First demonstration of positive allosteric-like modulation at the human wild type translocator protein (TSPO)

Article abstract of DOI:10.1021/acs.jmedchem.5b01288

We show that changing the number and position of nitrogen atoms in the heteroatomic core of a pyrazolopyrimidine acetamide is sufficient to induce complex binding to wild type human TSPO. Only compounds with this complex binding profile lacked intrinsic effect on glioblastoma proliferation but positively modulated the antiproliferative effects of a synthetic TSPO ligand. To the best of our knowledge this is the first demonstration of allosteric-like interaction at the wild type human TSPO.

Full text of DOI:10.1021/acs.jmedchem.5b01288

Brief Article
pubs.acs.org/jmc
First Demonstration of Positive Allosteric-like Modulation at the
Human Wild Type Translocator Protein (TSPO)
Rajeshwar Narlawar,†,# Eryn L. Werry,‡,# Alana M. Scarf,§ Raphy Hanani,† Sook Wern Chua,∥
Victoria A. King,§ Melissa L. Barron,‡ Ralph N. Martins,⊥ Lars M. Ittner,∥ Louis M. Rendina,†
and Michael Kassiou*,†,‡
†School of Chemistry, ‡Faculty of Health Sciences, and §School of Medical Sciences (Pharmacology), Bosch Institute, The University
of Sydney, Sydney, NSW 2006, Australia
∥Dementia Research Unit, School of Medical Sciences, University of New South Wales, Sydney, NSW 2052, Australia
⊥School of Medical Sciences, Edith Cowan University, Joondalup, WA 6027, Australia
S
* Supporting Information
ABSTRACT: We show that changing the number and position of nitrogen atoms in the heteroatomic core of a
pyrazolopyrimidine acetamide is sufficient to induce complex binding to wild type human TSPO. Only compounds with this
complex binding profile lacked intrinsic effect on glioblastoma proliferation but positively modulated the antiproliferative effects
of a synthetic TSPO ligand. To the best of our knowledge this is the first demonstration of allosteric-like interaction at the wild
type human TSPO.
important for binding of the prototypic benzodiazepine Ro 5-
4864 are not critical for binding of the first-generation synthetic
isoquinoline carboxamide ligand PK 11195.13
INTRODUCTION
■
The 18 kDa translocator protein (TSPO) is a highly conserved
five transmembrane protein most commonly found in the outer
mitochondrial membrane.1 It is suggested to be a part of the
mitochondrial permeability transition pore (MPTP) in a
complex with the voltage-dependent anion carrier (VDAC)
and the adenine nucleotide transporter (ANT).2,3 TSPO is
expressed in various tissues and organs throughout the body.4
Baseline TSPO expression levels in the brain are low, but
TSPOs are upregulated in reactive glia in areas damaged by
neurodegenerative diseases and also in cancers such as
glioma.5−9 Conversely, TSPO expression is downregulated in
lymphocytes and leukocytes in a number of anxiety disorders.9
As such, TSPO has become a target for development of drug
treatments and diagnostic imaging agents for use in cancer,
neurodegenerative diseases, and anxiety.5−7,9,10
Development of these drugs, however, has been hindered by
the emerging complexity of ligand interactions at the TSPO.
Several classes of ligands interact with the TSPO at distinct
binding sites. The endogenous ligand cholesterol binds to an
area of the cytosolic C-terminus defined as the cholesterol
recognition amino acid consensus (CRAAC) domain.11 Other
endogenous ligands such as protoporphyrin IX and many
synthetic ligands bind to loop 1 of the N-terminal.12 Site-
directed mutagenesis, however, suggests residues in this loop
Another well-documented influence on binding complexity
to the human TSPO is the rs6971 polymorphism. This
polymorphism located in exon 4 of the TSPO gene results in
the substitution of alanine with threonine at amino acid residue
147.14 Homozygous expression of Ala147 TSPO in human
brain tissue and leukocytes results in high affinity binding, while
homozygous expression of Thr147 TSPO results in low affinity
binding and heterozygous expression results in complex two-
site mixed affinity binding.15,16 This modulation of binding has
functional outcomes with Ala147 homozygous leukocytes
producing more pregnenalone than homozygous Thr147 and
heterozygous leukocytes.14 A phase II clinical trial of the TSPO
ligand XBD173 in generalized anxiety disorder failed, partly due
to the presence of this polymorphism in subjects (Clinical-
Trials.gov NCT00108836).
