). The para-alkyne (16d) was the
most potent and selective GRK5 inhibitor from this series (IC50
= 1.1 µM, 90-fold selectivity over GRK2 after 4 h incubation).
16d also demonstrated comparable inhibitory potency for
GRK5/6 when using light-activated ROS as a substrate instead
of tubulin (Figure S5).
Figure 2. Time-dependent inhibition of GRK5 by 5 and 16d but not CCG-
215022. Compounds were pre-incubated for times of 0 min (black), 100 min
(magenta), and 240 min (green). (A) CCG-215022, which has no covalent mod-
ifier, does not show a significant change in IC50 (380 nM) over time. (B) 5 and
(C) 16d exhibit the expected leftward shift in IC50 for covalent inhibitors as pre-
incubation times increase, as indicated by the blue arrows. Each curve is the
average of either 3 (215022 and 16d) or 4 (5) experiments. Error bars represent
standard deviation.
Interestingly, potency for GRK2 was lost for all compounds
with the para-substitution. It is possible that the slightly longer
AST-loop in the GRK2 subfamily allows it to reach further in
the active site, as seen for residues 476-479 in the GRK2-
Gβγ·GSK180736A co-crystal structure (PDB entry 4PNK),
which would collide with substituents in the para-position.
SEM deprotection. The meta series of compounds 16f-j were
accomplished through a modified route (Scheme S4). Coupling
partner 17 was achieved through reduction of the amide (18) to
the 3-amino-aniline, 19. Compound 19 was then protected with
Cbz-chloride to then yield 17 in 75% yield. Then, using cou-
pling partners 12 & 17, 13a′ was achieved via a standard Buch-
wald coupling. Precursor 14a′ was achieved by a reductive Cbz
deprotection. From 14a′ compound 15f-j and 16f-j were fur-
nished using the same coupling and deprotection conditions
used in Scheme S4.
Overall, the meta-substituted compounds (16g-j) tended to
have higher potency against GRK5 but lower or even reversed
selectivity versus GRK2 (the exception being the vinyl sulfone
16j). Small polar groups, specifically those with hydrogen bond
acceptor capability, were well tolerated in GRK5. For example,
both 16g and 16j inhibited GRK5, but the polar vinyl sulfone
of 16j was more potent (IC50 = 7.1 μM) than the more lipophilic
acrylamide of 16g (IC50 = 22 µM). The meta-alkyne (16i) had
no potency for GRK5, suggesting that this more rigid covalent
modifier may collide with the P-loop or AST-loop. The 16h
compound had low µM potency for GRK5.
We then determined IC50 values of the homologated com-
pounds after a 4 h incubation for GRK2, GRK5, and GRK6
(Table 1). GRK6 was included as a positive control for GRK4
subfamily selectivity. As in the non-homologated series, the
ethylamide compounds (16a and 16f) showed an SAR cliff,
with the para ethylamide unable to inhibit any of the GRKs
tested. The reactive para-substituted series (16b-e) were more
selective for GRK5/6 over GRK2 than their meta analogs with
the exception of 16e. GRK5 tolerated both the small substitu-
ents of the acrylamide 16b (IC50 = 78 µM) and vinyl sulfone,
16e (IC50 = 19 µM) but also the large N,N-dimethyl-butenoic
Although these compounds selectively inhibit GRK5, they
have only modest potency. Our two most potent analogs, 5 and
16d, have IC50 values of 0.22 and 1.1 µM, respectively, after 4
hour incubation using tubulin as a substrate (Table 1). We at-
tribute this to one of two reasons. Either the core scaffold has a
suboptimal engagement with the hinge region, or the entropic
cost of locking down the flexible AST loop via a covalent bond
is high enough to limit the binding affinity.
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Figure 4. Evidence for GRK5-Cys474 covalent engagement of 16d. (A) MS trace for GRK5, (B) MS trace for GRK5 incubated with 16d indicating covalent
modification, (C) MS trace for GRK5-C474S, (D) MS trace for GRK5-C474 incubated with 16d indicates that 16d cannot covalently modify GRK5-C474S. Each
peak shown is representative of n = 3 experiments.
Of the homologated series, only 16g, 16h and 16f showed
activity against GRK2. Our modeling suggested that shorter
warheads may be more easily accommodated within the shal-
low GRK2 ribose pocket. All the other meta-substituted mate-
rials have larger, less flexible warheads. Our most GRK2 selec-
tive analog, 16f, has only modest potency compared to previ-
ously developed GRK2 inhibitors, and we therefore are not pur-
suing it as a GRK2 lead compound.26,29
to install para-substituted linkers that can interact covalently
with the AST from GRK5 but not from GRK2. Additionally,
we have shown that Cys474 is selectively targeted by our cova-
lent warheads, validating our design strategy. Moving forward,
we aim to further improve the potency of these compounds
against GRK5 and GRK6, and to pursue crystal structures of
the GRK5/6·5 and 16d complexes to confirm their binding
poses and the effect of covalent modification on the overall con-
formation of GRK5. Before examining the effects of our com-
pounds in vivo on cardiomyocyte contractility and, ultimately,
to parse the role of GRK5 in heart failure, these compounds will
also need to be tested for selectivity against other protein ki-
nases.
