Elsevier

Neuropharmacology

Volume 164, 1 March 2020, 107902
Neuropharmacology

LSP5-2157 a new inhibitor of vesicular glutamate transporters

https://doi.org/10.1016/j.neuropharm.2019.107902Get rights and content

Abstract

Vesicular glutamate transporters (VGLUT1-3) mediate the uptake of glutamate into synaptic vesicles. VGLUTs are pivotal actors of excitatory transmission and of almost all brain functions. Their implication in various pathologies has been clearly documented. Despite their functional importance, the pharmacology of VGLUTs is limited to a few dyes such as Trypan Blue, Rose Bengal or Brilliant Yellow type. Here, we report the design and evaluation of new potent analogs based on Trypan Blue scaffold. Our best compound, named LSP5-2157, has an EC50 of 50 nM on glutamate vesicular uptake. Using a 3D homology model of VGLUT1 and docking experiments, we determined its putative binding subdomains within vesicular glutamate transporters and validated the structural requirement for VGLUT inhibition. To better estimate the specificity and potency of LSP5-2157, we also investigated its ability to block glutamatergic transmission in autaptic hippocampal cells. Neither glutamate receptors nor GABAergic transmission or transmission machinery were affected by LSP5-2157. Low doses of compound reversibly reduce glutamatergic neurotransmission in hippocampal autpases. LSP5-2157 had a low and depressing effect on synaptic efficacy in hippocampal slice. Furthermore, LSP5-2157 had no effect on NMDA-R- mediated fEPSP but reduce synaptic plasticity induced by 3 trains of 100 Hz. Finally, LSP5-2157 had the capacity to inhibit VGLUT3-dependent auditory synaptic transmission in the guinea pig cochlea. In this model, it abolished the compound action potential of auditory nerve at high concentration showing the limited permeation of LSP5-2157 in an in-vivo model. In summary, the new ligand LSP5-2157, has a high affinity and specificity for VGLUTs and shows some permeability in isolated neuron, tissue preparations or in vivo in the auditory system. These findings broaden the field of VGLUTs inhibitors and open the way to their use to assess glutamatergic functions in vitro and in vivo.

Introduction

Glutamate, the major excitatory neurotransmitter in the Central Nervous System (CNS), is involved in a vast majority of neurophysiological functions as well as neuropathologies. Before its exocytotic release, glutamate is accumulated in synaptic vesicles by vesicular glutamate transporters (VGLUT1-3). The three isoforms of VGLUTs share a high degree of sequence homology (more than 75%) and have almost complementary brain distributions (El Mestikawy et al., 2011). VGLUTs are pivotal functional and anatomical markers of glutamatergic transmission. Various studies suggest the involvement of VGLUTs in human neurological conditions such as Parkinson's disease, Alzheimer disease or epilepsy (Kashani et al., 2007, 2008; Kirvell et al., 2006; van der Hel et al., 2009) as well as psychiatric disorders (Eastwood and Harrison, 2010; Oni-Orisan et al., 2008; Sakae et al., 2015; Uezato et al., 2009). VGLUTs were suggested as potential target for the treatment of temporal lobe epilepsy (Van Liefferinge et al., 2013). Despite their key role in excitatory transmission the pharmacology of VGLUTs is notably under-developed. Pharmacological tools are therefore crucially needed to better understand the functional implication of VGLUT1-3 in normal and pathological conditions. Additionally, these compounds could be developed as radiomarkers to follow the fate of glutamatergic terminals in human pathologies of the CNS.

Few chemical compounds efficiently target this important family of glutamate transporters. Presently, three major types of VGLUT's inhibitors have been identified (Thompson et al., 2005): glutamate-like inhibitors (Thompson et al., 2005), substituted quinolines (Bartlett et al., 1998; Carrigan et al., 2002) and dyes (Kehrl et al., 2017; Ozkan and Ueda, 1998; Roseth et al., 1995, 1998; Tamura et al., 2014). Dyes have Ki in the 20nM-10μM range, substituted quinolines (DCQ) in the 40–300 μM range and glutamate analogs have IC50 above 230 μM. Glutamate-like inhibitors interact with glutamate receptors and transporters (Thompson et al., 2005). Dyes display higher affinities but a lower selectivity than quinolines (Shigeri et al., 2004). We previously reported that Rose Bengal was equally effective on monoamine vesicular accumulation and on VGLUTs (Pietrancosta et al., 2010). On the other hand, DCQ derivatives remain potential candidates for this pharmacological purpose. However, Carrigan et al. have shown their limit due to their moderate affinities (Carrigan et al., 2002; Laras et al., 2012).

