Structural determinants influencing the potency and selectivity of indazole-paroxetine hybrid G protein-coupled receptor kinase 2 inhibitors

Article abstract of DOI:10.1124/mol.117.110130

G protein-coupled receptor kinases (GRKs) phosphorylate activated receptors to promote arrestin binding, decoupling from heterotrimeric G proteins, and internalization. GRK2 and GRK5 are overexpressed in the failing heart and thus have become therapeutic

Full text of DOI:10.1124/mol.117.110130

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Structural Determinants Influencing the Potency and Selectivity of Indazole-Paroxetine
Hybrid G Protein-Coupled Receptor Kinase 2 Inhibitors
Renee Bouley, Helen V. Waldschmidt, M. Claire Cato, Alessandro Cannavo, Jianliang Song,
Joseph Y. Cheung, Xin-Qiu Yao, Walter J. Koch, Scott D. Larsen, and John J. G. Tesmer
Life Sciences Institute (R.B., H.V.W., M.C.C., and J.J.G.T.), Departments of Medicinal
Chemistry (H.V.W., S.D.L., J.J.G.T.), Pharmacology (R.B. and J.J.G.T.), Biological Chemistry
(
M.C.C., J.J.G.T.), and Vahlteich Medicinal Chemistry Core, College of Pharmacy (H.V.W. and
S.D.L.), University of Michigan, Ann Arbor, Michigan. Department of Chemistry, Georgia State
University (X.Y.), Atlanta, Georgia. Center for Translational Medicine, Temple University
(A.C., J.S., J.Y.C, and W.J.K.), Philadelphia, Pennsylvania.
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Running Title Page
a) Indazole-Paroxetine GRK2 Inhibitor Structural Analysis
b) Corresponding author:
John J. G. Tesmer
2
10 Washtenaw Ave Rm 3435
Ann Arbor, MI 48198
Tel. 734-615-9544
Fax 734-763-6492
Email: tesmerjj@umich.edu
c) Number of text pages: 33
Number of tables: 5
Number of figures: 5
Number of schemes: 3
Number of references: 32
Number of words in abstract: 244
Number of words in introduction: 750
Number of words in discussion: 877
d) List of nonstandard abbreviations: AUC, area under the curve; BSA, buried surface area;
GPCR, G protein-coupled receptor; GRK, G protein-coupled receptor kinase; MLM,
mouse liver microsomes; PCA, principal component analysis; PK, pharmacokinetics;
PKA, protein kinase A; ROCK1, Rho-associated coiled-coil containing kinase 1; r.m.s.d.,
root mean squared deviation.
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Abstract
G protein-coupled receptor kinases (GRKs) phosphorylate activated receptors to promote
arrestin binding, decoupling from heterotrimeric G proteins, and internalization. GRK2 and
GRK5 are overexpressed in the failing heart and thus have become therapeutic targets.
Previously, we discovered two classes of GRK2-selective inhibitors, one stemming from
GSK180736A, a ROCK1 inhibitor, and the other from paroxetine, a selective serotonin-reuptake
inhibitor. These two classes of compounds bind to the GRK2 active site in a similar
configuration, but contain different hinge-binding “warheads”: indazole and benzodioxole,
respectively. We surmised from our prior studies that an indazole would be the stronger hinge
binder, and would impart increased potency when substituted for benzodioxole in paroxetine
derivatives. To test this hypothesis, we synthesized a series of hybrid compounds that allowed us
to compare the effects of inhibitors that differ only in the identity of the warhead. The indazole-
paroxetine analogs were indeed more potent than their respective benzodioxole derivatives but
lost selectivity. To investigate how these two warheads dictate selectivity, we determined crystal
structures of three of the indazole hybrid compounds (CCG224061, CCG258284, and
CCG258748) in complex with GRK2–Gbg. Comparison of these structures with those of
analogous benzodioxole-containing complexes confirmed that the indazole-paroxetine hybrids
form stronger interactions with the hinge of the kinase, but also stabilize a distinct conformation
of the kinase domain of GRK2 compared to previous complexes with paroxetine analogs. This
conformation is analogous to that which can be assumed by GRK5, at least partially explaining
the loss in selectivity.
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Introduction
Heart failure is broadly described as an inability of the heart to effectively pump and
supply blood to the body. In response to insufficient blood flow, the sympathetic nervous system
produces increased amounts of catecholamines to stimulate β-adrenergic receptors, which are
part of the G protein-coupled receptor (GPCR) superfamily (Salazar et al., 2007; Belmonte and
Blaxall, 2011). However, the prolonged stimulation of GPCRs initiates the upregulation and
phosphorylation by GPCR kinases (GRKs), which targets these receptors for arrestin binding and
internalization (Ferguson et al., 1996; Ribas et al., 2007). Receptor internalization reduces the
number of receptors present on the cell surface, thus diminishing the responsiveness to agonists
(
Ferguson et al., 1996).
There are seven different GRKs found in humans, which are divided into the GRK1
GRK1 and 7), GRK2 (GRK2 and 3), and GRK4 (GRK4, 5, and 6) subfamilies, all of which
(
belong of the protein kinase A, G, and C (AGC) family of Ser/Thr protein kinases. GRKs 2 and 5
are the dominant GRKs in the myocardium and are upregulated during heart failure (Dzimiri et
al., 2004; Montó et al., 2012). A series of studies have shown that selective inhibition of GRK2
activity is a promising approach to improve cardiac function during heart failure (Rockman et
al., 1998; Raake et al., 2013; Schumacher et al., 2015).
