Discovery of the Translocator Protein (TSPO)

INTRODUCTION

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Discovery of the Translocator Protein (TSPO)

In humans exist two different forms of benzodiazepine receptors: central benzodiazepine receptors (CBRs) and peripheral benzodiazepine receptors (PBRs).

The first type (CBRs) is found in the brain and forms an allosteric site on the GABA

receptor complex. In fact, ligands acting at this allosteric site, such as diazepam and chlordiazepoxide, enhance the affinity of the γ- aminobutyric acid (GABA) toward the CBR and, in this way, influence chloride (Cl

) influx at the GABA

receptor pore, causing downstream effects on GABA-mediated inhibition.

Different studies have shown benzodiazepines (BZs) acting on CBRs to be responsible for different GABA

-induced effects: BZs are used clinically as muscle relaxants, anticonvulsants, anxiolytics and sedative-hypnotics.

The second type (PBRs) is first described in 1977 by Braestrup and Squires

as an alternative binding site in non-neuronal tissue for the diazepam, a centrally acting benzodiazepine. It was named peripheral according to this tissue distribution and benzodiazepine receptor because BZs is the class of ligands by which PBR was discovered. However, multiple other names have been used to refer to this protein, including mitochondrial benzodiazepine receptor (MBR), mitochondrial diazepam binding inhibitor (DBI) receptor complex (mDRC), PK11195 binding sites (PKBs), isoquinoline binding protein (IBP), Omega3 ( 3) receptor.

In 2006 Papadopoulos and colleagues

proposed the new name

Translocator Protein 18 kDa, abbreviated TSPO. Although the name PBR

was widely accepted - mainly for historical reasons - in the scientific

community, it didn’t take into account the new findings regarding its

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structure, subcellular roles and tissue distribution. It has also been shown that many ligands structurally different from BZs bind to this protein, which is not a receptor in the traditional sense, but rather a translocator of molecules such as cholesterol and protoporphyrins. Henceforth in this thesis work is adopted the abbreviation TSPO instead of PBR.

During their pharmacological studies, Braestrup and Squires

observed a specific binding of [

H] diazepam to receptors in rat kidney, liver and lung, but at the same time noted that binding in non-neuronal tissue appeared to be very different from the specific binding to brain membranes: first, while binding in kidney was associated with mitochondrial fraction, in brain it was associated with a membrane fraction; second, the affinity for receptors in kidney was lower than that for brain binding sites.

Further studies demonstrated that TSPO is also present in nervous tissue and particularly in 1982 Marangos et al. confirmed that TSPO has a brain regional distribution distinct from that of CBR and that it is not linked to GABA-regulated anion channels.

In this key contribution, receptor binding was assayed using diazepam which interacts only weakly with TSPO and the chlorinated analogue of diazepam Ro5-4864 (7-chloro-1,3- dihydro-1-methyl-5-(p-chlorophenyl)-2H-1,4-benzodiazepine-2-one),which has a nanomolar affinity for TSPO (7.3nM)

but a very low affinity for GABA

receptors (163 μM

in rat brain membranes) (Figure 1)

N N

O

Cl N

N O

Cl

Cl

Diazepam (1) Ro 5-4864 (2)

Figure 1. Diazepam (1), an anxiolytic drug with high affinity to CBR and Ro5-4864 (2), that differs from diazepam only by a chloride in the 5’ aromatic ring, substitution that dramatically reduces the activity at the CBR to TSPO.

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Subcellular studies have confirmed the abundant mitochondrial localization of TSPO, although it has also been detected in smaller concentrations in other subcellular compartments such as nuclei, Golgi apparatus, lysosomes, peroxisomes and plasma membrane.

In 1986 Anholt and coworkers investigated the submitochondrial localization of TSPO, using cytochrome oxidase as marker for the inner membrane and monoamine oxidase as outer membrane marker. Treating rat adrenal mitochondria with digitonin, a detergent able to separate the outer membrane from the inner membrane, they noticed that the release of TSPO increased parallel to the release of monoamine oxidase and provided evidence for the association of TSPO with the mitochondrial outer membrane.

Later, with the understanding of molecular structure and functions of TSPO, it has been found that it is particularly concentrated at contact sites between the outer/inner mitochondrial membrane, sites also known as the mitochondrial permeability transition pore (MPTP).

Molecular structure of TSPO

Using photolabeling studies in 1988 Antkiewicz-Michaluk et al.

identified the TSPO monomer, a protein with a molecular mass of 18 kDa, from the rat adrenal gland. It is a very hydrophobic and tryptophan-rich protein of 169 amino acids highly conserved throughout species.

Three-dimensional modeling has revealed a structure with five

transmembrane domains consisting of α-helices composed of 21 residues

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that span the entire membrane bilayer, with a carboxyl-terminal tail located outside the mitochondria and an amino terminal inside the mitochondria

(Figure 2). Subsequently, topographic studies have indicate that the 18 kDa TSPO protein is organized in clusters of four to six molecules to form a single pore, reflecting the recognized function of transporter in the mitochondrial membrane.

Figure 2. Molecular structure of 18 kDa TSPO and localization at the contact site be- tween the outer and inner mitochondrial membrane. It is also shown some proteins as- sociated with TSPO in the MPTP complex (VDAC, ANT and PRAX-1).⁽¹²⁾

In 1992 McEnery et al. reported that TSPO is strictly associated in a trimeric complex with VDAC, a voltage-dependent anion channel of 32 kDa located at sites of contact between outer and inner mitochondrial membrane that acting as a channel allowing passage of small molecules and ions into the mitochondria, and ANT, adenine nucleotide translocase, a specific antiporter of 30 kDa, located in the inner mitochondrial membrane, for the exchange of ATP and ADP as part of oxidative phosphorylation.

Together with other proteins, these three subunits constitute the mitochondrial permeability transition pore (MPTP).

