Small molecule inhibitors of regulators of G protein signaling (rgs) proteins

Article abstract of DOI:10.1021/ml200263y

Recently, regulators of G protein signaling (RGS) proteins have emerged as potential therapeutic targets since they provide an alternative method of modulating the activity of G protein-coupled receptors, the target of so many drugs. Inhibitors of RGS proteins must block a protein-protein interaction (RGS-Gα) but also be cell and, depending on the therapeutic target, blood-brain barrier permeable. A lead compound (1a) was identified as an inhibitor of RGS4 in a screening assay, and this has now been optimized for activity, selectivity, and solubility. The newly developed ligands (11b and 13) display substantial selectivity over the closely related RGS8 protein, lack the off-target calcium mobilization activity of the lead 1a, and have excellent aqueous solubility. They are currently being evaluated in vivo in rodent models of depression.

Full text of DOI:10.1021/ml200263y

Letter
pubs.acs.org/acsmedchemlett
Small Molecule Inhibitors of Regulators of G Protein Signaling
(RGS) Proteins
Emma M. Turner,† Levi L. Blazer,‡ Richard R. Neubig,‡ and Stephen M. Husbands*,†
†Department of Pharmacy and Pharmacology, University of Bath, Bath, BA2 7AY, United Kingdom
‡Department of Pharmacology, University of Michigan, Ann Arbor, Michigan 48109, United States
S
* Supporting Information
ABSTRACT: Recently, regulators of G protein signaling (RGS) proteins have emerged as
potential therapeutic targets since they provide an alternative method of modulating the activity of
G protein-coupled receptors, the target of so many drugs. Inhibitors of RGS proteins must block a
protein−protein interaction (RGS-Gα) but also be cell and, depending on the therapeutic target,
blood−brain barrier permeable. A lead compound (1a) was identified as an inhibitor of RGS4 in a
screening assay, and this has now been optimized for activity, selectivity, and solubility. The newly developed ligands (11b and
13) display substantial selectivity over the closely related RGS8 protein, lack the off-target calcium mobilization activity of the
lead 1a, and have excellent aqueous solubility. They are currently being evaluated in vivo in rodent models of depression.
KEYWORDS: RGS4, RGS protein, thiadiazolidinone, GPCR, protein−protein interaction
protein-coupled receptors (GPCRs) are widely distributed
throughout the body and are an important class of
must act by disrupting this interaction either directly or through
an allosteric mechanism. While a number of PPI inhibitors are
known against a variety of targets, most of these compounds are
large molecules that lack access to the CNS.5 The development of
cell- and blood−brain barrier-permeable small molecules that can
act as selective PPI inhibitors is a nontrivial task but one that
could ultimately provide significant clinical benefit.
Our group recently discovered small molecule inhibitors of
RGS412−15 using a high-throughput flow cytometry protein
interaction assay (FCPIA).16 As part of this screening process,
CCG-50014 (1a, Figure 1) was identified as a selective inhibitor of
G
therapeutic targets for drug discovery.1 The majority of GPCR-
targeted drugs act as orthosteric agonists or antagonists at the
canonical ligand binding site, but recent attention has focused on
drugs acting allosterically to modulate receptor activity.2 One
advantage of allosteric agents is their ability to modulate ongoing
physiological signaling. Other approaches to modulate GPCR-
mediated signal transduction may also provide significant
therapeutic benefit. To this end, we have focused on targeting a
class of proteins that negatively regulates GPCR signaling.
Regulators of G protein signaling (RGS) proteins are potent
negative modulators of GPCR signaling. They accelerate the
rate of GTP hydrolysis by Gα subunits of heterotrimeric G
proteins, shortening the duration and decreasing the magnitude
of signal after receptor activation.3 The important role of RGS
proteins in GPCR signaling has generated, in recent years,
significant interest in RGS proteins as therapeutic targets in
their own right.4,5 One of the attractions is that while many
receptors are expressed throughout the body, modulators of the
signaling pathway, including RGS proteins, are expressed in a
more tissue-specific manner. This may be of particular
importance for the development of centrally acting agonist
therapeutics. Of over 20 mammalian RGS proteins identified,
RGS4 is one of the most extensively characterized. It is broadly,
but heterogeneously, expressed in the central nervous system
(CNS),6,7 less so in peripheral tissues, and has been shown to
modulate activity of the μ opioid (MOP), δ opioid (DOP), and
M3 muscarinic receptors among others.8,9 The selectivity in
regional distribution, coupled with the finding that RGS4 can
selectively suppress signaling through DOP receptors as com-
pared to MOP receptors,10 suggests that a level of specificity for
RGS4 inhibitory action will be possible.