We previously identified paroxetine, an FDA-approved serotonin-reuptake inhibitor, and
GSK180736A (Figure 1) as GRK2 inhibitors (Thal et al., 2012; Homan et al., 2015). Co-crystal
complexes of these compounds bound to GRK2 revealed that they both bind similarly in the
active site (Figure 2a) (Thal et al., 2012; Homan et al., 2015). The A rings of both compounds
occupy the adenine subsite and bind the hinge (Figure 2a). The core of the scaffold (ring B),
either a dihydropyrimidine (GSK180736A) or piperidine (paroxetine), occupies the ribose
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subsite while the C ring occupies the polyphosphate subsite. However, neither occupy the
hydrophobic pocket of the active site as was observed with the Takeda compound (Figure 2b)
known as CMPD101 or compound 115h (Ikeda et al., 2007; Thal et al., 2011; Okawa et al.,
2
017). We subsequently used a rational drug design approach to generate derivatives of both
GSK180736A and paroxetine with substituents appended onto the C ring via an amide linker in
order to occupy the hydrophobic pocket (Waldschmidt et al., 2016, 2017).
We hypothesized that due to the similar binding modes of GSK180736A and paroxetine
(
Figure 2), their structure-activity relationships would be translatable. Although this turned out
generally not to be the case, overall the addition of amide-linked D-ring substituents to these
scaffolds did lead to increases in potency in each class. In the case of GSK180736A, several
substituents enabled us to build in selectivity for GRK2 over other kinases, especially Rho-
associated coiled-coil containing kinase 1 (ROCK1) for which this compound was originally
designed to inhibit (Sehon et al., 2008). We were also interested in understanding if the hinge-
binding moiety, an indazole in the case of GSK180736A and a benzodioxole in the case of
paroxetine, was interchangeable. However, it was observed in the case of the GSK180736A
scaffold that exchanging the indazole for a benzodioxole resulted in a severe loss of activity
(Waldschmidt et al., 2016).
We next hypothesized that by exchanging the benzodioxole moiety in the paroxetine
scaffold with an indazole would instead result in increased potency due to its stronger
interactions with the hinge (two hydrogen bonds for indazole versus a hydrogen bond and a
carbon-oxygen hydrogen bond for benzodioxole). Thus, we synthesized a series of paroxetine
hybrids in which the benzodioxole ring was exchanged for indazole. These indazole-paroxetine
hybrids in general showed an increase in GRK2 potency relative to their benzodioxole
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counterparts, however lost selectivity. To understand the molecular basis for enhanced potency
and loss of selectivity, we crystallized several of these hybrid compounds in complex with
GRK2–Gbg. These and prior crystallographic studies show that the indazole warhead locks the
kinase domain into a specific conformation that leads to one particular crystal form, whereas the
benzodioxole-based compounds lead to a variety of crystal forms with subtly different
conformations of the kinase domain. Our results indicate that the identity and strength of the
hinge-binding moiety is important for dictating the overall conformation of the GRK2 kinase
domain, and that weaker hinge interactions allow access to a broader distribution of subtly
different conformational states. The selectivity generally exhibited by benzodioxole-based
compounds for GRK2 may therefore result from the ability of its kinase domain to access
conformational states not readily available to closely related enzymes.
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Materials and Methods
1
Synthesis. Synthetic procedures are provided in the supplemental data. H NMR spectra
were taken in DMSO-d6, MeOD, or CDCl
3
at room temperature on Varian Inova 400 MHz or
1
Varian Inova 500 MHz instruments. Reported chemical shifts for the H NMR spectra were
recorded in parts per million (ppm) on the δ scale from an internal standard of residual
tetramethylsilane (0 ppm). Mass spectrometry data was measured using a Waters Corporation
Micromass LCT or Agilent6230 Q-TOF. HPLC was used to determine purity of biologically
tested compounds on an Agilent 1100 series with an Agilent Zorbax Eclipse Plus−C18 column.
A gradient of 10-90% acetonitrile/water over 6 min followed by 90% acetonitrile/water for 7 min
was used with detection at 254 nm. All tested compounds had purity >95%.
Kinase Assays. PKA and ROCK1 inhibition was assayed using the ADP-Glo Kinase
Assay system (Promega, Madison, WI) as previously described (Homan et al., 2015).
Compounds were tested against PKA in duplicate using an 8-point concentration range and the
experiment repeated in triplicate on separate days. Compounds were screened at 10 µM for
ROCK1 inhibition in triplicate and the experiment repeated in triplicate on separate days and the
percent inhibition was calculated. A BMG Labtech PHERAstar imaging system was used to
measure luminescence. Inhibition of GRK1, 2, and 5 was determined in 20 mM HEPES pH 7.0,
2
2
mM MgCl , and 0.025% DDM with 50 nM of the respective GRK and 500 nM tubulin. Kinetic
3
2
reactions were initiated by the addition of [g- P]ATP (500 µCi, 5 µM), allowed to proceed for 5
min and quenched by the addition of SDS loading buffer. Each compound was tested in duplicate
using an 8-point concentration range and the experiment repeated in triplicate on separate days.