Given the already documented complex nature of binding to
the TSPO and that multiple drug binding sites exist on the
TSPO, it may be possible that allosteric-like interactions occur
at the TSPO. In fact in rat brain cortical synaptosomes, a
Received: August 19, 2015
© XXXX American Chemical Society
A
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a
Table 1. Nature of Binding Interaction and TSPO Affinities for Compounds 1−5
a
nH indicates Hill slope value.
multiphasic Scatchard analysis of radioligand binding results
suggested complex, possibly allosteric, interactions between the
synthetic steroid stanozolol and PK 11195;17 however,
interpretation of binding nature from Scatchard plots can be
ambiguous, and this study did not characterize whether
stanozolol altered the potency of PK 11195. Furthermore, it
was not documented whether the TSPO in this study had the
Ala147Thr polymorphism and hence whether the results could
have been explained solely by the presence of the poly-
morphism. As such, the extent to which complex allosteric-like
interactions occur at the human Ala147 (wild type) TSPO is
currently unclear.
Pyrazolo[1,5-a]pyrimidine acetamides are a promising class
of TSPO ligands with recent in vitro and human imaging
studies suggesting derivatives of this class have a high affinity,
good bioavailability and are well-tolerated.2,18 However, the
number of nitrogen atoms in the central heteroatomic core of
TSPO ligands can impact TSPO binding affinity.19 In the
present study, we aimed to examine how complexity in
interaction with human Ala147 (wild type) TSPO is affected by
changing the number and position of nitrogen atoms in the
central heteroatomic core of a pyrazolopyrimidine acetamide
(4). We show that specific numbers and positions of nitrogen
atoms in the core heterocycle of TSPO ligands are sufficient to
prevent effects on glioblastoma proliferation by these ligands
themselves while enabling them to positively modulate the
antiproliferative effects of PK 11195.
RESULTS
■
Chemistry. A set of TSPO ligands were designed by
changing the number and position of the nitrogen atoms in the
heterobicyclic core of 4, resulting in various heterocylic
derivatives such as the indole 1, benzimidazole 2, imidazopyr-
idine 3, and purine 5 derivatives (Table 1). The synthesis of
these novel derivatives began with the indole 1 as outlined in
Scheme 1. Briefly, 3,5-dimethylphenylhydrazine 7 was synthe-
B
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a
11 was reduced using Zn powder and hydrazine hydrate in n-
butanol at reflux to afford the diamine 12 in good yield. 2-(4-
(Benzyloxy)phenyl)-4,6-dimethyl-1H-benzo[d]imidazole 14
was synthesized in one step from 3,5-dimethylbenzene-1,2-
diamine 12 and 4-benzyloxybenzaldehyde 13 in the presence of
ammonium acetate under microwave conditions. N-Alkylation
of benzimidazole 14 proceeded in good yield in the presence of
sodium hydride and chloro-N,N-diethylacetamide to give 15.
The benzyl group of 15 was deprotected under hydrogenolysis
using 10% Pd/C in methanol to afford the phenol 16. The
phenol 16 was alkylated using sodium hydride and methyl
iodide to give the corresponding desired aryl ether 2.
Scheme 1. Synthetic Route to Indole Derivative 1
The imidazopyridine derivative 3 was synthesized as outlined
in Scheme 3. Commercially available 2-amino-4,6-dimethyl-
pyridine 17 was regioselectively brominated at the doubly
activated 5-position and upon treatment with 0.5 equiv of 1,3-
dibromohydantoin at −50 °C afforded 18. Installation of the
bromine at the 5-position was essential for directing the
subsequent nitration at 3-position. Under standard nitration
conditions the nitropyridine derivative 19 was obtained in good
yield. Nitro compound 19 was then successfully converted into
the corresponding diamine 20 in 90% yield using a
combination of zinc and hydrazine monohydrate. The product
was sufficiently pure and was used in the next step without
further purification. Diamine 20 was cyclized under microwave
irradiation and in the presence of ammonium acetate and 4-
methoxybenzaldehyde to give imidazopyridine 21. N-Alkylation
of 21 was achieved using sodium hydride and chloro-N,N-
diethylacetamide resulting in 22. Finally, the debromination of
22 was carried out using n-BuLi in THF at −50 °C to afford
desired imidazopyridine derivative 3 in good yield.
a
Reagents and conditions: (a) (1) aq NaNO2, conc HCl, −10 °C, 30
min; (2) SnCl2, conc HCl, −5 °C to rt, 30 min; (3) 3 M KOH, DCM,
30 min, 40% over three steps; (b) (1) cat. AcOH, EtOH, reflux, 2 h;
(2) PPA, 120 °C, 30 min; (c) NaH, ClCH2CONEt2, DMF, 0 °C to rt,
overnight, 47%.
sized from commercially available 3,5-dimethylaniline 6.
