Identification of a Low–Molecular Weight Trkb Antagonist with Anxiolytic and Antidepressant Activity in Mice
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Identification of a low–molecular weight TrkB antagonist with anxiolytic and antidepressant activity in mice Maxime Cazorla, … , Christoph Kellendonk, Didier Rognan J Clin Invest. 2011;121(5):1846-1857. https://doi.org/10.1172/JCI43992. Research Article Neuroscience The neurotrophin brain-derived neurotrophic factor (BDNF) and its receptor tropomyosin-related kinase B (TrkB) have emerged as key mediators in the pathophysiology of several mood disorders, including anxiety and depression. However, therapeutic compounds that interact with TrkB receptors have been difficult to develop. Using a combination of structure- based in silico screening and high-capacity functional assays in recombinant and neuronal cells, we identified a low– molecular weight TrkB ligand (ANA-12) that prevented activation of the receptor by BDNF with a high potency. ANA-12 showed direct and selective binding to TrkB and inhibited processes downstream of TrkB without altering TrkA and TrkC functions. KIRA-ELISA analysis demonstrated that systemic administration of ANA-12 to adult mice decreased TrkB activity in the brain without affecting neuronal survival. Mice administered ANA-12 demonstrated reduced anxiety- and depression-related behaviors on a variety of tests predictive of anxiolytic and antidepressant properties in humans. This study demonstrates that structure-based virtual screening strategy can be an efficient method for discovering potent TrkB- selective ligands that are active in vivo. We further propose that ANA-12 may be a valuable tool for studying BDNF/TrkB signaling and may constitute a lead compound for developing the next generation of therapeutic agents for the treatment of mood disorders. Find the latest version: https://jci.me/43992/pdf Research article Identification of a low–molecular weight TrkB antagonist with anxiolytic and antidepressant activity in mice Maxime Cazorla,1,2 Joël Prémont,1 Andre Mann,3 Nicolas Girard,3 Christoph Kellendonk,2 and Didier Rognan3 1Neurobiology and Molecular Pharmacology, Centre de Psychiatrie et Neurosciences, UMR-894 INSERM/Université Paris Descartes, Paris, France. 2Department of Pharmacology and Psychiatry, Columbia University, New York, New York, USA. 3Structural Chemogenomics, Laboratory of Therapeutic Innovation, UMR-7200 CNRS/Université de Strasbourg, Illkirch, France. The neurotrophin brain-derived neurotrophic factor (BDNF) and its receptor tropomyosin-related kinase B (TrkB) have emerged as key mediators in the pathophysiology of several mood disorders, including anxiety and depression. However, therapeutic compounds that interact with TrkB receptors have been difficult to develop. Using a combination of structure-based in silico screening and high-capacity functional assays in recombinant and neuronal cells, we identified a low–molecular weight TrkB ligand (ANA-12) that prevented activation of the receptor by BDNF with a high potency. ANA-12 showed direct and selective binding to TrkB and inhibited processes downstream of TrkB without altering TrkA and TrkC functions. KIRA-ELISA analysis demonstrated that systemic administration of ANA-12 to adult mice decreased TrkB activity in the brain with- out affecting neuronal survival. Mice administered ANA-12 demonstrated reduced anxiety- and depression- related behaviors on a variety of tests predictive of anxiolytic and antidepressant properties in humans. This study demonstrates that structure-based virtual screening strategy can be an efficient method for discovering potent TrkB-selective ligands that are active in vivo. We further propose that ANA-12 may be a valuable tool for studying BDNF/TrkB signaling and may constitute a lead compound for developing the next generation of therapeutic agents for the treatment of mood disorders. Introduction changes in the reward circuitry of rodents that predict the facilita- Neurotrophins are a family of growth factor hormones that regu- tion of depression and stress-induced anxiety in humans (7, 12). late development and maintenance of the central and peripheral In these studies, preventing BDNF production in dopaminergic nervous systems by interacting with the tropomyosin-related regions or inhibiting BDNF/TrkB signaling in the nucleus accum- kinase (Trk) receptors. Nerve growth factor (NGF) binds preferen- bens reduced the anxiety toward former aggressors and induces tially to TrkA, brain-derived neurotrophic factor (BDNF) and neu- strong antidepressive effects in rodent, predicting that TrkB inhi- rotrophin-4/5 (NT-4/5) bind to TrkB, and neurotrophin-3 (NT-3) bition may lead to antidepressant action in humans (7, 12). In line binds to TrkC. All neurotrophins also interact with the nonse- with this