Samples were then separated by SDS-PAGE and gels were dried and exposed with a
phosphorimaging screen. The images were then scanned with a Typhoon imager and quantified
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using ImageQuant, as previously described (Thal et al., 2012). Data was fit to a three-parameter
dose-inhibitor response curve with a fixed Hill slope of 1 and the bottom constrained to 0 using
GraphPad Prism. A maximum of two outliers were removed per individual dataset and in some
cases an entire dataset was excluded from the analysis.
Mouse Liver Microsome Assays. Metabolic stability was determined using CD-1 mouse
liver microsomes. Reactions consisted of 1 µM compound, 0.5 mg/mL microsomes, 1.7 mM
NADPH in 0.1 M phosphate buffer pH 7.4 supplemented with 3.3 mM MgCl
2
, incubated at 37
°
C. Aliquots of 40 µL were taken at 0, 5, 10, 15, 30, 45, and 60 min and quenched in 3 volumes
of cold acetonitrile containing 100 ng/mL internal standard. Samples were centrifuged at 15,000
rpm for 10 min and the supernatant was analyzed by LC-MS/MS.
Protein Expression and Purification. Human GRK2 S670A with a C-terminal
hexahistidine tag was expressed in High-Five cells using the Bac-to-Bac insect cell expression
system (Life Technologies, Carlsbad, CA). Cells were harvested 48 h post infection and lysed.
GRK2 was purified from the clarified lysate as described previously for GRK1 using nickel-
nitrilotriacetic acid affinity and cation exchange chromatography (Singh et al., 2008). Fractions
containing GRK2 were pooled and further purified on a Sephadex 200 column into 20 mM
HEPES pH 7.5, 100 mM NaCl, and 1 mM DTT. Soluble human Gb
an N-terminal (on the Gb unit) hexahistidine tag was expressed using a dual-promoter insect
cell expression vector in High-Five cells. The cells were harvested 48 h post infection and lysed.
Gb was purified from the clarified lysate as described previously using nickel-nitrilotriacetic
acid affinity and anion exchange chromatography (Kozasa, 2004). Fractions containing Gb
1 2
g (C68S mutant) containing
1
1 2
g
1 2
g
were pooled and further purified on a Sephadex 200 column into 20 mM HEPES pH 8.0, 100
mM NaCl, and 1 mM DTT.
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Crystal Structure Determination. Purified GRK2 (S670A) and soluble Gb
were mixed in a 1.2:1 molar ratio with a final protein concentration of approximately 10 mg/mL.
Inhibitor (500 µM) and MgCl (2 mM) were added to the protein mixture and allowed to
1 2
g (C68S)
2
incubate on ice for 30 min prior to filtration through a 0.2 µm Nanosep centrifugal device (Pall
Laboratory, Port Washington, NY). Inhibitor complexes were crystallized as previously
described by hanging drop vapor diffusion at 4 °C with drops consisting of 0.8 µL protein and
0
.8 µL reservoir solution, which consisted of 50 mM MES pH 6.0, 0.8-1.2 M NaCl, and 8-16%
PEG3350 (Thal et al., 2011, 2012). Crystals generally appeared after 2-3 days and grew for 1-2
weeks. Crystals were harvested in a cryoprotectant solution consisting of the reservoir solution
supplemented with 25% ethylene glycol and 500 µM inhibitor before being flash frozen in liquid
nitrogen. Diffraction data was collected on the LS-CAT beamlines 21-ID-G and 21-ID-D at
wavelengths of 0.97857 and 1.0332 Å, respectively. Data integration and scaling was performed
with XDS (Sauter et al., 2013) or DIALS (Gildea et al., 2014). The structures were solved using
Phaser (McCoy et al., 2007) with PDB ID 4PNK as the search model. Reciprocal-space
refinement was performed with PHENIX (Afonine et al., 2012) and alternated with local real-
space refinement and model building using Coot (Emsley and Cowtan, 2004). Crystal refinement
statistics are listed in Supplementary Table 1. The final models were validated using
MolProbity (Chen et al., 2010) prior to deposition in the Protein Data Bank under accession
codes: 5WG3, 5WG4, and 5WG5.
Principal Component Analysis. Principal component analysis was previously performed
for the kinase domains of PKA, GRK1, GRK4 family members, and GRK2 that were previously
deposited on the Protein Data Bank (Yao et al., 2017). The kinase domains were aligned using
core residues which were identified to be structurally invariant. Previous structural analysis of 49
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GRK and 201 PKA structures revealed two distinct structural motions which were grouped into
PC1 (72% of the structural variance) and PC2 (approximately 10% of the structural variance).
These structural motions corresponded to an opening and closing of the kinase domain mediated
by the hinge connecting the small and large lobes (PC1) and a twisting motion between these
domains (PC2). The GRK2 structures from this study were incorporated into the same analysis to
benchmark their conformational states with respect to these previously published structures.
