logo SBA


Digital archive of theses discussed at the University of Pisa


Thesis etd-01232019-145802

Thesis type
Tesi di dottorato di ricerca
Thesis title
Neuroprotective strategies: steroidogenesis and Translocator Protein (TSPO, 18 kDa). The kinetic parameter Residence Time
Academic discipline
Course of study
tutor Prof.ssa Costa, Barbara
  • EAE
  • multiple sclerosis
  • neuroprotection
  • Residence Time
  • steroidogenesis
  • Translocator Protein
  • TSPO
Graduation session start date
The pharmacological stimulation of neurosteroidogenesis via TSPO ligands leads to benefic pleiotropic effects in several neurodegenerative conditions and psychiatric disorders. Generally, drug discovery processes are based on the paradigm of binding affinity (thermodynamic equilibrium constant, Ki); nonetheless, for TSPO ligands this approach led to inconsistent results due to the poor correlation between binding affinity and efficacy. In the last few years, the Residence Time of a drug on its target (the reciprocal of the dissociation constant, RT=1/koff), is emerging as a new parameter useful for lead optimization. Based on these observations, a kinetic method for the determination of TSPO ligands RT was recently set up and optimized in our laboratory. Such a method, called competition kinetic association assay, allows quantifying with high accuracy the RT of non-radiolabeled TSPO ligands through the competition with classical radiolabeled TSPO ligands, e.g. [3H]PK11195. In this thesis, using the competition kinetic association assay, the RT of different classes of TSPO ligands, including two well-known anxiolytic ligands, XBD173 and etifoxine, and a number of phenylindolylglyoxyilamide derivatives (PIGAs), was quantified. Then, the relationship between RT and in vitro steroidogenic activity was evaluated. To this aim, the pregnenolone (the first metabolite of steroidogenesis) production was assessed by ELISA in a well-described model (C6 rat glioma cells) following exposure with increasing ligand concentrations. Both etifoxine and XBD173 resulted highly steroidogenic, but only XBD173 showed a slow dissociating rate at [3H]PK11195 binding site, while etifoxine showed a fast dissociating profile. Such unexpected result led us to further investigate etifoxine-TSPO binding interaction, taking into consideration a different binding site, those of Ro5-4864. By the means of a Scatchard analysis in the presence and absence of etifoxine, a competitive interaction of etifoxine at [3H]Ro5-4864 binding site in rat kidney membranes was observed. Then, etifoxine RT was quantified at [3H]Ro5-4864 binding site and interestingly, it showed a slow dissociating rate. These data corroborated our hypothesis that RT predicts neurosteroidogenic efficacy more accurately than binding affinity and for this reason, it should be taken into consideration during drug discovery and lead optimization processes. Moreover, to further investigate the role of TSPO in steroidogenesis and the effects of its pharmacological stimulation, a small library of PIGA ligands were assessed in vitro using human astrocytic models (U87MG glioblastoma cells and normal human astrocytes). PIGA ligands, generally characterized by high affinity(nM or sub-nM) for TSPO but variable steroidogenic efficacy, were thus evaluated for their ability to increase the oxidative metabolism activity and proliferation (OMA/P) of human astrocytes, all resulting significantly active. Among them, the best performing compounds in terms of OMA/P index increase were then evaluated for their steroidogenic efficacy and RT. Remarkably, the most active compounds also demonstrated high steroidogenic efficacy and long RT, further confirming the beneficial effects of neurosteroidogenesis stimulation in the CNS and the pivotal role/the predictive potential of RT. Finally, seen the promising results obtained in vitro, we extended our investigation concerning the neuroprotective effectiveness of PIGAs in vivo. Previously reported studies have already described anxiolytic properties of PIGAs in rats. Based on the neuroprotective effect observed in astrocytes, two PIGAs, PIGA1138 and PIGA839, showing long and medium RT respectively, were then assessed in a mouse model of experimental autoimmune encephalomyelitis (EAE). Biochemical and immunohistochemical experiments are still ongoing; however, behavioural tests seem to suggest that the two PIGAs could produce amelioration of the clinical symptoms of EAE and that the amelioration is more pronounced for the PIGA1138 with respect to PIGA839 when compared at the best performing concentration (15 mg/kg).