observation, mice susceptible to stress-related anxiety and lective p75NTR receptor with similar affinities (1). Interaction of depression exhibit high levels of BDNF in the ventral striatum (13) BDNF with TrkB triggers receptor dimerization, transphosphory- and acutely stressed mice show a rapid and sustained increase in lation of intracellular tyrosine residues, and subsequent activation BDNF expression in the prefrontal cortex and in the hippocampus of the 3 major signaling pathways involving MAPK, PI3K, and (14, 15). In consequence, while inhibiting BDNF signaling may not phospholipase C-γ (2). lead to depression-like behaviors (16–18), reports have suggested Given its trophic effect on neurons and its contribution to the that inhibiting TrkB signaling may in contrast be a therapeutic regulation of synaptic transmission and plasticity (3, 4), the BDNF/ strategy for treating human mood disorders (5, 7, 13, 19). TrkB system has been proposed as a key element in the pathophysi- Despite their potential clinical significance, small-molecule ology of several mood disorders, including drug addiction, depres- antagonists of TrkB are not available. The development of low– sion, and anxiety (5–7). Accordingly, BDNF signaling in the stress molecular weight compounds (<500 Da) that can selectively antag- system of the brain, including the hippocampus and the hypothal- onize a large 145-kDa dimeric TrkB complex activated by a 27-kDa amo-pituitary-adrenocortical (HPA) axis, is thought to be a poten- dimeric ligand has been difficult to address. We recently developed tial target for antidepressant action (8–10). Positive modulators of a highly potent small peptide that allosterically inhibits TrkB sig- BDNF/TrkB signaling have therefore been proposed to have anti- naling in neurons and that is active in vivo after systemic delivery depressive potential in humans (11). However, recent studies have (20). Our in vivo study, together with others’ studies (21–23), dem- challenged this hypothesis, and the role of BDNF in the pathogen- onstrates the feasibility of developing heterocyclic ligands that can esis of depression and anxiety is still controversial. modulate TrkB activity in living animals. However, peptide-derived In contrast to its function on antidepressant efficacy in the ligands have the problem of being proteolytically unstable, which hippocampus and HPA axis, BDNF induces long-term neuronal potentially decreases their efficiency under therapeutic conditions. Here, we aimed at identifying small, proteolytically stable modula- Conflict of interest: The authors have declared that no conflict of interest exists. tors that are selective for TrkB and active in vivo. To this end, we Citation for this article: J Clin Invest. 2011;121(5):1846–1857. doi:10.1172/JCI43992. used a rational in silico screening approach based on the known 1846 The Journal of Clinical Investigation http://www.jci.org Volume 121 Number 5 May 2011 research article Figure 1 Computational modeling of the specificity patch. The structure-based virtual screening was performed by targeting the specificity interac- tion patch between the N-terminal segment of BDNF (N-TERM, red/ orange) and the d5 subdomain of TrkB (TrkB-d5, green). An alignment of the N-terminal amino acid sequence of rodent BDNF, NT-4/5, NT-3, and NGF is shown in the upper panel (the specific region is boxed; dashes represent gaps). The 3D model of the complex between the TrkB-d5 domain (green solid surface) and the BDNF N-terminal part (H1SDPAR6, orange sticks) is illustrated in the lower panel. compounds. Preliminary docking studies of the BDNF terminal epitope (H1SDPAR6) with various docking algorithms indicated that the FlexX program (32) was particularly well suited for dock- ing in the TrkB specificity site. All 78,045 compounds were directly docked with the FlexX v2.2 program into the specificity patch of the TrkB-d5 domain, yielding 295 virtual hits that were finally clustered by maximal common substructures using ClassPharmer (31). A representative compound (best FlexX score) of each of the 22 ClassPharmer classes described by at least 3 compounds was finally selected for purchase and biological evaluation. In silico screening identifies TrkB antagonists. Out of the 22 virtual hits, 12 molecules were obtained from commercial sources and tested in the presence or absence of BDNF in KIRA-ELISA assays, a method that rapidly quantifies TrkB activation in 96-well plates (20). In a first set of assays performed on recombinant human TrkB expressed in an inducible cell line (TetOn-rhTrkB), 2 com- pounds (N-T04 and N-T19; Figure 2A) were found to fully inhibit BDNF-induced TrkB phosphorylation and were selected for fur- structural interaction between neurotrophins and Trk receptors. ther pharmacological characterizations. Using a similar in silico screening strategy but based