Furthermore, it has been identified four cytosolic TSPO-associated proteins

which most of time play an important role in the biological processes where

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TSPO is involved. p10 is the first cytosolic protein associated to TSPO identified, but whose biochemical role has not been understood yet. It is a protein of 10 kDa that co-immunoprecipited with 18 kDa TSPO using the isoquinoline carboxamide radioactive probe PK14105, a ligand selective for mitochondrial benzodiazepine receptors, to photolabel rat mitochondrial preparations.

PBR associated protein-1 (PRAX-1) is isolated, cloning and characterized by Galiègue et al. in 1999.

It is a protein of 1857 amino acids with a molecular mass of 240 kDa, discovered using the yeast two hybrid screening strategy. Exhibiting various domains involved in protein-protein interaction, such as proline-rich domains, leucine-zipper motifs and Src ho- mology region 3-like (SH

-like) do domains, it has been suggested that PRAX-1 acts as an adaptor protein to recruit additional targets to the vicinity of TSPO so as to modulate its function. In addition, it was as- sumed that a single PRAX-1 protein interacts with the C-terminal end (14 amino acids) of several molecules of TSPO (at least two of them) (Figure 3).

Figure 3. Hypothetical model of the interaction between TSPO and PRAX-1. ⁽¹⁷⁾

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Another important protein functional associated to TSPO is Steroidogenic Acute Regulatory Protein (StAR), that mediates the flow of cholesterol from the outer to the inner mitochondrial membrane, permitting steroid formation in steroidogenic cells. Further studies indicated that StAR acts at the outer mitochondrial membrane and it is not needed to allow the entry of cholesterol into mitochondria. Therefore, Hauet et al. suggest that TSPO and StAR work in concert to bring cholesterol into mitochondria and in particular TSPO serves as a gatekeeper in cholesterol import into mitochondria and StAR plays the role of the hormone-induced activator.

PKA-associated protein 7 (PAP7) is a cytosolic protein with a molecular mass of 52 kDa involved in the hormonal regulation of steroid formation, interacting with both the cytosolic RIα subunit of PKA and TSPO.

Particularly, PAP7 targets the PKA isoenzyme, linked to increased steroid synthesis, which phosphorylating specific protein substrates induces the reorganization of TSPO topography and function.

TSPO distribution in Healty and pathological tissue

The expression of TSPO is ubiquitary, although it considerably varies

among tissues. An high density of TSPO is found in secretory and glandular

tissues, such as adrenal glands, pineal glands, salivary glands, olfactory

epithelium, ependyma and gonads. Intermediate levels of TSPO are

detected in renal and myocardial tissues; in contrast, the brain (in particular

glia, microglia and reactive astrocytes) and liver express in normal

condition relatively low levels of this protein. TSPO are also found among

all human peripheral blood leukocyte subsets, particularly in monocytes,

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polymorphonuclear cells and lymphocytes, and in mature human erythrocytes.

TSPO Altered Expression

TSPO basal expression is up-regulated in a number of human pathologies, including a variety of tumours and neuropathologies, such as gliomas and neurodegenerative disorders (Huntington’s and Alzheimer’s diseases), as well as various forms of brain injury and inflammation.

Furthermore, changes in TSPO receptor levels were found in anxiety and mood disorders.

Recent evidences suggest that TSPO on glial cells may regulate the biosynthesis of neurosteroids, leading to the hypothesis of its potential role as key determinant for the treatment of neuropathological conditions.

TSPO in Cancer

The recent literature on TSPO includes many reports dealing with TSPO and cancer. The rationale behind the potential application of TSPO-targeted therapies in cancer is based on two main features: I) alteration of TSPO expression in tumour cells and II) TSPO-dependent apoptosis modulations.

Considering TSPO expression, some of highest densities of TSPO are

observed in neoplastic tissues and cell lines. Ovarian, hepatic and colon

carcinomas, adenocarcinoma, and glioma all show increased TSPO

densities relative to untransformed tissues. Even higher levels of TSPO

density were observed in more rapidly proliferating breast cancer cells and

more aggressive breast cancer phenotypes. In further, an important

relationship between TSPO expression, proliferation rate and tumour

aggressivity was recently documented by Hardwich and coworkers.

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These data suggest that monitoring TSPO expression may be relevant in the clinic and that the protein could be used as a diagnostic and/or prognostic marker. Some TSPO ligands may have potential as antitumor molecules which would be related to their antiproliferative and pro-apoptotic properties. Consistent with this, FGIN-1-27

and PK 11195

were shown to have antitumoral activities and to induce apoptosis and cell cycle arrest in colorectal, or esophageal cancer cell lines and cancer primary cell cultures. These observations indicate that TSPO is an attractive target in cancer therapy.

TSPO in Inflammation and Auto-immune disease

TSPO is also involved in the regulation of immune responses. In the immune system, TSPO is widely expressed: in the thymus, where the protein is restricted to the medulla, in the white pulp of the spleen, in the lymph nodes, in the Peyer patches of the intestine and in all human peripheral blood leukocyte subsets. As observed for tumoral cell lines, changes in TSPO expression have already been associated with several inflammatory conditions: PK 11195 binding is up-regulated in experimental autoimmune encephalitis, motor neuron axotomy, sciatic nerve degeneration and regeneration.

TSPO ligands exhibit anti-inflammatory properties. They modulate monocyte chemotaxis and humoral responses such as macrophage production of reactive oxygen intermediates, TNF, or IL-1 and IL-6.

Both Ro 5-4864 and PK 11195 were show to limit the severity and

progression of different inflammatory processes. For instance, they were

highly potent at inhibiting inflammatory sings in two mouse models of

acute inflammation, where a treatment with carrageenan induced a paw

oedema formation and pleurisy.

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The potential therapeutic benefit of TSPO ligands is not restricted to arthritis treatment and may be enlarged to other autoimmune pathologies.

The protective effect of Ro 5-4864, PK 11195 and SSR 180575 was also demonstrated in a spontaneous inflammatory skin pathology developed in an in vivo model of lupus erythematosus.

These examples support the rationale that targeting TSPO may have beneficial effect in the treatment of inflammatory disorders and auto-immune pathologies.