Diazotization of aniline 6 using NaNO2 in HCl followed by
the reaction with SnCl2 in HCl gave the hydrazine hydro-
chloride as colorless solid. Phenylhydrazine hydrochloride was
then treated with 3 M KOH to afford the free phenylhydrazine
which then underwent the Fischer indole synthesis. Phenyl-
hydrazine 7, 4-methoxyacetophenone 8, and catalytic acetic
acid were refluxed in absolute ethanol to afford the phenyl-
hydrazone as a golden-colored solid. Crude hydrazone and
polyphosphoric acid mixture was heated at 120 °C to afford the
indole 9 in moderate yield. N-Alkylation of indole 9 was
achieved using 2-chloro-N,N-diethylacetamide and NaH in
DMF to afford the desired indole acetamide 1.
The pyrazolopyrimidine derivative 4 was synthesized
following a published procedure21 and was considered to be
The bezimidazole derivative 2 was synthesized as outlined in
Scheme 2. Commercially available 2,4-dimethylaniline 10 was
nitrated using a documented procedure.20 The nitro group of
1
of >95% purity according to H and 13C NMR spectroscopy
and elemental analysis.
a
Scheme 2. Synthetic Route to Benzimidazole Derivative 2
a
Reagents and conditions: (a) (i) Ac2O, conc HNO3, 0 °C, 10 min; (ii) conc HCl, ethanol, reflux, overnight, 78%; (b) NH2NH2.H2O, Zn powder, n-
butanol, reflux, 6 h, 91%; (c) NH4OAc, abs EtOH, microwave, 75 °C, 2.5 h, 81%; (d) NaH, ClCH2CONEt2, DMF, 0 °C to rt, overnight, 70%; (e)
H2, 10% Pd/C, MeOH, rt, 12 h, 91%; (f) MeI, NaH, DMF, 0 °C to rt, overnight 89%.
C
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a
Scheme 3. Synthetic Route to Imidazopyridine Derivative 3
a
Reagents and conditions: (a) 1,3-dibromo-5,5-dimethylimidazolidine-2,4-dione, CH2Cl2, −50 °C, 30 min, 76%; (b) conc HNO3, 6 N H2SO4, −10
°C to rt, 30 min, 91%; (c) NH2NH2.H2O, Zn powder, n-BuOH, reflux, overnight, 90%; (d) 4-methoxybenzaldehyde, NH4OAc, abs EtOH, 75 °C,
microwave, 2.5 h, 80%; (e) NaH, ClCH2CONEt2, DMF, 0 °C to rt, overnight, 75%; (f) n-BuLi, THF, −50 °C, 1 h, 89%.
a
Scheme 4. Synthetic Route to Purine Derivative 5
a
Reagents and conditions: (a) K2CO3, ClCH2CO2Et, DMF, 152 °C, microwave, 15 min; (b) Me3Al, Pd(PPh3)4, THF, reflux, overnight, 45% over
two steps; (c) 4-iodoanisole, Pd(OAc)2, CuI, Cs2CO3, DMF, 120 °C, 13 h, 58%.
Figure 1. Effect of changing the number of nitrogens in the heteratomic core of pyrazolopyrimidine acetamide derived compounds on competition
with [3H]PK 11195 at the human TSPO in HEK-293T cells (left) and T98G cells (right).
The purine derivative 5 was synthesized in three steps as
depicted in Scheme 4. The synthesis of the purine derivative
began with N-alkylation of commercially available 2,6- dichloro-
9H-purine 23. N-Alkylation was performed using sodium
hydride and chloro-N,N-diethylacetamide which resulted in an
inseparable mixture of two products believed to be
regioisomers of 24. Thus, the subsequent cross-coupling
reaction was performed using the mixture. The crude
regioisomeric mixture was utilized in a palladium-catalyzed
methylation in the presence of trimethylaluminum. tetrakis-
(triphenyphosphine)palladium(0). 2-(2,6-Dimethyl-9H-purin-
9-yl)-N,N-diethylacetamide 25 was isolated in pure form at
this stage and in moderate yield (45% over 2 steps). Direct C-2
arylation was employed using 4-iodoanisole, Pd(OAc)2, CuI,
and Cs2CO3 to obtain the desired purine derivative 5 in
moderate yield.