Myocyte Shortening Assays. Mouse cardiomyocytes were isolated from left-ventricular
free wall and septum of C57/Bl6 mice as previously described (Song et al., 2008). Cells were
used within 2-8 h of isolation and plated on laminin-coated coverslips and bathed in HEPES-
2
+
buffered (20 mM, pH 7.4) medium 199 containing 1.8 mM extracellular Ca . Coverslips were
then mounted in the Dvorak-Stotler chamber for recording and bathed in 0.7 mL of fresh
medium. Imaging was performed with a variable field-rate camera (Zeiss IM35, Ionoptix) using
edge detection and sarcomere length. Peak contraction was measured as the percentage of cell
shortening. Cells were paced at 1 Hz and treated with isoproterenol (0.5 µM) for 2 min, with
pretreatment of either PBS as vehicle or CCG224061 (0.5 and 1 µM) for 10 min, and the
contractions recorded (Thal et al., 2012).
Mouse Pharmacokinetic Studies. All animal experiments were approved and conducted
in accordance with standards set by the University of Michigan Committee on Use and Care of
Animals and Unit for Laboratory Animal Medicine (ULAM). Preliminary pharmacokinetics of
CCG224061 were determined in female CD-1 mice following intraperitoneal injection at 10
mg/kg. The compound was dissolved in 15% (v/v) DMSO, 15% (v/v) PEG-400, and 70% (v/v)
PBS. Blood samples of 50 µL were collected at 0.5, 2, 4, and 7 h, centrifuged at 3500 rpm for 10
min, and the plasma frozen at -80 °C for later analysis. Plasma compound concentrations were
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quantified using LC-MS/MS. The LC-MS/MS method consisted of a Shimadzu HPLC system
with a Waters Xbridge-C18 column (5 cm ´ 2.1 mm, 3.5 µm) for chromatographic separation of
the compound. An AB Sciex QTrap 4500 mass spectrometer equipped with an electrospray
ionization source (ABI-Sciex, Toronto, Canada) in the positive-ion multiple reaction monitoring
(MRM) mode for detection. All pharmacokinetic parameters were calculated by
noncompartmental methods using WinNonLin software, version 3.2 (Pharsight Corporation,
Mountain View, CA, USA).
Results
Synthesis.
Paroxetine hybrid compounds in which there is an indazole in place of the benzodioxole
were prepared by a convergent approach (Scheme 1). Intermediate 7b was synthesized from
commercially available 2-fluoro-5-methylphenol 1. Nitration of compound 1 proceeded first
through the nitroso via sulfuric acid and sodium nitrite then nitric acid was used to afford nitro 2
(Flaugh et al., 1979). Amine 3 was readily accessed via palladium reduction of nitro 2.
Acetylation followed by cyclization of the amine with isoamyl nitrite and subsequent hydrolysis
yields 6-fluoro-1H-indazol-5-ol 4b (Iwakubo et al., 2007). Silyl protection of the alcohol gives
5
b. In the presence of catalytic dimethylaminopyridine the indazole is then Boc-protected giving
regioisomers of 6b. Final silyl deprotection gives the free alcohol 7b. Intermediate 7a was
synthesized from the commercially available 1H-indazol-5-ol 4a as described for 7b.
Analogs 11a and 11b were synthesized from intermediate 8 which was obtained as
previously described (Scheme 2) (Waldschmidt et al., 2017). Mesylation of alcohol 8 followed
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by displacement by 7a or 7b gives intermediates 9a/b. Base mediated hydrolysis of 9a/b
followed by amide coupling and Boc deprotection yields analogs 11a/b.
Synthesis of the indazole substituted non-hybrid analogs are shown in Scheme 3. Alcohol
1
2, which has previously been reported (Waldschmidt et al., 2017), was then N-alkylated via two
different methods. The N-ethyl 13a was achieved through substitution using ethyl iodide under
basic conditions. The N-isopropyl 13b was prepared through reductive amination with acetone.
Respective N-substituted analogues (compound 14 is commercially available and 15 has been
previously reported) were then mesylated at the benzylic alcohol which was subsequently
displaced with alcohols 7a or 7b to give intermediates 16a-e. Final Boc deprotection yielded
analogs 17a-e.
Structure-Activity Relationships
To investigate whether binding of paroxetine hybrids could be improved, we exchanged
the benzodioxole moiety for an indazole, analogous to what is present in GSK180736A (Table
1
). CCG224061 showed a 20-fold increase in potency for GRK2 (IC50 = 66 nM) over paroxetine
(IC50 = 1.38 µM) (Thal et al., 2012). However, this change also resulted in increased activity
against GRK1 and 5, PKA, and ROCK1. Although CCG224061 was still selective for GRK2, the
selectivity factor over ROCK1 was only 5-fold, versus more than 50-fold selectivity observed
with paroxetine (Waldschmidt et al., 2017). In an effort to improve selectivity, 2,6-
dimethoxybenzylamide was appended to the fluorophenyl C-ring (CCG232406). Previously, this
adduct gave the largest improvement in the GSK180736A scaffold (Waldschmidt et al., 2016).
Compound CCG232406 indeed showed improvement in GRK2 selectivity over the other GRKs
and PKA, and approximately 20-fold selectivity over ROCK1.