Figure 1. Structure and activity of the lead compound, CCG50014.
RGS4 that acted by forming a covalent adduct to cysteine residues
in the RGS protein.15,17 With an IC50 of 30 nM, it is the most
potent RGS inhibitor reported to date. In an attempt to further
define the structural requirements for high-potency inhibition of this
protein, analogues of 1a have been synthesized with variation in
both the N2 and the N4 side chains. In addition, a set of these
newly synthesized RGS4 inhibitors have been evaluated for their
effects on calcium mobilization, an off-target activity displayed by 1a.
Received: November 4, 2011
Accepted: December 12, 2011
Published: December 12, 2011
The RGS-Gα site is a large, relatively featureless protein−
protein interaction (PPI) interface.11 Inhibitors of RGS proteins
© 2011 American Chemical Society
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ACS Medicinal Chemistry Letters
Letter
The synthesis of CCG-50014 (1a) and its thiadiazolidinone
(TDZD) analogues is shown in Scheme 1. Commercially
available isothiocyanates were reacted with isocyanates in the
presence of sulfuryl chloride (Scheme 1).18 This allowed a
range of R1 and R2 substituents, having varying lipophilic, elec-
tronic, and steric properties, to be evaluated. While chlorine
gas19 or N-chlorosuccinimide20 are also used in literature proce-
dures for making TDZDs, we found the use of sulfuryl chloride
straightforward and consistent. The resulting S-chloroisothio-
a
Scheme 1. Synthesis of CCG50014 and Analogues
carbamoyl chloride, proposed by Slomczyn
́
ska and Barany,18
was subsequently oxidized in atmospheric oxygen to the desired
products. This reaction was easily carried out in parallel, with
typically 11 different reactions running simultaneously. In total,
75 TDZD analogues were synthesized, with 39 (1a−i, 2a−i,
3−8, 9a,b, 10a−d, 11a−d, 12a−d, and 13) reported in Table 1.
Data for all compounds are reported in the Supporting
Information.
a
Conditions: (i) SO2Cl2, THF, 0 °C−room temperature, 18 h. (ii) Air,
30 min.
Table 1. Inhibition of Gαo Binding to RGS4 and RGS8
a
a
RGS4
RGS8
selectivity
R1
4-FBn
R2
IC50 (nM)
IC50 (μM)
RGS4/RGS8
1a
4-MePh
Ph
30.1
16.3
13.5
10.9
35.7
79.3
121
11.0
6.20
7.60
11.4
17.2
16.2
7.10
12.8
39.8
7.5
366
380
1b
1c
4-FBn
4-FBn
4-FBn
4-FBn
4-FBn
4-FBn
4-FBn
4-FBn
Bn
4-ClPh
4-MeOPh
3,4-diClPh
3-CF3Ph
3-MePh
3-ClPh
4-MeBn
4-MePh
Ph
564
1d
1e
1f
1050
481
204
1g
1h
1i
59
52.3
12.9
14.4
23.5
28.7
23.9
88.9
57.4
38.2
32.5
7.20
5.40
8.60
17.4
14.5
176
245
3090
519
2a
2b
2c
Bn
5.60
5.20
12.3
13.2
16.4
21.3
10.9
20.4
11.8
11.6
17.5
9.90
312
239
Bn
4-ClPh
4-MeOPh
3,4-diClPh
3-CF3Ph
3-MePh
3-ClPh
4-MeBn
4-MePh
4-MePh
4-MePh
4-MePh
4-MePh
4-MePh
n-Bu
183
2d
2e
2f
Bn
515
Bn
149
Bn
286
2g
2h
2i
Bn
558
Bn
335
Bn
2840
2170
1340
1005
679
3
4-ClBn
4-MeBn
3-ClBn
3-MeBn
4-MeOBn
3,4-diClBn
4-FBn
4-FBn
Me
4
5
6
7
1780
460
8
34.2
15.6
22.3
18.9
22.3
23.5
27.8
19.7
14.4
29.8
53.6
14.0
26.3
38.6
29.1
54.3
>100000
93300
15.7
31.6
18.7
8.40
37.0
28.4
56.0
9.50
83.5
122
9a
2020
842
9b
10a
10b
10c
10d
11a
11b
11c
11d
12a
12b
12c
12d
13
14
15
Et
4-MePh
Et
445
Me
1660
1210
2020
483
Me
n-Bu
Me
t-Bu
n-Bu
4-MePh
Et
n-Bu
5810
4110
2220
550
n-Bu
n-Bu
n-Bu
t-Bu
119
i-Bu
4-MePh
Et
7.70
70.6
98.0
194
i-Bu
2680
2540
6660
665
i-Bu
n-Bu
i-Bu
t-Bu
MeOCH2CH2
Et
36.1
>100
0.0
b
b
4-MeBn
4-MePh
N/A
c
c
Bn
4-MePh
0
a
b
Values are an average from two independent experiments. The calculated ΔpIC50 gave a mean error of 0.2 for RGS4 and 0.14 for RGS8. Scheme 2
c
for the structure of 14. Scheme 3 for the structure of 15.