The purity of all the compounds used in subsequent
biological evaluation was determined to be greater than 95%
by elemental analysis.
Sequencing for rs6971. Sequencing was carried out on
HEK 293T and T98G cells to establish the nature of their
genetic sequence at the 439th nucleotide in the human TSPO
gene. Both cell lines expressed a guanine at this position in the
gene, indicating they both have alanine as their 147th TSPO
amino acid and hence do not have the rs6971 polymorphism.
Binding Interactions. The TSPO binding nature of 1−5
was determined by competition radioligand binding against
[3H]PK 11195 in HEK 293T and T98G cell membranes.
Results are displayed in Table 1 and Figure 1. In general,
compounds with 3 nitrogen atoms in the heterocyclic core (3,
4) interacted with the TSPO in a one-site manner with a Hill
slope of 1 at the same site as [3H]PK 11195, while compounds
D
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with less than 3 nitrogen atoms displayed complex Hill slopes.
Compound 1 had a Hill slope more negative than −1 at HEK
293T and T98G membranes (p < 0.05), while 2 had a Hill
slope more positive than −1 at T98G membranes (p < 0.05). A
binding model could not be fitted for 5, as it did not bind to the
TSPO, even up to 10 μM.
In HEK 293T cells, all assessed compounds displayed TSPO
binding affinity in the low nM range apart from the non-
binding purine derivative (5). All compounds except the
pyrazolopyrimidine acetamide (4) showed a lowering of affinity
to TSPO in T98G cells compared to in HEK 293T cells,
particularly the indole (1). Excluding 5, affinity order in T98G
cells followed the number of nitrogen atoms present in the
heterocyclic core, with compounds containing 3 nitrogen atoms
showing the highest affinity (4 followed by 3) and the
compound with one nitrogen atom in the heterocyclic core
showing lowest affinity (1).
EC10 concentration of PK 11195 (Table 1 in Supporting
Information).
Cell viability was not significantly affected by 1−5 in the
absence or presence of an EC10 concentration of PK 11195.
DISCUSSION AND CONCLUSIONS
■
In this study, we aimed to explore the impact on complexity of
TSPO interaction by altering the heterocyclic core of a high
affinity pyrazolopyrimidine acetamide (4). Initially we exam-
ined the impact of nitrogen atom position on TSPO interaction
by comparing the affinity and efficacy of the lead pyrazolo-
pyrimidine 4 and the imidazopyridine derivative 3. Template
hopping of 4 leading to the imidazalopyridine derivative 3
decreased TSPO affinity; however, both compounds showed
normal one-site binding, and these compounds did not
modulate PK 11195 antiproliferative effects.
We then explored the impact of changing the number of
nitrogen atoms in the heterocyclic core on TSPO interaction.
Increasing the number of nitrogen atoms to make the purine
derivative 1 removed the ability of the compound to bind to
TSPO, suggesting the number of nitrogen atoms in the
heterocyclic core is critical for affinity. This in agreement with a
previous study whereby N,N-dialkyl(2-phenylindol-3-yl)-
glyoxylamides demonstrated decreased TSPO affinity com-
pared to N,N-dialkyl-4-phenylquinazoline-2-carboxamides.19
Given this sensitivity to TSPO affinity found by increasing
the number of nitrogen atoms in the heterocyclic core, we
examined the impact of decreasing the number of nitrogen
atoms on TSPO interaction by screening an indole and a
benzimidazole derivative. The indole derivative 1 showed a Hill
slope more negative than −1 in competition with the synthetic
TSPO ligand [3H]PK 11195. In classical receptor pharmacol-
ogy, a Hill slope of −1 indicates binding interactions that obey
the law of mass action, while a Hill slope more negative than
−1 suggests positive cooperativity, where the binding of the
novel compound at one site enhances the affinity of the
radiolabeled compound at another site.22 One way that this
may manifest functionally is in positive allosteric modulation,
where a ligand may have no intrinsic efficacy but may enhance
the functional effect of a second ligand.23 Through assays
indexing antiproliferative potential of these novel compounds
we showed that the indole derivative did not affect proliferation
by itself, but rather it enhanced PK 11195’s anti-proliferative
affects. Given that this complexity of interaction occurred in the
verified absence of the rs6791 polymorphism, which is the most
well-documented cause of affinity and potency change at the
TSPO, it is possible that this complex interaction is allosteric-
like behavior.
