title: TSPO ligand residence time influences human glioblastoma multiforme cell death/life balance.
abstract: Ligands addressed to the mitochondrial Translocator Protein (TSPO) have been suggested as cell death/life and steroidogenesis modulators. Thus, TSPO ligands have been proposed as drug candidates in several diseases; nevertheless, a correlation between their binding affinity and in vitro efficacy has not been demonstrated yet, questioning the specificity of the observed effects. Since drug-target residence time is an emerging parameter able to influence drug pharmacological features, herein, the interaction between TSPO and irDE-MPIGA, a covalent TSPO ligand, was investigated in order to explore TSPO control on death/life processes in a standardized glioblastoma cell setting. After 90 min irDE-MPIGA cell treatment, 25 nM ligand concentration saturated irreversibly all TSPO binding sites; after 24 h, TSPO de-novo synthesis occurred and about 40 % TSPO binding sites resulted covalently bound to irDE-MPIGA. During cell culture treatments, several dynamic events were observed: (a) early apoptotic markers appeared, such as mitochondrial membrane potential collapse (at 3 h) and externalization of phosphatidylserine (at 6 h); (b) cell viability was reduced (at 6 h), without cell cycle arrest. After digitonin-permeabilized cell suspension treatment, a modulation of mitochondrial permeability transition pore was evidenced. Similar effects were elicited by the reversible TSPO ligand PIGA only when applied at micromolar dose. Interestingly, after 6 h, irDE-MPIGA cell exposure restored cell survival parameters. These results highlighted the ligand-target residence time and the cellular setting are crucial parameters that should be taken into account to understand the drug binding affinity and efficacy correlation and, above all, to translate efficiently cellular drug responses from bench to bedside.
authors.affiliation: Department of Pharmacy, University of Pisa, via Bonanno, 6-56126, Pisa, Italy, bcosta@farm.unipi.it.
authors.forename: Barbara
authors.initials: B
authors.lastname: Costa
authors.forename: Eleonora
authors.initials: E
authors.lastname: Da Pozzo
authors.forename: Chiara
authors.initials: C
authors.lastname: Giacomelli
authors.forename: Sabrina
authors.initials: S
authors.lastname: Taliani
authors.forename: Sara
authors.initials: S
authors.lastname: Bendinelli
authors.forename: Elisabetta
authors.initials: E
authors.lastname: Barresi
authors.forename: Federico
authors.initials: F
authors.lastname: Da Settimo
authors.forename: Claudia
authors.initials: C
authors.lastname: Martini
mesh_terms: D014151:Anti-Anxiety Agents / Q000737:chemistry / Q000494:pharmacology*; D000970:Antineoplastic Agents / Q000737:chemistry / Q000494:pharmacology*; D001665:Binding Sites; D002453:Cell Cycle / Q000187:drug effects / Q000235:genetics; D016923:Cell Death / Q000187:drug effects; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D002986:Clinical Trials as Topic; D018756:GABA Antagonists / Q000737:chemistry / Q000494:pharmacology*; D015870:Gene Expression; D006801:Humans; D007211:Indoles / Q000737:chemistry / Q000494:pharmacology*; D007700:Kinetics; D008024:Ligands; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D009457:Neuroglia / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D011485:Protein Binding; D018079:Receptors, GABA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013861:Thiocyanates / Q000737:chemistry / Q000494:pharmacology*
chemical_list: D014151:Anti-Anxiety Agents; D000970:Antineoplastic Agents; D018756:GABA Antagonists; D007211:Indoles; D008024:Ligands; C503726:N,N-Di-n-butyl-5-chloro-2-(4-chlorophenyl)indol-3-ylglyoxylamide; D018079:Receptors, GABA; C499229:TSPO protein, human; D013861:Thiocyanates