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In a parallel attempt to improve selectivity, we appended two favorable adducts, 2-
pyridylmethylamide and 3-pyrazolylmethylamide, discovered from our previous efforts using the
paroxetine scaffold (Waldschmidt et al., 2017) to generate CCG257284 and CCG258748,
respectively. CCG257284 showed no improvement in GRK2 potency relative to CCG224061 but
resulted in pan-GRK inhibition with potencies similar to the analogous GSK180736A-based
analog (CCG215022) (Waldschmidt et al., 2016). CCG258748 exhibited our most potent GRK2
inhibition to date with an IC50 of 8 nM. Although this compound displayed greater than 30-fold
selectivity over the other GRKs and PKA, the IC50 for GRK5 was still quite potent at 240 nM.
Lastly, we investigated alkylating the piperidine nitrogen of CCG224061 to improve
selectivity (Table 2). N-methylation (CCG258001) resulted in a 4-fold decrease in GRK2
potency but much more dramatic decreases in potency for GRK1, GRK5, and PKA, resulting in
>
100 fold-selectivity for GRK2 over all three kinases. Increasing the size of the methyl to ethyl
and isopropyl (CCG258211 and CCG258746, respectively) was not as well tolerated, in that the
GRK2 potency dropped with increasing substituent size. Therefore, larger alkyl groups were not
explored.
Metabolic Stability
In addition to improving the in vitro potency and selectivity for GRK2 we also aimed to
generate analogs with more favorable pharmacokinetic (PK) properties and thus hopefully
efficacy in vivo because the paroxetine scaffold is much better tolerated than GSK180736A
(
Waldschmidt et al., 2017). The compounds were incubated with mouse liver microsomes
MLM) in order to test for metabolic stability (Table 1). The initial hit compound, paroxetine,
(
had a half-life of 24.1 min in this MLM assay (Waldschmidt et al., 2017). Replacing the
benzodioxole ring with an indazole (CCG224061) resulted in a longer half-life of 30.3 min.
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However, CCG257584 and CCG258748 did not show any improvement in metabolic stability
compared to their respective benzodioxole analogs (CCG211998 and CCG258208). We also
explored fluorination of the C6 position of the indazole ring to improve potency and improve
metabolic stability as this was previously shown to be successful with similar compounds
(Goodman et al., 2007; Sehon et al., 2008). However, addition of a fluoro substituent to the A
ring (CCG258002 and CCG258003) did not improve GRK2 potency and did not have a
significant effect on the metabolic stability (Table 1).
Crystallography
Previously we were successful in obtaining crystals of GRK2–Gbg complexes bound to
three paroxetine analogs containing a benzodioxole warhead and paroxetine (Thal et al., 2012;
Waldschmidt et al., 2017). These complexes crystallized in two different crystal forms (two C2
and one P2), whereas inhibitors based off the GSK180736A scaffold (which contain an indazole
1
warhead) all crystallized in a C222 crystal form (Waldschmidt et al., 2016) (Table S1). A
previously reported benzolactam derivative of paroxetine (PDB ID: 4MK0), also crystallized
with the same packing as the GSK180736A-based compounds (Homan et al., 2013). Therefore,
we hypothesized that the identity of the hinge-binding moiety is critical for not only determining
potency towards GRK2 in these compounds but also dictating the conformation of the kinase
domain and consequently the crystal form. The indazole and benzolactam moieties seem to be
able to form stronger hinge interactions via multiple conventional hydrogen bonds, thereby
stabilizing a consistent kinase conformation that allows GRK2 to crystallize in a reproducible
way (Thal et al., 2012; Homan et al., 2013, 2015).
Consistent with this reasoning, the three indazole-paroxetine hybrids (CCG224061,
CCG258284, and CCG258748) co-crystallized with GRK2–Gbg in this study also crystallized in
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1
the C222 space group. In all three complexes, the indazole forms two hydrogen bonds to the
carbonyl oxygen of Asp272 and the backbone nitrogen of Met274 in the hinge (Figure 3). As
previously observed in other paroxetine complexes, there is an additional highly conserved
hydrogen bond between the piperidine nitrogen of the paroxetine scaffold and the backbone
carbonyl of Ala321. As expected, the D-ring amide linker of CCG258748 and CCG257284 made
additional interactions. In the case of CCG258748, the carbonyl oxygen and amide nitrogen
formed hydrogen bonds with the backbone nitrogen of Gly201 and the sidechain of Asp335,
respectively (Figure 3c). In the case of CCG257284 a hydrogen bond with the sidechain of
Asp335 was not observed due to this residue being shifted slightly away from the ligand. Finally,
in the case of CCG258748 the pyrazole is able to make additional hydrogen bonds with the
sidechains of Glu239 and Lys220 that could explain its high potency (Figure 3c). The pyridine
nitrogen of CCG257284 does not appear to be able to make any interactions with the protein and
is modeled with the nitrogen pointing out of the active site so that it can favorably interact with
solvent (Figure 3b).
Structural Comparisons with Analogous GRK2-Inhibitor Complexes
We have previously published complexes with paroxetine, CCG211998, and CCG258208
(
PDB IDs: 3V5W, 5UKK, and 5UKM, respectively), which are the benzodioxole analogs of the
complexes of CCG224061, CCG257284, and CCG258784, respectively, that we report here
Thal et al., 2012; Waldschmidt et al., 2017). Comparison of these structures thus give insights
(
into how substituting indazole for benzodioxole in the paroxetine scaffold influences the overall
conformation of the small and large lobes of the kinase domain, and therefore clues into the
molecular basis for the remarkable selectivity underlying the benzodioxole-based compounds.
