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A long residence time at the neurosteroidogenic 18 kDa Translocator Protein characterizes the anxiolytic ligand XBD173 Barbara Costa, Eleonora Da Pozzo, Chiara Cavallini, Sabrina Taliani, Federico Da Settimo, and Claudia Martini ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.6b00149 • Publication Date (Web): 06 Jul 2016 Downloaded from http://pubs.acs.org on July 8, 2016
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ACS Chemical Neuroscience
A long residence time at the neurosteroidogenic 18 kDa Translocator Protein characterizes the anxiolytic ligand XBD173 Barbara Costa 1*§, Eleonora Da Pozzo1§, Chiara Cavallini 1, Sabrina Taliani1, Federico Da Settimo1, Claudia Martini1.
1
Department of Pharmacy, University of Pisa, via Bonanno, 6-56126 Pisa, Italy.
*:Corresponding author: Barbara Costa, Department of Pharmacy, University of Pisa, via Bonanno, 6-56126 Pisa, Italy; E-mail address:
[email protected] §
These authors contributed equally to this work
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Abstract Recent data have demonstrated a positive correlation between the Residence Time (RT) and neurosteroidogenic efficacy of a ligand at the Translocator Protein (TSPO), an attractive anxyolitic target. To explore the potential impact of RT on TSPO ligand anxiolytic activity, the RT and the steroidogenic activity of XBD173, a ligand exerting anxiolytic activity in humans, were retrospectively evaluated. To this aim, XBD173 association and dissociation rate constants were measured (1.23x107 M-1 min-1 and 0.0079 min-1, respectively). XBD173 resulted to have a long RT (127 min) and to stimulate efficaciously neurosteroidogenesis, in terms of pregnenolone production. The present findings corroborate the importance of TSPO ligand RT to predict their effective neurosteroidogenic activity and promising anxiolytic action. These positive results prompted us to set up a fast and high-throughput kinetic method to improve the efficiency of RT-based TSPO drugdiscovery process.
Key words: 18 kDa Translocator Protein; Residence Time; TSPO ligands; anxiolytic activity; neurosteroids; kinetic binding assay. 2 ACS Paragon Plus Environment
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INTRODUCTION Ligands of 18 kDa Translocator Protein (TSPO) are emerging as fast-acting anxiolytics with favorable safety, tolerability, and pharmacokinetic profiles.1-4 Among the most promising TSPO ligands, XBD173 (also called AC-5216 or Emapunil) has exerted anti-anxiety and anti-panic effects in rodents and humans without the typical undesirable side-effects of benzodiazepines (ClinicalTrials.gov identifier: NCT00108836).1,5,6 The anxiolytic activities of TSPO ligands are proposed to be mediated through the stimulation of neurosteroidogenesis7, a process that produces molecules able to modulate nervous system activities, termed “neuroactive steroids”.8 In nervous system, such molecules are synthesized starting from the initial substrate cholesterol, or processing metabolites produced in the peripheral adrenal and gonadal sources.8 TSPO, highly expressed in steroid-synthesizing tissues, plays a crucial role in the neuroactive steroid biosynthesis, as converging data have suggested that it is involved in the first rate-limiting step of steroidogenesis.9 Specifically, TSPO, mainly located in the outer mitochondrial membrane, translocates cholesterol from cytoplasm to the inner mitochondrial membrane, where the cytochrome P450 enzyme CYP11A1 converts it into pregnenolone, the precursor of all neurosteroids.9 Neuroactive steroids exert important pleiotropic effects for brain functioning, including regulation of the physiological response to stress.8 They rapidly modulate neuronal network excitability through their interaction with neurotransmitter receptors, including type A receptor of the main brain inhibitory neurotransmitter γ-amino-butyric acid.8 An impairment of neurosteroid homeostatic activities has been proposed in psychiatric disorders, including anxiety disorders, as dysregulated neurosteroid levels have been found.8 Consistent with altered levels of neurosteroids, reduced levels of TSPO have been documented in patients with generalized social phobia, adult separation anxiety, obsessive compulsive, and post-traumatic stress disorders.10-14 The ability of a TSPO ligand to promote the neurosteroidogenesis could restore concentrations of specific neuroactive steroids, contributing to the positive therapeutic effects observed in anxiety models. Very recently, we have reported that the period a drug is bound to TSPO (defined as Residence Time) is a better measure to estimate the steroidogenic efficacy of a ligand, with respect to the binding affinity parameter used during traditional drug discovery programs.15 Specifically, by means of TSPO ligands belonging to phenylindolylglyoxylamides (PIGAs)16-18 and classical TSPO ligands, our data demonstrated that a long Residence Time implicates a high steroidogenic efficacy, regardless of compounds’ binding affinity. With the aim to explore whether Residence Time could be a metric of promising anxiolytic activities of a TSPO ligand, in the present work, the retrospective evaluation of Residence Time of 3 ACS Paragon Plus Environment
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XBD173 and the relationship with its steroidogenic efficacy were explored. In addition, a fast and high-throughput kinetic method suitable for a more efficient Residence Time-based TSPO ligand screening process was designed and setup.