Trypan Blue or Evans Blue are well known universal cellular dyes that lack selectivity. Chemical variations of these dyes are not easily accessible. Furthermore, these compounds have the ability to inhibit glutamate vesicular accumulation as well as excitatory synaptic transmission (Neale et al., 2014).

In the present study, we synthesized and characterized a derivative of Trypan Blue. We thoroughly evaluated the capacity of one new compound (named LSP5-2157) to selectively inhibit VGLUT activity as well as glutamatergic synaptic transmission in hippocampal autapses and slices. In addition, we evaluated the ability of LSP5-2157 to modulate auditory input in vivo in the guinea pig cochlea. This study establishes that LSP5-2157 has a high affinity/high selectivity for VGLUTS and furthermore is partially permeant to the blood brain barrier and to cell membranes.

Section snippets

Synthesis

Compounds were synthesized according to procedure already described (Favre-Besse et al., 2014) with minor modifications (Supplementary Material and Methods).

Glutamate vesicular uptake

Vesicular Glutamate uptake was assayed as previously described (Kish and Ueda, 1989; Naito and Ueda, 1985; Pietrancosta et al., 2010) with minor modifications. Aliquots of crude rat synaptic vesicles obtained by differential centrifugation of rat cortex were diluted in a solution containing 4 mM HEPES-KOH, pH 7,4 and 0,32 M sucrose. Test

Effect of VGLUTs inhibitors on glutamate vesicular accumulation

Compounds 1 to 8 (LSP5-2157, LSP9-2103, LSP9-1148, LSP9-3058, LSP5-2105, LSP9-2106, LSP9-3076) were synthetized and characterized as described in Supplementary Material and Methods. Other compounds (FCFB-7b, FCFB-4j, FCFB-4i, FCFB-7c, FCFB-4n, FCFB-4o, FCFB-7d, FCFB-4i, FCFB-4p, FCFB-7e, FCFB-4m) were obtained as previously reported (Favre-Besse et al., 2014).

Thirteen analogs of Trypan Blue dose-dependently inhibited ATP-dependent [3H]-Glutamate vesicular accumulation. Sigmoidal curves were

Discussion

In the present study, we described new VGLUTs inhibitors. To better understand the potency of various ligands we combined molecular modeling, synthesis, synaptic vesicular uptake, electrophysiology and in vivo recordings. Our best compound, LSP5-2157, is structurally analogous to Trypan Blue. Its activity was evaluated not only in vesicular uptake of [3H]-glutamate but also upon glutamatergic transmission in autaptic hippocampal neurons and hippocampal slices. In addition, we show for the first

Authors contributions

LEM, MAH, JW performed experiments and analyzed data. MAH, CR designed experiments. OP, MAK, MK, BP, SM, JW, FCFB, YL, HOB, PD, NP performed experiments. FA, JLP, BG, JE, PD, SD, SEM, NP designed the experiments. NP, PD and SEM wrote the paper with the help of LEM, MAH, JW, FA, JPP, JLP, BG, JE, CR and SD.

Declaration of competing interest

The authors declare no conflict of interest.

Acknowledgments

This worked was supported by following Funding sources: ANR (ANR-09-MNPS-033, ANR-13-SAMA-0005-01), 2011–2014 ANR/FRSQ (MALZ VGLUTAD), Fondation pour la Recherche Médicale (Équipe FRM DEQ20130326486), FRC, Brain Canada Multi-Investigator Research Initiative, INSERM, CNRS and Sorbonne Université. CNRS (80|PRIME  - 2019). The research team of SEM is member of the Bio-Psy Labex; this work was therefore supported by French state funds managed by the ANR within the/Investissements d'Avenir/program

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