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Alignment of the small lobes (residues 185-271) of the CCG224061 (indazole) and
paroxetine (benzodioxole) complexes yielded a root mean squared deviation (r.m.s.d.) value of
.32 Å for the Ca atoms. The two compounds superimpose almost exactly. Their A rings are
slightly offset but make analogous hydrogen bonds with the hinge (to Asp272 and Met274)
Figure 4a). However, in the case of paroxetine the interaction with Asp272 is weaker because it
0
(
is a CH—O hydrogen bond. In the CCG224061 complex, the backbone of the hinge is pushed
out a maximum of 0.6 Å at Asn275 compared to the paroxetine complex, and the P-loop (b1-b2
turn) exhibits a more closed conformation, exhibiting a 2.1 Å difference at the Ca of Gly201.
Differences were also observed in the b2-b3 and b4-b5 loops of up to 0.6 Å and 0.8 Å,
respectively.
Comparison of the CCG257284 (indazole) and CCG211998 (benzodioxole) complexes
yielded the largest r.m.s.d. value of the three pairs of complexes (0.70 Å). This likely reflects the
unusual space group of the CCG211998 complex (P2). However, the two compounds bind
similarly and their protein-ligand interactions are conserved (Figure 4b). The largest differences
are observed in the A and D rings. The indazole forms stronger interactions with the hinge via
shorter hydrogen bonds with the backbone of Met274 and Asp272 (3.3 and 2.8 Å versus 3.6 and
3
.4 Å, respectively). The tighter interaction between the indazole and hinge is paired with a shift
of the Ca of Asp272 by 0.7 Å. This propagates into a shift of the b2-b3 and b4-b5 loops of 1.0 Å
and 0.9 Å, respectively. The P-loop is shifted upwards in the CCG257284 complex by up to 1.0
Å (for the Ca of Gly201) and the aB-aC loop is pushed outwards by a maximum of 1.3 Å.
Additionally, a large portion of the active-site tether (AST, residues 493-500) is ordered in the
CCG257284 complex relative to the CCG211998 complex.
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The CCG258748 (indazole) and CCG258208 (benzodioxole) complexes displayed a
r.m.s.d. deviation of 0.41 Å for the Ca atoms of the small lobe. The compounds bind similarly
Figure 4c), although the hydrogen bonds with the hinge are again stronger (shorter) in the case
(
of CCG258748 (distances of 3.7 and 2.9 Å versus 3.8 and 3.1 Å, respectively). The hinge is
displaced a maximum of 0.5 Å away from the active site in CCG258748 compared to the
CCG2528208 complex. Additionally, the 3-pyrazolylmethylamide shows slightly different
conformation and interactions in the hydrophobic subsite. Although in both CCG258748 and
CCG258208 the two pyrazole nitrogens form hydrogen bonds with Lys220 and Glu239,
CCG258208 forms an additional contact with Asp335 via its amide linker. The P-loop is shifted
up to 0.6 Å at Gly201 away from the active site in the CCG258748 complex. Hinge interactions
formed by CCG258748 also induce up to a 0.8 Å change of the b4-b5 loop, and a large shift in
the b2-b3 loop (1.2 Å for the Ca of Asp212). There is a smaller change in the aB-aC loop
compared to other analogous complexes, with a 0.7 Å shift for the Ca of Glu233. Additionally,
the highly mobile AST is more ordered in the CCG258748 complex, allowing for an additional 5
residues (491-496) to be modelled.
The buried surface area (BSA) of each of these six ligands and their hinge-binding
moieties was then compared (Table 5). In general, the BSA of the indazole warhead was greater
than that of benzodioxole. However, the entire BSA of CCG258748 and CCG257284 was less
than observed with their respective benzodioxole analogs (CCG258208 and CCG211998,
respectively), due to the differences in packing of the D rings and outward movements of the
aB-aC loop.
Principal component analysis (PCA) was then performed to plot the conformation of the
new ligand complexes in the 2D space spanned by PC1 and PC2, in which PC1 represents a
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concerted opening/closing of the kinase domain describing the largest variance across published
GRK structures and PC2 is a twisting motion representing smaller structural variance (Yao et al.,
2
017). This approach allows us to analyze in an unbiased way the global conformational changes
of the kinase domain in response to binding different classes of inhibitors (in this case indazole
vs. benzodioxole warheads). As expected, this analysis showed that the GRK2 complexes with
the indazole-paroxetine hybrids (CCG224061, CCG257284, and CCG258748) all cluster closely
together with several previously determined indazole-containing complexes (Figure 5). More
specifically, all GRK2 complexes with indazole-containing compounds display similar PC1
compositions but exhibit greater variance along the PC2 coordinate (especially for entries 5HE0
and 5HE3). The GRK2 complexes with CCG224061 and CCG257284 showed almost identical
PC1/PC2 compositions and ended up very close to the CCG224406 (a GSK analog, entry 5HE2)
and GSK180736A (entry 4PNK) complexes. The GRK2 complex with CCG258748 was closest
to 4MK0 (complex with a benzolactam-paroxetine analog) in PC space. On the other hand, the
various benzodioxole complexes inhabit a much broader spectrum of conformational space. The
CCG211998 (5UKK) complex showed the smallest displacement from the mean along PC1,
perhaps consistent with the fact that this was the only complex thus far to crystallize in the space
group P2.