TSPO in Neurodegenerative Diseases

A dramatic increase in TSPO density has been observed following experimental injuries of the CNS and in pathological situations. In most lesions, the increase in TSPO density was associated with the inflammatory reaction of the nervous system. These conditions included inflammation,

metabolic stress,

traumatic, or ischemic chemically induced brain injury in animal models.

Similar changes in TSPO density are observed in acute and chronic neurodegenerative states in humans.

For example, temporal cortex obtained from patients with Alzheimer’s

disease, show an increase in TSPO density. A highly significant increase in

TSPO density was also observed in the putamen and a moderate but

significant increase in the frontal cortex of patients suffering from

Huntington’s disease.

Finally, Vowinckel and co-workers observed a

correlation between [

H]PK 11195 binding and inflammation both in vitro

and in vivo in multiple sclerosis and experimental autoimmune

encephalomyelitis.

Given these modulations, labelled PK 11195 and

positron emission tomography (PET) have been used for in vivo imaging of

neuroinflammation in a variety of brain diseases and at different disease

stages to I) detect in patients inflammatory changes and II) monitor the

progression of neuroinflammation as a marker of the disease activity.

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Consistent with these modulations and considering both the steroidogenic potential of TSPO ligands and the antiapoptotic property of the protein, it has been hypothesized that the modulation of TSPO functions could play neurotrophic and neoroprotective roles during neuronal damage. TSPO ligands might promote neuronal survival by acting at different levels, through the regulation of apoptosis which favoured the survival of glial cells and/or the production of mediators such as neurosteroids, cytokines or other neurotrophic factors that support nerve survival. For example in a same study the ligand SSR 180575 and Ro 5-4864 promoted neuronal survival and repair following axotomy.

TSPO in Anxiety

In the brain, neurosteroids such as allopregnanolone and pregnenolone, acting as positive modulators of γ-aminobutyric type A (GABA

) receptors, exert anxiolytic activity. Specific ligands targeting TSPO increase neurosteroid production and for this reason have been suggested to play an important role in anxiety modulation. Unlike benzodiazepines (BZs), which represent the most common anti-anxiety drugs administered around the world, selective TSPO ligands have shown anxiolytic effects in animal models without any of the side effects associated with BZs.

The specific TSPO ligands that are able to promote neurosteroidogenesis may represent the future of therapeutic treatment of anxiety disorders.

Furthermore, TSPO expression levels are altered in several different

psychiatric disorders in which anxiety is the main symptom.

Anxiety is a

normal reaction to stress, which helps people deal with daily difficulties and

cope with them. When anxiety is excessive and disabling, it may become

pathological and fall under the classification of an anxiety disorder

according to the diagnostic manual definition.

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Anxiety disorders are the most common and frequent mental disorders, affecting a significant percentage of the world's population (8-13%), and often overlapping with mood disorders and drug abuse.

Anxiety disorders markedly affect the living conditions of patients. In general, anxiety disorders are treated with medication, specific types of psychotherapy, or both. Treatment choices depend on the problem and the persons preference. The diagnosis is difficult and complex because of the variety of possible causes and symptoms, often arising from highly personalized experiences.

Anxiety disorders are classified as panic disorder (PD), obsessive- compulsive disorder (OCD), post-traumatic stress disorder (PTSD), generalized anxiety disorder (GAD) and social and specific phobias. A combination of pharmacological and genetic manipulation studies has provided forceful evidences that the GABAergic system is strongly linked to the development of anxiety behaviours.

The GABA

receptor is one of two ligand-gated ion channels responsible for mediating the effects of GABA, the major inhibitory neurotransmitter in the brain. In addition to the GABA binding site, the GABA

receptor complex appears to have distinct allosteric binding sites for benzodiazepines, barbiturates, ethanol, inhaled anaesthetics, furosemide, kavalactones, neuroactive steroids, and picrotoxin. The receptor is a multimeric transmembrane receptor consisting of five subunits (the most common type in the brain is a pentamer comprising two α, two β, and a γ subunit (α

β

γ)), arranged around a central pore.

Once bound by endogenous GABA, the protein receptor changes

conformation within the membrane, opening the pore to allow chloride ions

to pass down the electrochemical gradient. Activation of GABA

receptors

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tends to stabilize the resting potential. In that condition, the excitatory neurotransmitters cannot depolarize the neuron generating the action potential, so the net effect is typically inhibitory, reducing the activity of the neuron.

The BZs are a class of psychoactive drugs with anxiolytic, sedative, hypnotic, anticonvulsant, and muscle relaxant properties. BZs bind at the interface of the α and γ subunits on the GABA

receptor (BZR site). The long-term use of BZs can cause physical dependence with risk of withdrawal symptoms and rebound syndrome. However, BZs are the most commonly used medications to treat states of anxiety, and are ever prescribed for short term relief of severe anxiety disorders.

Common medications are lorazepam, clonazepam, alprazolam, and diazepam. Once bound to the BZR, the BZ ligand locks the BZR into a conformation in which the GABA neurotransmitter has a much higher affinity for its receptor. This increases the frequency of opening of the associated chloride ion channel, thereby hyperpolarizing the membrane of the associated neuron.

Neurosteroids synthesised de novo in the brain, modulate neuronal functioning and could play an important pathophysiological role.

Interestingly, a variety of neurological and psychiatric disorders, such as anxiety, depression, and schizophrenia, have been associated with both abnormal levels of neurosteroids and perturbations of neurotransmission.

Some of the endogenous neurosteroid GABA

modulators are

allopregnanolone (3α,5α-tetrahydroprogesterone, ALLO), pregnenolone

(PREG), and tetrahydrodeoxycorticosterone (THDOC). In addition,

dehydroepiandrosterone sulphate (DHEAS), the most abundant steroid

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found in the body, exhibits a bimodal effect on GABA

receptors: it demonstrates positive allosteric modulation at low nanomolar concentrations, and negative allosteric modulation at high micromolar concentrations. The anxiolytic properties of DHEAS may also occur since it is metabolised to androsterone and androstanediol, which are positive modulators of the GABA

receptor.