Effects on T98G Proliferation and Viability. A number
of TSPO ligands, including PK 11195, have shown anti-
proliferative effects on glioma cell lines.7 We examined whether
our novel compounds affected proliferation of the human
glioma cell line T98G and whether they enhanced the
antiproliferative effect of PK 11195. Compounds 1−5 did not
affect the basal proliferation of T98G cells (1−100 μM).
Nonetheless, 1 significantly increased the antiproliferative effect
of an EC10 concentration of PK 11195 at ∼10 μM and higher
(p < 0.05; Figure 2). The EC50 for this potentiating effect was
14.73
1.1 μM. Also 2 significantly increased the
antiproliferative effect of an EC10 concentration of PK 11195
at ∼62.5 μM (p < 0.05; Figure 2). Up to 100 μM, 3, 4, and 5
did not significantly modulate the antiproliferative effect of an
It is unclear, however, the extent to which the observed
behavior of the indole derivative can be termed “classical”
allosterism. Allosterism is most widely characterized in
receptors, ion channels, and enzymes and the TSPO does not
fit within these classes, so it is unknown as to the extent it obeys
normal pharmacological interactions seen in these more widely
studied protein categories. Further, a classic allosteric
modulator enhances the potency of an orthosteric ligand, that
is, a ligand that binds to the same site as an endogenous ligand.
PK 11195 is not an endogenous ligand and may bind at
different sites to the endogenous TSPO ligands cholesterol and
protoporphyrin IX.12,24 Whether the indole derivate binds to
the orthosteric TSPO sites to facilitate the effects of PK
1119513 is an important question to resolve. Furthermore,
Figure 2. Positive modulation of the antiproliferative effect of an EC10
concentration of PK 11195 by 1 (A) and 2 (B) in T98G cells. Dose−
response curve of the antiproliferative effect of PK 11195 is included
for comparison. Values represent mean
SD of a representative
experiment, repeated in triplicate.
E
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whether the indole derivative also facilitates the effects of TSPO
on the endogenous TSPO ligands remains to be examined.
The benzimidazole derivative 2 also enhanced PK 11195
antiproliferative affects without inducing antiproliferative effects
on its own. The benzimidazole also had a Hill slope that
deviated from −1 in competition radioligand binding studies
with [3H]PK 11195, but unexpectedly the Hill slope was more
shallow than −1. In classical receptor pharmacology, this would
predict negative modulation of PK 11195,22 the opposite of the
functional effects observed in this study. As discussed above,
this may suggest that the TSPO does not operate within
classical definitions of cooperativity and allosteric receptor
pharmacology. There are, however, two alternative explan-
ations. A Hill slope more shallow than −1 in competition
radioligand binding may also suggest that the binding occurs at
heterogeneous receptors that may bind the ligand with different
affinities.22 TSPO is known to exist in several forms: as a
monomer, dimer, and polymer.25,26 Recent structural resolution
of a bacterial analogue of TSPO led to predictions that
potential dimerization of the TSPO from monomeric form
could bring separated binding sites into closer proximity,
creating the potential for cooperative interactions,12 suggesting
the monomeric, dimeric, and polymeric forms of TSPO could
be thought of as heterogeneous receptors. In support of this
prediction, human chorionic gonadotropin can increase binding
of PK 11195 to TSPO in mouse MA-10 Leydig testicular tumor
cells in a manner than temporally correlates with its ability to
induce rapid polymerization of TSPO.27,28 It is important then
to examine the effect of the benzimidazole 2 on the equilibrium
between monomeric, dimeric, and polymeric forms of the
TSPO in T98G cells to determine if the positive modulation
could be due to disruption of this equilibrium.
A second explanation for the shallow Hill slope induced by
the benzimidazole 2 could relate to the existence of TSPO in a
complex with VDAC and ANT. A shallow Hill slope can be
seen when insufficient accessory proteins are available to
associate with a protein that usually exists in a complex.22
TSPO is predominantly found in the mitochondria1 where
there is ample VDAC and ANT but can translocate to the
nucleus in some cancers.29 It may be that in cancer cells,
nuclear and cytoplasmic TSPOs do not have enough VDAC
and ANT for adequate complex formation. This may explain
why a shallow Hill slope was only induced by the
benzimidazole 2 on binding to TSPO from glioma cells and
not from the mitochondria of HEK cells.