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RESULTS AND DISCUSSION Thermodynamic, Residence Time parameters and steroidogenic efficacy of XBD173. Research programs aimed to TSPO drug discovery have employed the traditional approach based on in vitro measurement of binding affinity, in terms of thermodynamic parameter at equilibrium (inhibitory constant, Ki or dissociation constant, Kd).19 This strategy has allowed the identification of several high affinity TSPO ligands, which have been further tested in a number of in vitro and in vivo experimental models.8 However, a recurrent issue about TSPO ligands concerns a poor relationship between binding affinity and steroidogenic efficacy.20,21 The recently found positive correlation between Residence Time (RT) of a TSPO ligand and its steroidogenic efficacy,15 prompted us to evaluate this parameter for the promising anxiolytic candidate XBD173. For this TSPO ligand, the affinity binding parameter is known4; however, to the best of our knowledge, data about its binding kinetics are not yet available. We have recently set up and optimized an experimental approach, named “competition kinetic association”, for the TSPO ligand RT quantification.15 This method is based on a mathematical model developed by Motulsky and Mahan22, where an unlabeled competitor is added simultaneously with a radioligand to the receptor preparation of interest. Then, the rate of specific radioligand binding can be modelled to provide the association (kon) and dissociation (koff) rates of the unlabeled compound. The RT value (expressed in minutes) is calculated from the reciprocal of koff. Thus, the RT parameter at TSPO [3H]PK11195 binding site has been determined for a number of our PIGA compounds, and the classical TSPO ligands PK11195 and Ro5-4864.15 A positive correlation between RT and ligand steroidogenic efficacy has been found, with the longest RT values (PIGA823= 127 min, PIGA839= 109 min, PIGA1138= 141 min) observed for the most effective compounds, in terms of pregnenolone production.15 Conversely, RT values of few minutes, approximately ranging from 10 to 30 min, have been obtained for TSPO ligands with low steroidogenic efficacy.15 In the present paper, for a correct application of the kinetic method for the XBD173 RT evaluation, as a first step, the XBD173 Ki was estimated in our TSPO-rich tissue model, consisting of rat kidney membranes. The obtained XBD173 Ki value was 2.41 nM, in agreement with previously reported data (3.04 nM in rat glioma cells4). Then, the “competition kinetic association” was applied to XBD173, tested at 3-fold its Ki, and data were fitted using the equation reported in ref. 15 to simultaneously calculate kon and koff parameters. The experimentally derived kon and koff values of XBD173 were 1.23 x 107 M-1 min-1 and 0.0079 min-1, respectively (Fig. 1A). The calculated XBD173 RT was 127 min. 5 ACS Paragon Plus Environment
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In order to investigate if XBD173 data obtained using kidney membranes could extend to steroidogenic tissues, radioligand binding assays were performed using rat adrenal membranes, too. To this aim, the above described experimental strategy, consisting of Ki determination followed by kinetic parameter evaluation of XBD173, was undertaken. In adrenal membranes, the obtained Ki was 2.2 nM and the kinetic data were the following: kon=1.15 x 107 M-1 min-1, koff = 0.0083 min-1; RT= 120 min. In figure 1B, the XBD173 “competition kinetic association” curve was shown. These results were comparable to those observed in kidney, suggesting that the data obtained with kidney membranes is applicable to the “classical” steroid synthesizing tissues. The obtained XBD173 RT was comparable with those showed for PIGAs with long RT and highly steroidogenic. In order to explore whether the phenomenon “long RT-high steroidogenic efficacy” occurred also for XBD173, its steroidogenic efficacy was measured in terms of in vitro production of pregnenolone. For such an assessment, the steroidogenic cell model was exposed to increasing ligand concentrations, for a fixed incubation time, in the presence of specific inhibitors of pregnenolone further metabolism. The XBD173 steroidogenic efficacy (Emax value, relative to the highest tested concentration) was calculated with respect to control sample, corresponding to basal pregnenolone production (fixed at 100% value). In parallel experiments, a “long RT/high steroidogenic” ligand (PIGA1138), and two “short RT/low steroidogenic” ligands (PK11195 and Ro5-4864) were included (Fig. 2). The XBD173 Emax (calculated at 40 µM, maximum concentration permitted in the assay salt medium) was 245 ± 17 %; the Emax values of PIGA1138, PK11195 and Ro54864 (calculated at 100 µM) were 275 ± 5 %, 150 ± 4 % and 150 ± 2 %, respectively. Thus, to make a comparative analysis among the tested TSPO ligands, the steroidogenic stimulation mean values were compared at the concentration of 40 µM: XBD173, 245 ± 17 %; PIGA1138, 266 ± 6 %; PK11195, 148 ± 6 %; Ro5-4864, 129 ± 1 %. The obtained values showed no significant difference between XBD173 and PIGA 1138 (P=0.352; t=0.953), whereas difference was significant with classical TSPO ligands (XBD173 vs PK11195: P=0.0108, t=2.86; XBD173 vs Ro5-4864: P