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In addition to the large series of TDZD compounds
synthesized, an imidazolidine-2,4-dione (14) and a maleimide
(15) were both prepared to compare their activity to the
TDZDs. The synthesis of 14 was carried out in three steps,
starting from ethyl bromoacetate and p-toluidine (Scheme 2a).
resulting in each compound having near 3 orders of magnitude
selectivity. In fact, of the compounds discussed so far, that is,
retaining a benzyl or substituted benzyl at R1, 1i and 2i were
the most selective.
Variation in the aryl groups of R1 and R2 has therefore led to
the discovery of a number of ligands with high potency and
excellent selectivity. However, the uniformly high lipophilicity
(Clog P typically >4) of these ligands resulted in only moderate
solubility in aqueous solution, and they were therefore less than
ideal for consideration for more in-depth study. To address this
problem, analogues in which one or both R groups were
replaced with short alkyl chains were prepared. In the former
series, where one aryl group was replaced by alkyl (9a,b, 10a,
11a, and 12a), potency and selectivity at RGS4 were retained.
Making both R groups short alkyl chains (10b−d, 11b−d, and
12b−d) substantially improved solubility (complete solubility
at 500 μM) while also providing the most consistently selective
group of compounds yet developed (all >1000-fold selective).
The potency at RGS4 (IC50 14.4 nM), near 6000-fold selec-
tivity, and high solubility of 11b mean that it is an ideal can-
didate for further evaluation, including in vivo studies. As a means
to even further enhance solubility of this compound, analogues
containing ether side chains were considered, and the ether
analogue of 11b was prepared. This compound (13) retained
good potency (56 nM) and excellent selectivity (>600-fold).
The effects of 1a, 11b, and 13 were tested on the Ca2+
transient induced by M3 muscarinic receptors in HEK293T
cells. Compound 1a at 10 μM nearly completely abolished the
carbachol-induced Ca2+ transient (Figure 2), while 11b and 13
a
Scheme 2. Synthesis of the non-TDZD Analogues
a
Conditions: (i) NaOAc, EtOH, 80 °C, 1 h. (ii) Methyl benzyl
isocyanate, toluene, reflux, 5 h. (iii) NaH, THF, 0 °C−room
temperature, 18 h. (iv) Benzylamine, AcOH, 50 °C, 18 h. (v) p-
Tolylboronic acid, CsF, Cl2Pd(dppf)·CH2Cl2, dioxane, 40 °C, 1 h.
After reaction in the presence of sodium acetate, the resulting
amino acetate 16 was stirred at reflux in the presence of p-
methylbenzyl isocyanate to provide the corresponding ureido
acetate 17. This was cyclized using sodium hydride, providing
14. Compound 15 was synthesized by first reacting bromo-
maleic anhydride with benzyl amine in the presence of acetic
acid. The desired product resulted from a Suzuki reaction cou-
pling p-tolyl boronic acid to 18.
All compounds were evaluated using the FCPIA assay as a
primary screen to determine IC50 values for inhibition of Gαo
binding to both RGS4 and RGS8 (the closest relative to RGS4
based upon sequence homology). In preliminary studies, 1a
was also found to suppress Ca2+ responses to GPCRs in a
manner unrelated to its activity at RGS proteins. The most
interesting of the new ligands was also assessed for this off-
target effect.
CCG-50014 (1a) was confirmed as a potent inhibitor of
RGS4 with excellent selectivity over RGS8. Retaining the 4-
fluorobenzyl R1 group and varying R2 led to both more (e.g.,
1d) and less (e.g., 1g) potent and selective compounds. It
appeared that a 3-substituent on the R2 aryl ring was associated
with reduced RGS4 potency as compared to unsubstituted and
4-substituted analogues (e.g., 1f, 1g, 1h cf. 1b, 1c, 1d). While
not completely consistent, this trend is repeated across a
number of the series where R1 is held constant and R2 is varied.