EXPERIMENTAL SECTION
■
Chemistry. All compounds synthesized as per the Results and
Supporting Information sections were analyzed by H and 13C NMR
1
and high-resolution electrospray ionization mass spectrometry and
were found to be >95% pure. Full experimental procedures and results
of analyses can be found in the Supporting Information.
ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jmed-
chem.5b01288.
Synthetic procedure and spectral data of compounds,
biological procedures, supplementary Table 1 (PDF)
AUTHOR INFORMATION
■
Corresponding Author
*Phone: +612 9351 2745. E-mail: michael.kassiou@sydney.
edu.au.
Author Contributions
#R.N. and E.L.W. are joint first authors.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors acknowledge the support received from the Bosch
Institute Molecular Biology Facility and the expert help of
facility staff, especially Dr. Donna Lai and Dr. Sheng Hua. Work
performed at The University of Sydney and presented herein
was supported in part by the European Union’s Seventh
Framework Programme [FP7/2007-2013] INMiND (Grant
HEALTH-F2-2011-278850).
ABBREVIATIONS USED
■
ANT, adenine nucleotide transporter; CRAAC, cholesterol
recognition amino acid consensus; HEK, human embryonic
kidney; MPTP, mitochondrial permeability transition pore;
TSPO, translocator protein; VDAC, voltage-dependent anion
carrier
REFERENCES
■
(1) Papadopoulos, V.; Baraldi, M.; Guilarte, T. R.; Knudsen, T. B.;
Lacapere, J. J.; Lindemann, P.; Norenberg, M. D.; Nutt, D.; Weizman,
A.; Zhang, M. R.; Gavish, M. Translocator protein (18 kDa): new
nomenclature for the peripheral-type benzodiazepine receptor based
on its structure and molecular function. Trends Pharmacol. Sci. 2006,
27, 402−9.
(2) Scarf, A. M.; Ittner, L. M.; Kassiou, M. The translocator protein
(18 kDa): central nervous system disease and drug design. J. Med.
Chem. 2009, 52, 581−92.
(3) McEnery, M. W.; Snowman, A. M.; Trifiletti, R. R.; Snyder, S. H.
Isolation of the mitochondrial benzodiazepine receptor: association
with the voltage-dependent anion channel and the adenine nucleotide
carrier. Proc. Natl. Acad. Sci. U. S. A. 1992, 89, 3170−4.
(4) Gavish, M.; Bachman, I.; Shoukrun, R.; Katz, Y.; Veenman, L.;
Weisinger, G.; Weizman, A. Enigma of the peripheral benzodiazepine
receptor. Pharmacol Rev. 1999, 51, 629−50.
(5) Politis, M.; Su, P.; Piccini, P. Imaging of microglia in patients with
neurodegenerative disorders. Front. Pharmacol. 2012, 3, 96.
(6) Chua, S. W.; Kassiou, M.; Ittner, L. M. The translocator protein
as a drug target in Alzheimer’s disease. Expert Rev. Neurother. 2014, 14,
439−48.
Considering neither compound with three nitrogens in the
heterocyclic core showed allosteric-like behavior and the indolic
derivative 1 had more pronounced positive modulation than
the benzimidazole 2, these results suggest a lower number of
nitrogen atoms in the heteroatomic core is likely the key to
inducing allosteric-like effects of pyrazolopyrimidine acetamide
derivatives. We plan to further explore this by examining the
allosteric potential of isoindole and indazole derivatives.
In conclusion, we have identified an indole 1 and
benzimidazole derivative 2 of pyrazolopyrimidine acetamide 4
which show complex binding, lack of intrinsic activity, and
positive modulation of the antiproliferative effects of PK 11195.
To the best of our knowledge, this is the first demonstration of
allosteric-like behavior at the wild-type human TSPO. Although
the mechanisms behind this potentiation are as yet undefined,
identification of such compounds may open the way for
development of novel allosteric-like anxiolytics, neuroprotective
agents, and anticancer drugs that target the TSPO.
