4
LIST OF ABBREVIATION
GABA, γ-aminobutyric acid
BZ, benzodiazepine
TSPO, translocator protein
CBR, central benzodiazepine receptor
PBR, peripheral benzodiazepine receptor
PNS, peripheral nervous system
CNS, central nervous system
VDAC, voltage-dependent anion channel
ANT, adenine nucleotide translocase
MPTP, mitochondrial permeability transition pore
PRAX-1, PBR associated protein-1
StAR, steroidogenic acute regulatory protein
PAP7, PKA-associated protein 7
P450scc, cytochrome P450 side-chain cleavage
CRAC, cholesterol recognition amino acid consensus
hCG, human choriogonadotrophin
ROS, reactive oxygen species
DBI, diazepam binding inhibitor
SAR, structure-activity relationship
PET, positron emission tomography
LOR, line of response
MBR, mitochondrial benzodiazepine receptor
IBP, isoquinoline binding protein
mDRC, mitochondrial diazepam receptor complex
PKBs, PK11195 binding sites
ANT, adenine nucleotide translocase
PPA, polyphosphoric acid
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FIGURES AND TABLES
Figures
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.
9
Figure 2. Molecular structure of 18kDa TSPO and localization at the contact site
between the outer and inner mitochondrial membrane. It is also shown some proteins
associated with TSPO in the MPTP complex (VDAC, ANT and PRAX-1).(12)
11
Figure 3. Hypothetical model of the interaction between TSPO and PRAX-1.(17)
12
Figure 4. Cholesterol metabolism in hepatic and steroidogenic cells. Cholesterolbinds 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.
22
Figure 5. Schematic representation of TSPO-mediated regulation of neurosteroid
bio-synthesis and its role in neurological disorders.(18)
23
Figure 6. Schematic structure of the MPTP.(13)
24
Figure 7. TSPO regulation of the mitochondrial apoptosis pathway.(52)26
Figure 8. Chemical structures of protoporphyrin IX (3) and heme (4).29
Figure 9. Structures of the some representative ligands of the most important classesof compounds selective for TSPO.
33
Figure 10. General formula of 2-phenylimidazo[1,2-α] pyridine derivatives CB.
34
Figure 11. Imidazopyridines tested in vivo as anxiolytics.34
Figure 12. Design of DAA derivatives starting from Ro 5-4864.35
Figure 13. Structure of DAA 1106 and DAA 1097 derivatives.36
Figure 14. TSPO pharmacophore/topological model.41
Figure 15. Energetic Transitions of Electrons During Fluorescence.51
Figure 16. Stokes shift52
Figure 17. 7-nitrobenzofurazane (NBD), dansyl and the naphthalimide.54
Figure18. Pharmacophore/topological model (13) and structure of IIIf (14)55
Figure 19. Rat C6 glioma cells stained with 10 μM IIIf (A,B); cells stained with 10μM IIIf in the presence of 10 μM PK 11195 (C); cells stained with 10 μM IIIf in the presence of 10 μM Io (D); and cells stained with 10 μM IIIf in the presence of 50 μM PK 11195 (E). (70)
56
Figure 20. Positron emission and annihilation. (72)
59
Figure 21. Coincidence detection (author Jens Lang)60
Figure 22. Chemical structures of various PET tracers for imaging of the TSPO.62
Figure 23. TSPO pharmacophore/topological model.65
Figure 24. N,N-dialkyl-2-phenylindol-3-ylglyoxylamides (I).70
Figure 25. Diagram of a generic radiolabeled probe: ligand acts as a carrier for
specific biological target, thanks to its selective affinity in receptor binding (biological target) and a linker spacer (linker) was inserted between ligand and the chelator bifunctional.
72
6
Figure 26. Final compounds.
72-73
Figure 27. Displacement of [3H]Ro 5-4864 by compound 14 in rat kidney mitochondria membranes. Bound radioactivity is expressed as a percentage of specific binding in the presence of competitor molecule. Points represent the mean values ± SEM of triplicate determinations pooled from three independent experiments.
87
Figure 28. Time course of compound 14 binding to receptor. The semi-logarithmic
representation of the decrease in [3H]Ro 5-4864 binding to TSPO by pretreatment (0,
10, 30, 60, 90, and 180 min) with various concentrations of compound 14 is shown. Points represent the mean values ± SEM of duplicate determinations pooled from three independent experiments.
88
Figure 29. Human glioma cell staining with the irreversibile fluorescent TSPO ligand
14. The cells were incubated with compound 14 for 90 min, in the absence or presence of Ro 5-4864 (grey bars). Data were obtained from 14 independent assays and are shown as mean values of Fluorescence Intensity ± SEM.
90
Tables
Table 1. TSPO binding affinity of N,N-dialkylindolylglyoxylamide derivatives
Ia-Iaah.
43
Table 2. Properties of commonly used positron emitting radioisotopes.(72)
58
Table 3. TSPO binding affinity of N,N-dialkylindolylglyoxylamide derivatives 16-26compared to those of the unmethylated counterparts (Id, Ie, Ig, Ih, Ij, In, Ip, Iv, Isss,
Ittt, Iuuu, Izzz, Iaab).
67
Table 4. Values of IC50 and Ki for Compound 14 obtained from Three Independent
Equilibrium Binding Assays (A).