It has been recognized that neurosteroids are synthesized from cholesterol in the CNS by a cascade of enzymatic processes, controlled by both P450 and non-P450 cytochrome enzymes.

The first step in the neurosteroid synthesis is cholesterol conversion to pregnenolone, which occurs in mitochondria and is facilitated by TSPO.

This step is the rate-limiting reaction of neurosteroid synthesis and appears to regulate the neurosteroid levels in the brain. The binding of TSPO ligands induces cholesterol transport and steroid formation, leading to the formation of active neurosteroids (ALLO), which positively modulate the activity of the GABA

receptor potentiating GABAergic transmission and thus explaining the anxiolytic activity, as primarily shown in animal models of anxiety.

This evidence has suggested TSPO involvement in the secretion of neurosteroids, whose levels have been reported to be changed in several diseases and to be implicated in the pathogenic mechanisms of anxiety.

Indeed, the important involvement of TSPO in anxiety in humans has been

extensively demonstrated by numerous studies in which TSPO expression

levels have been reported to be changed. Specifically, TSPO density is up-

regulated in acute stress conditions and down-regulated in chronic or

repeated stress. A decrease of TSPO density has been shown in blood cells

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(platelets or lymphocytes) of patients affected by different psychiatric disorders, mainly characterized by anxiety, such as generalized anxiety disorder, generalized social phobia, post-traumatic stress disorder, panic disorder, chronic obsessive–compulsive disorder and also in suicidal patients,

as well as in healthy subjects with high trait anxiety levels.

Has been demonstrated that TSPO expression was significantly decreased in lymphocytes of patients with post-traumatic stress disorder,

and in platelets of patients with panic disorder or major depressive disorder associated with severe separation anxiety symptoms.

Functional roles of TSPO in Biological Processes

Regulation of Steroidogenesis

The most extensively characterized function of TSPO concerns its role in steroid biosynthesis. Two important observations suggest that TSPO plays an important role in steroidogenesis: I), the location of this protein on the outer mitochondrial membrane and II), the extremely high density in steroidogenic endocrine tissues, such as adrenocortical cells and Leydig cells. Moreover, different publications report that TSPO ligands stimulate steroid biosynthesis in adrenal, placental, testicular, ovarian and glial systems.

The biosynthesis of steroids begins with the enzymatic transformation of

cholesterol into pregnenolone, which occurs through cholesterol side-chain

cleavage by the cytochrome P450scc (also known as CYP11A1) and

auxiliary electron transferring proteins, localized on the matrix side of the

inner mitochondrial membrane (Figure 4).

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Pregnenolone then leaves mitochondria to move to the endoplasmic reticulum, where it is transformed in the final steroid products.

The rate-limiting step is the translocation of cholesterol from the cellular stores across the aqueous intermembrane space to the inner mitochondrial membrane and P-450scc. The fundamental role of TSPO in this mitochondrial cholesterol transport and thus in steroids synthesis is supported by numerous data, and particularly knockout and antisense experiments in vitro have demonstrated that down-regulation of TSPO causes a decrease in steroid synthesis.

The topographic study of TSPO in the mitochondrial membranes have also shown that after treatment of Leydig cells with a steroidogenesis stimulator, such as choriogonadotrophin hormone (hCG), there are various morphological changes, such as a formation of large complexes of 15-25 molecules of TSPO and a rapid reorganization of their localization in the mitochondrial membrane, as well as a rapid increase in TSPO ligand binding.

Moreover, amino acids deletion, site-directed mutagenesis and structural studies have permitted to identified a cholesterol recognition amino acid consensus (CRAC) sequence in the cytosolic carboxy-terminal domain of the TSPO that could be part of the binding site for the uptake and

Figure 4. Cholesterol metabolism in hepatic and steroidogenic cells. Cholesterol binds to TSPO and its transported up on binding of endozepine also called DBI. In hepatic cells, cholesterol is hydroxylated by cytochrome

P450 (CYP27A1) to give 27-

hydroxycholesterol, whereas in steroidogenic cells, cholesterol is cleavage by another cytochrome P450 (CYP11A1) that cleave side chain to give pregnenolone.

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translocation of cholesterol (channel-like interaction) through a channel delimited by five α-helixes of TSPO. However, for cholesterol delivery into mitochondria TSPO-mediated is also required the interaction of TSPO with StAR, as referred above, a protein acting as hormone-induced activator.

It has been observed a relationship between steroid levels, TSPO levels and anxiety, principally due to the fact that neurosteroids are endogenous modulators of the GABA

receptor (Figure 5).

In general TSPO levels, determined by radioligand binding to platelets, decrease in patients with anxiety disorders. Therefore, ligands binding to TSPO located in glial cells, such as N,N-dialkyl-2-phenylindol-3- ylglyoxylamides synthesized by Da Settimo et al.

, provide the cholesterol necessary to restore neurosteroid synthesis and increase pregnenolone formation, which is then metabolize to form allopregnanolone, a potent allosteric modulator of the GABA

exerting anxiolytic effects. In this sense TSPO could be considering a promising target for the psychiatric disorders that involve dysfunction in steroid biosynthesis.

Figure 5. Schematic representation of TSPO-mediated regulation of neurosteroid bio- synthesis and its role in neurological disorders.⁽¹⁸⁾

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Regulation of apoptosis

TSPO is a component of the mitochondrial permeability transition pore (MPTP), a multiprotein complex located at the contact site between the inner and outer mitochondrial membranes. MPTP plays an important role in the modulation of signaling pathways mediating apoptotic and necrotic cell death.

The exact composition of the MPTP is not yet established, but it has been recognized various proteins implicated in pore formation and its regulation (Figure 6): an hexokinase, in the cytosol; a trimeric complex constituted by VDAC, ANC and TSPO (see “Molecular Structure of TSPO”); a creatine kinase in the intermembrane space and cyclophilin D in the matrix (for more information see ref. 51).