F
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Journal of Medicinal Chemistry
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(7) Austin, C. J.; Kahlert, J.; Kassiou, M.; Rendina, L. M. The
translocator protein (TSPO): a novel target for cancer chemotherapy.
Int. J. Biochem. Cell Biol. 2013, 45, 1212−6.
droxy-4,4′,6,6′-tetramethyl-1,1′-biphenyl. Tetrahedron: Asymmetry
2003, 14, 1267−1273.
(21) Banister, S. D.; Wilkinson, S. M.; Hanani, R.; Reynolds, A. J.;
Hibbs, D. E.; Kassiou, M. A practical, multigram synthesis of the 2-(2-
(4-alkoxyphenyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-yl)-
acetamide (DPA) class of high affinity translocator protein (TSPO)
ligands. Tetrahedron Lett. 2012, 53, 3780−3783.
(22) Motulsky, H.; Christopoulos, A. Fitting Models to Biological Data
Using Linear and Nonlinear Regression: A Practical Guide to Curve
Fitting; Oxford University Press: Oxford, U.K., 2004; 351 pp.
(23) Christopoulos, A.; Kenakin, T. G protein-coupled receptor
allosterism and complexing. Pharmacol Rev. 2002, 54, 323−74.
(24) Jaremko, L.; Jaremko, M.; Giller, K.; Becker, S.; Zweckstetter,
M. Structure of the mitochondrial translocator protein in complex with
a diagnostic ligand. Science 2014, 343, 1363−6.
(25) Delavoie, F.; Li, H.; Hardwick, M.; Robert, J. C.; Giatzakis, C.;
Peranzi, G.; Yao, Z. X.; Maccario, J.; Lacapere, J. J.; Papadopoulos, V.
In vivo and in vitro peripheral-type benzodiazepine receptor
polymerization: functional significance in drug ligand and cholesterol
binding. Biochemistry 2003, 42, 4506−19.
(26) Sakai, M.; Ferraz-de-Paula, V.; Pinheiro, M. L.; Ribeiro, A.;
Quinteiro-Filho, W. M.; Rone, M. B.; Martinez-Arguelles, D. B.; Dagli,
M. L.; Papadopoulos, V.; Palermo-Neto, J. Translocator protein (18
kDa) mediates the pro-growth effects of diazepam on Ehrlich tumor
cells in vivo. Eur. J. Pharmacol. 2010, 626, 131−8.
(27) Boujrad, N.; Gaillard, J. L.; Garnier, M.; Papadopoulos, V. Acute
action of choriogonadotropin on Leydig tumor cells: induction of a
higher affinity benzodiazepine-binding site related to steroid biosyn-
thesis. Endocrinology 1994, 135, 1576−83.
(28) Boujrad, N.; Vidic, B.; Papadopoulos, V. Acute action of
choriogonadotropin on Leydig tumor cells: changes in the topography
of the mitochondrial peripheral-type benzodiazepine receptor.
Endocrinology 1996, 137, 5727−30.
(29) Brown, R. C.; Degenhardt, B.; Kotoula, M.; Papadopoulous, V.
Location-dependent role of the human glioma cell peripheral-type
benzodiazepine receptor in proliferation and steroid biosynthesis.
Cancer Lett. 2000, 156, 125−32.
(8) Lacapere, J. J.; Papadopoulos, V. Peripheral-type benzodiazepine
receptor: structure and function of a cholesterol-binding protein in
steroid and bile acid biosynthesis. Steroids 2003, 68, 569−85.
(9) Rupprecht, R.; Papadopoulos, V.; Rammes, G.; Baghai, T. C.;
Fan, J.; Akula, N.; Groyer, G.; Adams, D.; Schumacher, M.
Translocator protein (18 kDa) (TSPO) as a therapeutic target for
neurological and psychiatric disorders. Nat. Rev. Drug Discovery 2010,
9, 971−88.
(10) Rupprecht, R.; Rammes, G.; Eser, D.; Baghai, T. C.; Schule, C.;
Nothdurfter, C.; Troxler, T.; Gentsch, C.; Kalkman, H. O.; Chaperon,
F.; Uzunov, V.; McAllister, K. H.; Bertaina-Anglade, V.; La Rochelle,
C. D.; Tuerck, D.; Floesser, A.; Kiese, B.; Schumacher, M.; Landgraf,
R.; Holsboer, F.; Kucher, K. Translocator protein (18 kD) as target for
anxiolytics without benzodiazepine-like side effects. Science 2009, 325,
490−3.