Myocyte Shortening Assays
To investigate how indazole-paroxetine hybrids perform in a more in vivo environment,
we tested CCG224061 with mouse cardiomyocytes and measured their ability to produce a
contraction. The cardiomyocytes were harvested from adult mice and incubated with varying
does of the inhibitors followed by a dose of the b-AR agonist isoproterenol. Paroxetine needed to
be given at a dose of 10 µM in order to produce a significant response, whereas GSK180736A
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showed a similar response at 1 µM (Thal et al., 2012; Waldschmidt et al., 2016) consistent with
its 10-fold improvement in potency over paroxetine measured in vitro. Similarly, CCG224061
produced a significant increase in the maximum contraction response when dosed at 1 µM,
consistent with its 20-fold higher potency for GRK2 compared to paroxetine (Table 3).
Mouse Pharmacokinetic Studies
CCG224061 was then evaluated in a rapid mouse pharmacokinetics study (Table 4). In
this study, the compound was injected intraperitoneally at 10 mg/kg and plasma samples
collected after 30 min, 2, 4, and 7 h. The plasma levels of compound were quantified using
MS/MS and a calibration curve. At 7 h the levels of CCG224061 were still quantifiable at 13
ng/mL (0.04 µM), however this is below the IC50 of 0.066 µM. This compound showed a much
lower area under the curve (AUC) than CCG258208, which was previously evaluated
(Waldschmidt et al., 2017). However, in the mouse liver microsome assays CCG224061 showed
a 3-fold longer half-life than CCG258208. This suggests that CCG224061 may be cleared renally
in addition to undergoing CYP-mediated metabolism.
Discussion
Understanding the molecular bases for potency and selectivity among closely related
protein kinases is key to the rational design of improved GRK chemical probes. In this study, we
hypothesized that substituting the benzodioxole moiety of paroxetine or its derivatives for an
indazole would result in higher potency for GRK2. The hope was that they would retain the same
or better selectivity as mediated by other substituents in each compound (namely the B, C and D
rings). Indeed, these compounds showed corresponding increases in GRK2 potency.
Additionally, this class of indazole-paroxetine hybrids showed similar metabolic stability and
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efficacy in a mouse cardiomyocyte assay compared to previous paroxetine analogs. However,
when CCG224061 was tested in mice it showed worse PK parameters than a previous
benzodioxole analog (CCG258208) suggesting a renal clearance mechanism. The expected
increase in potency was also met with corresponding increases in GRK5, PKA, and ROCK1
potency and the indazole series of compounds suffered from a 200- to 20-fold loss in selectivity
compared to their benzodioxole-containing analogs.
To investigate the reasons for these changes in potency and selectivity, we determined
crystal structures of GRK2–Gbg bound to three pairs of analogs in each series. The stronger
hydrogen bond interactions and small increase in BSA for the indazole moiety likely leads to
stronger interactions in the adenine binding pocket next to the hinge, leading to higher stability
and a well-defined kinase domain conformation that consistently leads to the same crystal form
(
Table S2). The indazole-paroxetine analogs that contained amide-linked D rings also appeared
to further stabilize the AST region, which passes over the active site. On the other hand,
selectivity conferred by the benzodioxole hinge-binding moiety seems to be due to looser
interactions in the adenine pocket, as typified by weaker hydrogen bonds to the hinge (including
one CH-O bond) and less BSA. GRK2 is able to alter its configuration in order to conform to
each particular analog, yielding three distinct crystal forms when in complex with benzodioxole
warhead ligands (one paroxetine analog not reported in this paper was observed to crystallize in
1
C222 , the same crystal form as the indazole-hybrids). Given their highly conserved active sites,
the molecular basis for selectivity for GRK2 over other GRKs and AGC kinases is therefore
most easily explained by the innate ability of GRK2 to accommodate these conformational
changes.
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Changing the identity of the hinge-binding moiety also affects how the D-ring packs,
thereby influencing the conformation of residues in the aC helix and aB-aC loop that contacts
the D ring, if present. Indeed, it was observed that there was a consistent shift of the aB-aC loop
away from the active site in the indazole-hybrid complexes relative to those of the benzodioxole
analogs. However, in the case of the CCG258748 complex this change in the aB-aC loop is
subtler, which is most likely due to the interactions formed with the pyrazole D-ring of these
compounds. In particular, the hydrogen bond formed between a pyrazole nitrogen and Glu239 in
aC would restrict the movement of this loop. The outward movement of the aB-aC loop would
create a slightly broader active site that could accommodate larger substituents, which could
explain why bulky D-ring substituents like a 2,6-dimethoxybenzylamide showed very high
potency in the GSK180736A series but not in the benzodioxole-paroxetine compounds
(Waldschmidt et al., 2016, 2017). Additionally, this shift of aB-aC loop may contribute to a
kinase domain conformation that is more similar to that observed with other kinases such as
GRK5 or PKA.