Figure 6. Schematic structure of the MPTP.⁽¹³⁾

MPTP allows the transfer of solutes, including ATP/ADP exchange, from

the mitochondrial matrix to cytosol, through the VDAC/ANC conduit, and

therefore facilitates the crossing of the highly impermeable mitochondrial

inner membrane. This periodic transient increase in permeability by the

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MPTP allows the pumping of protons from the inner membrane by the electron transport chain and creates the transmembrane electrochemical gradient that drives ATP synthesis.

Several factors cause the opening of the MPTP: high [Ca

] is the fundamental trigger but alone is not enough. Other important MPTP sensitizers are low adenine nucleotide concentrations, high phosphate concentrations, oxidative stress and pro-apoptotic proteins such as Bcl-2.

Pore opening leads to the dissipation of transmembrane electrochemical gradient, uncoupling of mitochondria and swelling, resulting in the release of cytochrome c and apoptosis inducing factor (AIF) into the cytosol. Once in the cytosol, the first induces the caspase cascade ending in the destruction of cell nucleus, cytoskeleton and plasma membrane; the second principally leads to nuclear chromatin condensation, DNA fragmentation and then to cell death

.

TSPO is an endogenous modulator of apoptotic process but the exact

molecular events involved have not yet been definitively clarified. A

possible mechanism for regulating the apoptotic pathway by TSPO is

schematically illustrated in Figure 7. In 2008 Veenman et al.

suggested

that interaction between VDAC and TSPO is fundamental to initiate the

mitochondrial apoptosis pathway. The intermediary agent between TSPO

and VDAC was supposed to be provided by mitochondrial ROS (reactive

oxygen species) generation under the control of the same TSPO. ROS lead

first to dissociation of cytochrome c, from oxidized cardiolipins located at

the inner mitochondrial membrane and, subsequently to its release in the

cytosol via formation of a pore due to assemblage of VDAC molecules.

26 Figure 7. TSPO regulation of the mitochondrial apoptosis pathway.⁽⁵²⁾

Another possible mechanism was supposed in 2007 by Azarashvili et al.

that provided evidence for TSPO involvement in MPTP opening, controlling the Ca

-induced Ca

efflux and AIF release from mitochondria, important stage of initiation of programmed cell death.

It has also been observed a modulation by TSPO of interactions between

VDAC or ANT and pro-apoptotic or anti-apoptotic proteins (Bcl-2 and Bax,

respectively).

Therefore, it has been designed TSPO ligands with pro-

apoptotic effects acting as anticancer agents. In 2005 Chelli et al.

proposed PIGA, a ligand chosen from the N,N-dialkyl-2-phenylindol-3-

ylglyoxylamide series, as a novel pro-apoptotic compound with therapeutic

potential against glial tumours. TSPO-binding ligands have also been

widely explored as carrier for receptor-mediated drug delivery, such as

TSPO ligand-Ara-C conjugates

and Pt complexes.

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Immunomodulation

TSPO plays an important role in host defense mechanisms and inflammatory response. In fact, it is expressed in a wide range of cells involved in the regulation of the immune responses such as microglia, blood monocytes and leukocytes. It has been shown that some TSPO ligands, specifically benzodiazepines, have an immunosuppressive action, inhibiting the capacity of macrophages to produce ROS and inflammatory cytokines such as IL-1 (interleukin-1), TNFα (tumor necrosis factor alpha) and IL-6, and regulating phagocyte oxidative metabolism required for elimination of foreign antigens.

This anti inflammatory action TSPO- mediated is also due to its capacity of stimulate glucocorticoid synthesis in steroidogenic organs and in local tissue damage.

In CNS, TSPO is primarily expressed in microglia and in astrocytes.

Several studies have demonstrated that under conditions resulting in glial activation, such as inflammation and metabolic stress, basal levels of TSPO in these cells can increase in a time-dependent manner. This up-regulation of TSPO has been observed in traumatic, ischemic and chemically-induced brain injury, in neurodegenerative disorders, i.e., in the temporal cortex from patients with Alzheimer’s disease and in the putamen of patients suffering from Huntington’s disease, in the epilepsy, multiple sclerosis and experimental autoimmune encephalomyelitis.

Activation of microglia consists in migration to the site of damage,

proliferation, production and release of potent neuroinflammatory cytokines

(TNFα, IL-1β), arachidonic acid derivatives such as cyclooxygenase-2,

excitatory amino acids, and ROS. As inflammatory responses mediated by

the activation of microglia may aggravate in neuronal damage, and TSPO

has proved to be a promising marker for activated microglia, it has

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attempted to develop specific TSPO ligands able to prevent or limit neuroinflammation or to label the same activated microglia to track the progression of neuroinflammation.

Other functions

Other functions, some of which are linked to those previously mentioned,

have been attributed to TSPO including protein import, important for

membrane biogenesis and confirmed by the observation that TSPO is

necessary for the import of StAR protein into mitochondria; TSPO binding

of dicarboxylic porphyrin and transport into mitochondria have been

reported as well as a relationship between TSPO and heme biosynthesis

pathway; ion transport and calcium homeostasis, since it has been shown

that TSPO regulates the Ca

flow into the cell. TSPO plays an important

role in cellular respiration and mitochondrial oxidation, and affects cellular

proliferation and differentiation in a number of cell types.

Several studies

have also shown that TSPO can modulate responses to viral infections. In

fact, pathogenic virus targets TSPO to inhibit dissipation of the

transmembrane mitochondrial potential and prevents the release of

cytochrome c, resulting in the blockade of apoptosis and overcoming of the

host responses against viral infections.

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TSPO Endogenous and Synthetic Ligands

TSPO Endogenous Ligands

A wide variety of endogenous molecules with affinity for the TSPO and different chemical structures have been identified.

The Protoporphyrins (protoporphyrins IX, mesoporphyrins IX, deuteroporphyrins IX, hemin) exhibit a very high affinity for TSPO. As several steroidogenic tissues, such as the adrenal gland and testis, show high TSPO and porphyrin levels, it has been suggested a physiological role for the interaction of these two molecules. Furthermore, having a plane of symmetry (Figure 8), these molecules could bind dimerized form of TSPO, confirming in this way the postulated two-binding site model.

(3) (4)

Figure 8. Chemical structures of protoporphyrin IX (3) and heme (4).