(11) Li, H.; Papadopoulos, V. Peripheral-type benzodiazepine
receptor function in cholesterol transport. Identification of a putative
cholesterol recognition/interaction amino acid sequence and con-
sensus pattern. Endocrinology 1998, 139, 4991−7.
(12) Li, F.; Xia, Y.; Meiler, J.; Ferguson-Miller, S. Characterization
and modeling of the oligomeric state and ligand binding behavior of
purified translocator protein 18 kDa from Rhodobacter sphaeroides.
Biochemistry 2013, 52, 5884−99.
(13) Farges, R.; Joseph-Liauzun, E.; Shire, D.; Caput, D.; Le Fur, G.;
Ferrara, P. Site-directed mutagenesis of the peripheral benzodiazepine
receptor: identification of amino acids implicated in the binding site of
Ro5-4864. Mol. Pharmacol. 1994, 46, 1160−7.
(14) Costa, B.; Pini, S.; Gabelloni, P.; Da Pozzo, E.; Abelli, M.; Lari,
L.; Preve, M.; Lucacchini, A.; Cassano, G. B.; Martini, C. The
spontaneous Ala147Thr amino acid substitution within the trans-
locator protein influences pregnenolone production in lymphomono-
cytes of healthy individuals. Endocrinology 2009, 150, 5438−45.
(15) Kreisl, W. C.; Jenko, K. J.; Hines, C. S.; Lyoo, C. H.; Corona,
W.; Morse, C. L.; Zoghbi, S. S.; Hyde, T.; Kleinman, J. E.; Pike, V. W.;
McMahon, F. J.; Innis, R. B.; Biomarkers Consortium PET
Radioligand Project Team. A genetic polymorphism for translocator
protein 18 kDa affects both in vitro and in vivo radioligand binding in
human brain to this putative biomarker of neuroinflammation. J. Cereb.
Blood Flow Metab. 2013, 33, 53−8.
(16) Owen, D. R.; Yeo, A. J.; Gunn, R. N.; Song, K.; Wadsworth, G.;
Lewis, A.; Rhodes, C.; Pulford, D. J.; Bennacef, I.; Parker, C. A.; StJean,
P. L.; Cardon, L. R.; Mooser, V. E.; Matthews, P. M.; Rabiner, E. A.;
Rubio, J. P. An 18-kDa translocator protein (TSPO) polymorphism
explains differences in binding affinity of the PET radioligand PBR28.
J. Cereb. Blood Flow Metab. 2012, 32, 1−5.
(17) Masonis, A. E.; McCarthy, M. P. Direct interactions of
androgenic/anabolic steroids with the peripheral benzodiazepine
receptor in rat brain: implications for the psychological and
physiological manifestations of androgenic/anabolic steroid abuse. J.
Steroid Biochem. Mol. Biol. 1996, 58, 551−5.
(18) Corcia, P.; Tauber, C.; Vercoullie, J.; Arlicot, N.; Prunier, C.;
Praline, J.; Nicolas, G.; Venel, Y.; Hommet, C.; Baulieu, J. L.; Cottier, J.
P.; Roussel, C.; Kassiou, M.; Guilloteau, D.; Ribeiro, M. J. Molecular
imaging of microglial activation in amyotrophic lateral sclerosis. PLoS
One 2012, 7, e52941.
(19) Castellano, S.; Taliani, S.; Viviano, M.; Milite, C.; Da Pozzo, E.;
Costa, B.; Barresi, E.; Bruno, A.; Cosconati, S.; Marinelli, L.; Greco, G.;
Novellino, E.; Sbardella, G.; Da Settimo, F.; Martini, C. Structure-
activity relationship refinement and further assessment of 4-phenyl-
quinazoline-2-carboxamide translocator protein ligands as antiprolifer-
ative agents in human glioblastoma tumors. J. Med. Chem. 2014, 57,
2413−28.
(20) Liang, Y.; Gao, S.; Wan, H.; Wang, J.; Chen, H.; Zheng, Z.; Hu,
X. Syntheses and resolutions of new chiral biphenyl backbones: 2-
amino-2′-hydroxy-6,6′-dimethyl-1,1′-biphenyl and 2-amino-2′-hy-
G
DOI: 10.1021/acs.jmedchem.5b01288
J. Med. Chem. XXXX, XXX, XXX−XXX