PCA revealed that the GRK2–Gbg complexes with indazole-paroxetine hybrid analogs
adopt similar kinase domain conformations as complexes with GSK180736A analogs and the
1
benzolactam-paroxetine analog (which all crystallized in the C222 crystal form). Interestingly,
the kinase domain conformation in these complexes is also closely related to that of GRK5
bound to sangivamycin (PDB entry 4TNB) (Komolov et al., 2015). This result could help
explain why indazole-paroxetine hybrids are able to inhibit GRK5 more potently than the
corresponding benzodioxole analogs, as they are able to trap the kinase domains of GRK2 and
GRK5 in a similar conformational state. It should however be noted that the structure of GRK5
in complex with another indazole-based GSK180736A analog, CCG215022 (PDB entry 4WNK),
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which is a pan-GRK inhibitor, adopted a distinct kinase conformation (Figure 5). Thus, the
structure of a complex between GRK5 and an indazole-paroxetine hybrid would help to further
elaborate on how GRK5 potency is gained. On the other hand, the benzodioxole analogs allow
more conformational flexibility in the GRK2 kinase domain, as evidenced by the wider spread in
PC space of 3V5W, 5UKK, 5UKM, and 5UKL, especially along the PC1 axis. This indicates
once again that with respect to benzodioxole moieties, selectivity is likely driven by the
apparently unique ability of GRK2 to mold itself to these ligands. Notably, another highly
selective GRK2 inhibitor, CMPD101 (115h), also forms relatively weak interactions with the
hinge via a pyridine moiety, and crystallized in complex with GRK2 in one of the C2 crystal
forms (Thal et al., 2011; Okawa et al., 2017). Given that the unique conformational space
sampled by the kinase domains of GRK2 and GRK5 seems to be a major determinant of
selectivity, it will be important to take their differing landscapes into account as one looks
towards the rational design of GRK5-selective inhibitors.
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Acknowledgments
The mouse liver microsome stability and short pharmacokinetic studies were executed by the
University of Michigan Pharmacokinetics Core.
Authorship Contributions
Bouley and Waldschmidt contributed equally.
Participated in research design: Koch, Larsen, Tesmer
Conducted experiments: Bouley, Waldschmidt, Cato, Cannavo, Yao
Contributed new reagents of analytic tools: Song, Cheung
Performed data analysis: Bouley, Waldschmidt, Cato, Cannavo, Yao
Wrote or contributed to the writing of the manuscript: Bouley, Waldschmidt, Tesmer
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Footnotes
The work at University of Michigan was supported by the National Institutes of Health National
Heart, Lung, and Blood Institute [Grants HL071818 and HL122416] to J.J.G.T.; and the
American Heart Association [Grant 15PRE22730028] to H.V.W.; and the University of
Michigan Chemistry-Biology Interface (CBI) training program National Institutes of Health
[Grant 5T32GM008597] to M.C.C.. The work at Temple University was supported by the
National Institutes of Health [Grants R37 HL061690, P01 HL075443, P01 HL108806, and P01
HL091799] to W.J.K. Use of the Advanced Photon Source was supported by the US Department
of Energy, Office of Science, Office of Basic Energy Sciences, under Contract No. DE-AC02-
0
6CH11357; and the use of LS-CAT Sector 12 was supported by the Michigan Economic
Development Corporation and Michigan Technology Tri-Corridor Grant [085P1000817].
R.B. and H.V.W. made equal contributions.
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Figure Legends
Figure 1. Chemical structures of GRK2 inhibitor scaffolds and activity against GRK2, GRK5,
and ROCK1, expressed as either IC50 or percent inhibition.
Figure 2. Comparison of GRK2 inhibitor scaffolds bound in the GRK2 active site. A) Overlay of
paroxetine (gray carbons) and GSK180736A (purple carbons) bound to the GRK2–Gbg complex
(PDB IDs: 3V5W and 4PNK, respectively). B) Overlay of paroxetine (gray carbons) and Takeda
CMPD101 (light blue carbons) bound to the GRK2–Gbg complex (PDB IDs: 3V5W and 3PVW,
respectively). Ring systems are labeled according to the A, B, C, and D convention shown in
Figure 1.
o c
Figure 3. Electron density |F | - |F | omit maps contoured at 3.0 s and binding modes for ligands
co-crystallized with GRK2–Gbg. (A) GRK2–Gbg·CCG2224061, carbon atoms for ligand are
shown in cyan. (B) GRK2–Gbg·CCG257284, carbon atoms for ligand are shown in light blue.
(C) GRK2–Gbg·CCG258748, carbon atoms for ligand are shown in green. Hydrogen bonds are
shown as black dashed lines and backbone nitrogens are shown as blue spheres.
Figure 4. Structural comparison of analogous GRK2–Gbg complexes with indazole- or
benzodioxole-substituted paroxetine derivatives. Key structural changes in the P-loop and/or aB-
aC loop are highlighted with red circles and black dashed lines linking Ca carbons with the
greatest displacement in each region discussed in the text. A) GRK2–Gbg·CCG224061 is shown
in cyan and GRK2–Gbg·paroxetine (PDB ID: 3V5W) in gray. B) GRK2–Gbg·CCG257284 is
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