Another endogenous ligand is cholesterol, as previously reported discussing

the fundamental role of TSPO in the regulation of cholesterol transport and

thus in the steroidogenesis. Again it has been shown that a dimeric form of

TSPO possesses an enhanced binding capacity to cholesterol.

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In 1984 Corda et al. isolated and purified a neuropeptide able to inhibit diazepam binding to BZ binding site on brain membranes. This 11 kDa polypeptide, named DBI (diazepam binding inhibitor), is constituted by 86 amino acids and binds with low affinity both TSPO and CBR, while a short fragment of DBI, triakontatetra-neuropeptide, is more selective for TSPO.

Furthermore, DBI promotes loading of cholesterol to the cytochrome P- 450scc and stimulates steroidogenesis by directly interacting with TSPO.

Finally, anthralin, a 16 kDa protein that has been demonstrated interact with both TSPO and dihydropyridine binding site, and phospholipase A2 have also been proposed as endogenous ligands for TSPO.

TSPO Synthetic Ligands

Synthetic TSPO selective ligands have been developed with the aim to deepen the knowledge of the exact pharmacological role of TSPO, to define its involvement in several patho-physiological conditions and to establish the structural requirements needed for an optimum of affinity.

Initially, the most of these ligands have been designed starting from classical selective CBR ligands, such as benzodiazepines, making the necessary structural modifies in order to shift the affinity toward TSPO.

Until to date, the prototypic ligands, used as reference compounds in the

development of TSPO pharmacophore models and in SAR studies, are the

benzodiazepine Ro 5-4864 (2, Figure 1) and the isoquinolinecarboxamide

PK11195 (5, Figure 9), classified by LeFur and coworkers the first as a

TSPO agonist or partial agonist and the second as an antagonist.

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Subsequently, a number of diversified structures have been developed obtaining in some cases good results in term of affinity and selectivity, and leading to drawing various TSPO pharmacophore models useful for designing novel synthetic derivatives. Therefore, the topology of the TSPO binding cleft has not yet been completely defined.

TSPO specific synthetic ligands do not have a typical shared structure and thus can be distinguished nine different classes of compounds: (Figure 9)

I) In the class of benzodiazepines the selectivity toward the TSPO or CBR results very sensitive even to slight structural modifications: in fact, as described earlier in Figure 1, in the case of Ro 5-4864 (2) the insertion of a chlorine at the para position of the pendant phenyl rings of the diazepam, equipotent at the two receptors, shifts the selectivity toward TSPO. Ro 5- 4864 has been used in a number of studies aimed at characterizing TSPO, and particularly by Gavioli and colleagues to study the putative role of TSPO as a target for the treatment of psychiatric disorders.

II) Benzothiazepines, a class of TSPO ligands featuring a 6,7 bicyclic nucleus, were initially developed by Campiani and coworkers as selective ligands for CBR and GABA receptor subtypes. Some pyrrolobenzothiazepines derivatives possess an unexpected significant inhibitory activity at L-type calcium channels, equal to or higher than those of reference calcium antagonists such as verapamil and (+)-cis-diltiazem.

Some examples are THIA-66 (R=COCH

; R'=H; X=OCH

), THIA-67

(R=SO

CH

; R'=H; X=OCH

), displayed nonomolar affinity and notable

selectivity for TSPO binding sites; however they were less potent than PK

11195. To date, THIA-4i (R=CON(Et)

; R'=4-Cl; X=H) has been reported

as the most potent TSPO ligand from this class with an affinity comparable

to that of the gold standard TSPO ligand PK 11195. Although these ligands

32

might be useful probes for in vitro studies, further evaluation is required to elucidate their in vivo pharmacological profile.

(11, Figure 9)

III) On the basis of pyrrolobenzothiazepine skeleton, the pyrrolobenzoxazepine scaffold has been developed, featuring the replacement of the endocyclic sulfur (S5) with an oxygen (O5), that increases affinity by 2-3 fold. A number of these compounds displayed K

values in the subnanomolar range. OXA-17f (R=OCON(Et)

; X=H) and OXA-17j (R=OCON(Et)

; X=CH

) were reported as the most potent TSPO ligands from this series with K

values of 0.26 and 0.36 nM, respectively compared to K

value of 0.78 nM for PK 11195 in the same assay. All of these benzodiazepine-like compounds were shown to stimulate neurosteroids production with comparable potency to PK 11195 and Ro 5- 4864 in mouse Y-1 adrenocortical cell line.

(12, Figure 9)

IV) Isoquinolinecarboxamide PK 11195 (5) is the first non-

benzothiazepine ligand binding the TSPO with nanomolar affinity and it is

widely used for studies aimed to define and map the binding site. In 2008,

Chelli et al.

showed that treatment of a human astrocytoma cell line

(ADF) with PK 11195 actives an autophagic pathway followed by

apoptosis mediated by mitochondrial potential dissipation. Moreover, has

been showed that PK 11195 has a multidrug resistance modulating activity

increasing the efficacy of a daunorubicin treatment on human multidrug-

resistant leukemia cell line in vitro by 5-7 fold and blocking p-glycoprotein

efflux, a transporter whose activity contributes to limit antitumor drug

efficacy.

33

N Cl

N O

N N

Cl

N

O O

Cl N

O O

PK 11195 (5) Alpidem (6) DAA 1097 (7)

N N N

N O

O F

X N

H

R R1

N O

R₂ R₃

N N N

H O N

O

DPA 714 (8) FGIN-1 (9) SSR180585 (10)

S

R' ^OR

N

X

O R N

X

Figure 9. Structures of the some representative ligands of the most important classes of compounds selective for TSPO.

V) Imidazopyridines and Phenoxyphenyl-Acetamides are the pro- genitor of the class of Imidazopyridines has to be considered Alpidem (6, Figure 9), known to bind both TSPO and BZR with nanomolar affinity (K

0.5-7 nM and 1-28 nM, respectively).