As expected, inhibition of GRK6 was similar to that of GRK5
in most cases, the exception being 16d, which was unable to
inhibit GRK6. It is unclear whether this represents true intra-
subfamily selectivity or a vagary of the experimental conditions
for this combination. The most potent compound 16d was then
additionally tested at 0 and 100 min incubations to assess if
there was a time-dependent decrease in IC50, the hallmark of
covalent inhibition. Indeed, 16d displayed a marked increase in
apparent potency as a function of time (Figure 2). Further con-
firmation of covalent inhibition of GRK5 was achieved through
intact protein mass spectrometry (MS). Compounds 5 and 16a-
e were tested, but only 5, 16b and 16d showed significant
amounts of covalent linkage after a 3 h incubation (Figure 3,
S1), consistent with the results of our radiometric assays. Com-
pounds 16f-j were also tested but showed no covalent inhibition
(Figure S2).
ASSOCIATED CONTENT
Supporting Information
Experimental procedures and supplementary figures are pro-
vided in the Supporting Information.
The Supporting Information is available free of charge on the
ACS Publications website.
AUTHOR INFORMATION
Corresponding Author
For 5, 16b and 16d, we also tested whether inhibition is af-
fected when Cys474 is mutated to serine (GRK5-C474S). A de-
crease in GRK5-C474S potency relative to wild-type GRK5
would thus be consistent with a covalent inhibition mechanism.
Compound 5 lost all potency against the mutant protein,
whereas 16b and 16d retained comparable activity. The reason
is unclear, but a structure of GRK5-C474S bound to 16d would
help resolve that discrepancy by illuminating how this particu-
lar compound interacts with the Ser474 point mutation.
*Phone: 734-647-7374; E-mail: whitandd@umich.edu
Present Addresses
†HVW current address: 130 Scripps Way, Jupiter, FL 33458,
USA
Author Contributions
The manuscript was written primarily by RAR, MCC, and
JGGT. All authors have given approval to the final version of
the manuscript. HVW synthesized compounds 4-9 and deter-
mined their IC50 values for GRKs 1, 2, and 5. RAR synthe-
sized compounds 16a-j and performed MS experiments. MCC
determined IC50 values for GRK2, 5, 6, and GRK5-C474S.
MCC purified GRK2. RAB purified GRK5, 6, and GRK5-
C474S and contributed some of the IC50 dose response experi-
ments. QC and LA performed ROS phosphorylation assays.
We next tested whether 16d engaged GRK5 in a covalent
bond specifically at Cys474 using intact protein MS and
showed that GRK5-C474S mutant does not react (Figure
4).Using tandem mass spectrometry (MS/MS) we further ob-
served that 16d labels Cys474 but also a cysteine located in a
solvent-exposed position in its regulator of G protein signaling
homology domain, remote from the active site (Figure S3-4).
This additional labelling event is indicative of a concentration-
dependent covalent engagement and we do not believe that its
presence is indicative of a biologically relevant interaction.
Funding Sources
This work was supported by NIH grants HL071818 and
HL122416 and the Walther Cancer Foundation (to JJGT),
American Heart Association grants 15PRE22730028 (to
HVW) and 19POST3445019 (to QC), and a pilot grant from
the University of Michigan Center for Discovery of New Med-
icines (to ADW). MCC acknowledges training grant support
In summary, we report what we believe to be the first exam-
ples of covalent inhibitors of GRK5, including some that are
GRK5 sub-family selective (5 and 16d) with high nM to low
µM potency. We have leveraged the pyrrolopyrimidine scaffold
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from the University of Michigan Chemistry–Biology Interface
(CBI) training program (NIH grant 5T32GM008597).
(10)
Lohse, M. J.; Engelhardt, S.; Eschenhagen, T. What Is
the Role of β-Adrenergic Signaling in Heart Failure?
Circ. Res. 2003, 896–906.
https://doi.org/10.1161/01.RES.0000102042.83024.C
A.
Triposkiadis, F.; Karayannis, G.; Giamouzis, G.; Skou-
larigis, J.; Louridas, G.; Butler, J. The Sympathetic
Nervous System in Heart Failure. J. Am. Coll. Cardiol.
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Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENT
We thank Dr. Venky Bashar, of the University of Michigan Pro-
teomic Resource Facility for assistance with tandem mass spec-
trometry efforts. We also thank Dr. Pil Lee for her technical
assistance in building a GRK5 homology model.
2009,
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https://doi.org/10.1016/j.jacc.2009.05.015.
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Port, J. D.; Bristow, M. R. Altered Beta-Adrenergic Re-
ceptor Gene Regulation and Signaling in Chronic Heart
Failure. J. Mol. Cell. Cardiol. 2001, 33 (5), 887–905.
https://doi.org/10.1006/jmcc.2001.1358.
Rockman, H. A.; Chien, K. R.; Choi, D.-J.; Iaccarino,
G.; Hunter, J. J.; Ross, J.; Lefkowitz, R. J.; Koch, W. J.
Expression of a -Adrenergic Receptor Kinase 1 Inhibi-
tor Prevents the Development of Myocardial Failure in
Gene-Targeted Mice. Proc. Natl. Acad. Sci. 1998, 95
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ABBREVIATIONS
GPCR, G protein-coupled receptor; GRK, G protein-coupled
receptor kinase; βAR, β-adrenergic receptor; AST, active site
tether; P-loop, phosphate-binding loop; MS, mass spectrometry
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