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.

Classical and Novel TSPO Ligands for the Mitochondrial TSPO can Modulate Nuclear Gene Expression: Implications for Mitochondrial Retrograde Signaling

“…PK 11195 is a specific TSPO ligand, known to affect numerous TSPO functions, similar to the effects of TSPO knockdown by genetic manipulation , applying the covalent TSPO ligand irDE-MPIGA to GBM cells, showed that it takes more than 90 min for irDE-MPIGA (25 nM) to irreversibly saturate all TSPO binding sites; 3 hrs after ligand application ΔΨm collapse was observed; 6 hrs after ligand application externalization of phosphatidylserine was observed and cell viability was reduced. After 24 hrs, de-novo TSPO synthesis was observed [40]. The present study shows that modulation of gene expression in U118MG cells due to PK 11195 occurs hours before these previously reported physiological and phenotypic changes [5,14,35,37,40,52].…”

“…already at 15 minutes. This is at least two hours before changes in physiological responses that are typically associated with TSPO function [40]. Secondly, we found that within 15 minutes the classical TSPO ligand PK 11195 induces considerable changes in gene expression associated with the canonical pathway for modulation of gene expression in general.…”

The changing landscape in translocator protein (TSPO) function

“…Although a role for TSPO was not discussed in the new model, it remains to be explained how TSPO-binding chemicals could modulate cell death processes. The pharmacological evidence supporting a TSPO link to the MPTP appears well documented [22][23][24][25]. Developments in the field of the MPT were extensively described in a recent review on this topic [26].…”

TSPO as a target for glioblastoma therapeutics

“…To address these concerns, Chelli et al [36] showed that impairments in GM cell line viability and mitochondrial membrane potential induced by PK 11195 were prevented when TSPO was silenced with siRNA. Costa et al [52] also showed GM cell line viability impairment induced by a high-affinity TSPO ligand was decreased by knocking down TSPO levels. The authors showed this compound did not affect ATP synthase activity in GM cells.…”

“…These studies suggest, at least in vitro, the discrepancy between affinity and potency of anti-GM effects may be attributable to a mechanism other than nonspecificity. Recently, insight into what this mechanism may be was provided by Costa et al [52], who identified potency could be increased by increasing ligand residence time. A reversibly binding TSPO ligand that showed micromolar potency for impairing GM cell line viability could be transformed into a ligand that produced effects at low nanomolar doses by engineering it to covalently bind to TSPO.…”