Alpidem possesses anxiolytic activity with a profile that is substantially different from that of BZs. Was released in 1991 (Ananxyl

, Sanofi- Aventis) as a highly active anxiolytic agent and it was generally prescribed to patients with moderate to severe anxiety, particularly when these patients

THIA-66; 67; 4i (11) OXA-17f; 17j (12)

34

exhibited either sensitivity or resistance to benzodiazepine therapy.

Alpidem was withdrawn from the market in most of the world following reports of severe liver damage caused by Ananxyl.

In 1997, Trapani’s research group reported a wide SAR study on alpidem, developing a series of 2-phenylimidazo[1,2-a]pyridine derivatives (CB) that showed various degrees of affinity and selectivity for TSPO with respect to BZR, dependent on the value of n and the nature of the various substituents R

, R

, X, Y, Z.

(Figure 10)

N N

Z X

Y

( )n CONR1R2 1

2

4 3 5 6 7

8

Three lead compounds selected from this series, CB 34, CB 50, and CB 54 were investigated for their ability to stimulate central and peripheral steroidogenesis and to produce anticonflict action in rats.

(Figure 11)

N N

Cl Cl

Cl

CON(n-C₃H₇)₂

N N

Cl Cl

CON(n-C₃H₇)₂

N N

Br

CH₃

CON(n-C₃H₇)₂

CB 34 CB 50 CB 54 Figure 11. Imidazopyridines tested in vivo as anxiolytics.

In in vitro experiments performed on cerebral cortex membrane, all three compounds potently inhibited [

H]PK 11195 binding in the order CB 34 (IC

1.03 nM) > CB 54 (IC

1.54 nM) > CB 50 (IC

3.04 nM), without substantially affecting [

H]flunitrazepam binding to BZRs.

Figure 10. General formula of 2-phenylimidazo[1,2-a]

pyridine derivatives CB.

35

Intraperitoneal administration of CB compounds in rats resulted in significant dose-dependently increased concentrations of neuroactive steroids, such as pregnenolone, progesterone, allopregnanolone, and allotetrahydrodeoxycorticosterone (THDOC) in plasma and brain.

CB 34 also increased the brain concentration of neuroactive steroids in adrenalectomized-orchiectomized rats, although to a lesser extent than in sham-operated animals. The effect of these ligands on neurosteroid concentration was attributable to increased steroidogenesis in the brain, rather than to an increased supply from peripheral sources. In this view, CB 34 appeared to be a promising compound, which, acting as TSPO agonist, increased neuroactive steroid concentration, and proved beneficial for the treatment of stress and anxiety related diseases.

In 2004, the research group of Nakazato and colleagues from the Taisho Pharmaceutical and Co. described a novel class of selective TSPO ligands derived from a structural simplification of the bicyclic structure of the benzodiazepine Ro 5-4864, which is the opening of the diazepine ring. The resulting Phenoxyphenyl-Acetamide derivatives with general formulae DAA, were synthesized and evaluated for their affinity and selectivity for TSPO in an extensive SAR study.

(Figure 12)

N N H₃C O

Cl

Cl

N

O X₁

CH₃

CH₃ O

N

O X₁

Y

R₁ O

Ar₂ Ar₁

Ro 5-4864 DAA-I DAA

Figure 12. Design of DAA derivatives starting from Ro 5-4864.

36

The authors focused their attention on chemical modification of Ar

, Ar

, R

, X

, and Y, obtaining a number of compounds with remarkable affinity and selectivity, so that to define the most important structural features for this class of compounds in binding to TSPO.

Two phenoxyphenylacetamide derivatives, (DAA 1106 and DAA 1097) (Figure 13), have been extensively characterized respect to their receptor binding and behavioural profile, as well as to their effect on steroidogenesis.

N

O CH₃ O

OCH₃ H₃CO

F N

O CH₃ O O

Cl H₃C

CH₃

DAA 1106 DAA 1097

Figure 13. Structure of DAA 1106 and DAA 1097 derivatives

Both DAA 1106 and DAA 1097 inhibited [

H]PK 11195 and [

H]Ro 5- 4864 binding to crude mitochondrial preparations of rat whole brain with IC

values in the subnanomolar range (0.92 and 0.28 nM, and 0.64 and 0.21 nM, respectively).

These compounds were also highly selective, as they did not inhibit the binding of BzR ligand [

H]flunitrazepam (IC

>

10,000 nM) in the same preparation, and showed weak or negligible affinity (IC

values are approximately 10,000 nM) for 58 other receptors, including those of related neurotransmitters, ion channels, uptake/transporter and second messengers.

In a separate study the same research group performed an exhaustive

characterization of the binding of DAA 1106 in the mitochondrial fractions

37

of rat brain by means of its [

H]radiolabelled analogue. The binding of [

H]DAA 1106 was saturable, with a dissociation constant (K

) determined from a Scatchard plot analysis of 1.2 nM. The binding was inhibited by several TSPO ligands in an order similar to that observed for [

H]PK 11195, with DAA 1106 being the most potent inhibitor. In addition, [

H]DAA 1106 was highly TSPO selective, as its binding was not affected by several neurotransmitter-related compounds, including adrenoceptor, GABA, dopamine, 5-hydroxytriptamine (5-HT), acetylcholine, histamine, glutamate and BZR ligands even at a concentration of 10 μM.

When tested on mitochondrial fractions of the rhesus monkey cerebral cortex, [

H]DAA 1106 showed a high affinity for TSPO (K

0.426 nM), with its binding being potently inhibited by DAA 1106 and PK 11195, but not by Ro 5-4864, even at 1 μM. The results indicated that this ligand was not species dependent, similar to PK 11195 and contrary to Ro 5-4864.

Autoradiography and biochemical studies on rat brain showed that the highest binding of [

H]DAA 1106 is localized in the olfactory bulb and ventricular structures related to the secretion of cerebrospinal fluid (choroid plexus), followed by the cerebellum and cerebral cortex.

In in vivo studies, DAA 1106 and DAA 1097 showed potent anxiolytic-like properties in laboratory animals, in both the light/dark exploration test in mice and the elevated plus-maze test in rats.