A 3,4-dichlorophenyl group as R2 generally resulted in low
potency at RGS4 and relatively low selectivity (e.g., 1e, 2e). In
contrast, a 4-methyl substituent was more often associated with
high affinity and high selectivity at RGS4, with a number dis-
playing >1000-fold selectivity versus RGS8 (e.g., 3, 4, 5, and 7).
Replacement of the phenyl group by benzyl at R2 (1i, 2i) did
not improve activity at RGS4 but did reduce RGS8 activity,
Figure 2. Effect of compounds on carbachol-simulated Ca++
responses. HEK-293 cells stably transfected with the human M3
muscarinic receptor were plated in black, clear-bottomed, 96-well
plates overnight. They were loaded with Fluo4-NW according to the
manufacturer's instructions. After 30 min of loading at 37 °C, the
indicated compounds were added at a concentration of 10 μM (with
1% DMSO). After 30−45 min, the baseline fluorescence was measured
in a Flex-3 plate reader (Molecular Devices). Then, carbachol (10 nM
final) was injected into the wells, and the increase in intracellular Ca++
was measured and is expressed as the percentage of the baseline Ca++
level. Values are the mean
SD of triplicate determinations
(compounds) and 16 determinations (DMSO).
had no effect. The action of 1a on this response cannot be
through effects on RGS proteins since HEK cells express
minimal levels of functional RGS proteins.21
We have previously published our studies that indicate that
the lead compound (1a) reacts to form an adduct with a cys-
teine residue on the RGS protein through disulfide bond forma-
tion.15 The proposed mechanism (Scheme 3, 19b) is analogous
to that proposed by Nasim and Crooks for the ring-opening of
TDZDs with PPh3.20 To help confirm the importance of
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ACS Medicinal Chemistry Letters
Letter
Funding
Scheme 3. Proposed Mechanism of Reaction of a Thiol
with 1a
This work was funded by NIH Grant DA R01-023252
(R.R.N.).
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Susan Wade for production of the stable M3
muscarinic receptor expressing the HEK-293 cell line.
disulfide bond formation to the activity of this series of ligands,
analogues 14 and 15 were prepared. Compound 14 is the
imidazolidine-2,4-dione analogue of 4, while 15 is the
maleimide analogue of 2i; 4 and 2i being two of the most
potent inhibitors discovered. As expected, neither 14 or 15
displayed activity at RGS4. Also supporting the disulfide bond-
forming mechanism, the reaction of propane thiol with 1a
appears to give efficiently and cleanly the expected adduct 19a
(Scheme 3). Importantly, 1a is not a general cysteine alkylator,
failing to inhibit the cysteine protease papain, suggesting selec-
tivity for RGS4.15
Previously, thiadiazolidine-3,5-diones have been reported as
having a number of biological effects,22−24 including being gly-
cogen synthase kinase 3β (GSK-3β) inhibitors with activities in
the micromolar range.19 This latter activity has been suggested
to account, at least in part, for the antidepressant-like effects in
mice of the TDZD NP031115.25 Interestingly, 11b was evalua-
ted as part of that study and was found to be one of the weaker
inhibitors (GSK-3β IC50 70 μM) meaning that it has significant
selectivity (almost 5000-fold) for RGS4 over GSK-3β. As such,
11b should prove to be an invaluable tool in defining the
physiological role of RGS4 in vivo, including a potential role in
5-HT1A-mediated antidepressant effects.26
In summary, a series of RGS4 inhibitors have been
synthesized with improved selectivity over RGS8 and lacking
the off-target calcium mobilization activity of the lead 1a. One
compound, 11b, combines potency (RGS4 IC50 14 nM) and
selectivity (5800-fold over RGS8 and no calcium transient)
with excellent aqueous solubility and should prove an
invaluable tool for better defining the role of RGS4 and its
potential as a therapeutic target. Its ether analogue (13) had
further improved solubility while retaining good potency and
selectivity. Analogues 11b and 13 are now being evaluated in
vivo with positive preliminary data, and the results of this latter
work will be reported separately.
ABBREVIATIONS
■
GPCR, G protein-coupled receptor; RGS, regulators of G
protein signaling; CNS, central nervous system; MOP, μ opioid
receptor; DOP, δ opioid receptor; PPI, protein−protein
interaction; FCPIA, flow cytometry protein interaction assay;
TDZD, thiadiazolidinone
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ASSOCIATED CONTENT
■
S
* Supporting Information
Tabulated pharmacological data for all compounds, representa-
tive synthetic procedures, 1H, 13C NMR for all new
compounds, and elemental analysis data for key compounds.
This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Tel: (0)1225 383103. Fax: (0)1225 386114. E-mail: s.m.husbands@
bath.ac.uk.
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
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