Even though these two phenoxyphenylacetamides have a similar structure,

potently bind to TSPO and exert anxiolytic effects, their effects on

steroidogenesis appeared to be opposite. Culty and colleagues

examined

the effects of these two ligands on steroidogenic response using MA-10

Leydig tumour cells, C6-2B glioma cells, and rat brain mitochondria.

38

It has been observed that DAA 1097 activated steroidogenesis in all the three preparations in a similar fashion to that described for PK 11195, and more efficiently on brain than Leydig cells. Surprisingly, DAA 1106 did not activate steroidogenesis, despite its high affinity for TSPO. It has been suggested that the DAA 1097 and DAA 1106 binding sites on TSPO share a common domain with that of PK 11195, but also appear to contain additional motif(s) that do not interact efficiently with PK 11195 site.

The different effect on steroidogenesis has been rationalized by hypothesizing that the binding of DAA 1097 induce changes in the receptor similar to that triggered by PK 11195, allowing steroidogenesis activation.

On the contrary, the binding of DAA 1097 leads to conformational changes that do not permit or antagonize TSPO steroidogenic function. It has been thus concluded that DAA 1097 appears to be an agonist and DAA 1106 may act as a competitive antagonist.

VI) Pyrazolopyrimidineacetamides, i.e. DPA714 (8, figure 9) Binding assays, using [

H]PK 11195 as the radioligand and membranes from rat kidney tissue as receptor source, showed that a subset of ligands from this pyrazolopyrimidine series displayed high binding affinity

K

) for the TSPO ranging from 0.8-6.1 nM. Some of these compounds were shown to stimulate steroid biosynthesis in C6 glioma rat cells with a few capable of increasing pregnenolone synthesis with similar potency to Ro 5-4864 and PK 11195.

VII) Indoleacetamide derivatives, collectively named FGIN-1 (9, figure

9) SAR studies allowed the authors to identify the structural features of

FGIN-1 derivatives essential for high affinity: I) the presence of two alkyl

groups on the amide nitrogen; II) the length of such alkyl chains, with the

N,N-dihexyl groups conferring an optimum of affinity, twenty fold higher

39

than the corresponding N,N-dimethyl analogues; III) halogenation of the pendant 2-aryl groups and the indole benzofused ring (X and R

= Cl, F).

Broad screening studies revealed that these FGIN-1 derivatives were highly TSPO selective, as they failed to bind with any significant affinity (K

>

1000 nM) to other receptor systems, including the GABA

, GABA

, glycine, glutamate, dopamine, serotonin, opiate, cholecystokinin, beta adrenergic, cannabinoid, and sigma receptors. Compounds exhibiting the highest TSPO binding affinity were selected to evaluate their ability to stimulate pregnenolone formation from the mitochondria of C6-2B glioma cells.

In a recent study SSR180575 (10, figure 9), an indoleacetamide compound, was shown to have neuroprotective properties in different models of progressive degeneration of the PNS and CNS: precisely it promotes neuronal survival and repair following axotomy through the regulation of apoptosis of glial cells and/or the production of mediators such as neurosteroids, cytokines or other neurotrophic factors that support nerve survival.

The increase of pregnenolone in the brain implying the therapeutic effects may be mediated through its ability to stimulate steroidogenesis.

VIII) Indol-3-ylglyoxylamides (for more information see next section).

N,N-Dialkyl-2-Phenylindol-3-Ylglyoxylamides

In 2004 Primofiore et al.

prepared and tested a series of N,N-dialkyl-2-

phenylindol-3-ylglyoxylamide derivatives I (Table 1) as TSPO ligands,

designed as conformationally constrained analogues of the

40

indoleacetamides of the FGIN (9, figure 9) series previously described by Kozikowski and coworkers.

They observed that most of these compounds showed a high affinity for TSPO in the nanomolar to subnanomolar range and a high selectivity for TSPO over CBR. The TSPO/CBR selectivity was evaluated by binding studies using membranes from rat brain tissues and [

H]flumazenil as radioligand. For some of these TSPO ligands with high affinity, the ability to stimulate pregnenolone formation from rat C6 glioma cells was evaluated.

In 2008 Da Settimo et al.

refined TSPO pharmacophore/topological model through the synthesis and the biological evaluation of indole derivatives with the general formula I, bearing different combinations of substituents R

-R

. The SARs of these compounds were rationalized in light of a pharmacophore/topological model of TSPO binding site made up of three lipophilic pockets (L

, L

and L

) and a H-bond donor group (H

) (Figure 14). Specifically:

I) The second carbonyl group of the oxalyl bridge engages an H-bond with the donor site H

;

II) The two lipophilic substituents R

and R

(linear or ramified alkyl, arylalkyl group) on the amide nitrogen interact hydrophobically with the L

or L

lipophilic pockets;

III) The 2-phenyl group establishes π-stacking interactions within the L

pocket

41

N

R₆ R₅

R₄

R₃

O N

O

R₂ R₁

H₁

L₁

L₄

L₃

PK 11195 N,N-dialkyl-2-phenylindol-3-ylglyoxylamides Figure 14. TSPO pharmacophore/topological model.

In Table 1 (pages 43-46) has been reported the binding affinity of compounds Ia-Iaah (data taken from ref. 49, 65) and of the standard TSPO ligands Ro5-4864 (2), PK 11195 (5) and alpidem (6), expressed as K

values.

It has been observed that, among the unsubstitued derivatives Ia-Im (R

= R

= R

= R

= H), N-monosubstitued compounds Ia-Ib show the lowest affinity probably because they cannot occupy both the L

and L

lipophilic pockets. The N,N-disubstitued indolylglyoxylamides instead exhibit a high affinity and in particular compound Ig, bearing two n-hexyl groups, is the most potent, with a K

of 1,4 nM. Therefore, increasing the length of the linear N-alkyl groups has been observed an enhancement of affinity, due to the better filling of L

and L

lipophilic pockets.

Subsequently, the three derivatives Id, Ie, Ig and Ij has been selected as leads for further affinity optimization efforts. It has been observed that

:

N Cl

N O

H₁

L₁

L₃

L₄