σ2 receptor and its role in cancer with focus on a multitarget directed ligand (Mtdl) approach

Sigma-2 (σ2) is an endoplasmic receptor identified as the Endoplasmic Reticulum (ER) transmembrane protein TMEM97. Despite its controversial identity, which was only recently solved, this protein has gained scientific interest because of its role in the proliferative status of cells; many tumor cells from different organs overexpress the σ2 receptor, and many σ2 ligands display cytotoxic actions in (resistant) cancer cells. These properties have shed light on the σ2 receptor as a potential druggable target to be bound/activated for the diagnosis or therapy of tumors. Additionally, diverse groups have shown how the σ2 receptor can be exploited for the targeted delivery of the anticancer drugs to tumors. As the cancer disease is a multifactorial pathology with multiple cell populations, a polypharmacological approach is very often needed. Instead of the simultaneous administration of different classes of drugs, the use of one molecule that interacts with diverse pharmacological targets, namely MultiTarget Directed Ligand (MTDL), is a promising and currently pursued strategy, that may overcome the pharmacokinetic problems associated with the administration of multiple molecules. This review aims to point out the progress regarding the σ2 ligands in the oncology field, with a focus on MTDLs directed towards σ2 receptors as promising weapons against (resistant) cancer diseases

Persistent modification of forebrain networks and metabolism in rats following adolescent exposure to a 5-HT7 receptor agonist

RATIONALE: The serotonin 7 receptor (5-HT7-R) is part of a neuro-transmission system with a proposed role in neural plasticity and in mood, cognitive or sleep regulation. OBJECTIVES: We investigated long-term consequences of sub-chronic treatment, during adolescence (43-45 to 47-49 days old) in rats, with a novel 5-HT7-R agonist (LP-211, 0 or 0.250 mg/kg/day). METHODS: We evaluated behavioural changes as well as forebrain structural/functional modifications by in vivo magnetic resonance (MR) in a 4.7 T system, followed by ex vivo histology. RESULTS: Adult rats pre-treated during adolescence showed reduced anxiety-related behaviour, in terms of reduced avoidance in the light/dark test and a less fragmented pattern of exploration in the novel object recognition test. Diffusion tensor imaging (DTI) revealed decreased mean diffusivity (MD) in the amygdala, increased fractional anisotropy (FA) in the hippocampus (Hip) and reduced axial (D||) together with increased radial (D⊥) diffusivity in the nucleus accumbens (NAcc). An increased neural dendritic arborization was confirmed in the NAcc by ex vivo histology. Seed-based functional MR imaging (fMRI) identified increased strength of connectivity within and between "limbic" and "cortical" loops, with affected cross-correlations between amygdala, NAcc and Hip. The latter displayed enhanced connections through the dorsal striatum (dStr) to dorso-lateral prefrontal cortex (dl-PFC) and cerebellum. Functional connection also increased between amygdala and limbic elements such as NAcc, orbito-frontal cortex (OFC) and hypothalamus. MR spectroscopy (1H-MRS) indicated that adolescent LP-211 exposure increased glutamate and total creatine in the adult Hip. CONCLUSIONS: Persistent MR-detectable modifications indicate a rearrangement within forebrain networks, accounting for long-lasting behavioural changes as a function of developmental 5-HT7-R stimulatio

Activation of 5-HT7 receptor stimulates neurite elongation through mTOR, Cdc42 and actin filaments dynamics.

Recent studies have indicated that the serotonin receptor subtype 7 (5-HT7R) plays a crucial role in shaping neuronal morphology during embryonic and early postnatal life. Here we show that pharmacological stimulation of 5-HT7R using a highly selective agonist, LP-211, enhances neurite outgrowth in neuronal primary cultures from the cortex, hippocampus and striatal complex of embryonic mouse brain, through multiple signal transduction pathways. All these signaling systems, involving mTOR, the Rho GTPase Cdc42, Cdk5, and ERK, are known to converge on the reorganization of cytoskeletal proteins that subserve neurite outgrowth. Indeed, our data indicate that neurite elongation stimulated by 5-HT7R is modulated by drugs affecting actin polymerization. In addition, we show, by 2D Western blot analyses, that treatment of neuronal cultures with LP-211 alters the expression profile of cofilin, an actin binding protein involved in microfilaments dynamics. Furthermore, by using microfluidic chambers that physically separate axons from the soma and dendrites, we demonstrate that agonist-dependent activation of 5-HT7R stimulates axonal elongation. Our results identify for the first time several signal transduction pathways, activated by stimulation of 5-HT7R, that converge to promote cytoskeleton reorganization and consequent modulation of axonal elongation. Therefore, the activation of 5-HT7R might represent one of the key elements regulating CNS connectivity and plasticity during development

The arylpiperazine derivatives N-(4-cyanophenylmethyl)- 4-(2-diphenyl)-1-piperazinehexanamide and N-benzyl-4- (2-diphenyl)-1-piperazinehexanamide exert a long-lasting inhibition of human serotonin 5-HT 7 receptor binding and cAMP signaling

Abstract We performed a detailed in vitro pharmacological characterization of two arylpiperazine derivatives, compound N-(4-cyanophenylmethyl)-4-(2-diphenyl)-1-piperazinehexanamide (LP-211) previously identified as a high-affinity brain penetrant ligand for 5-hydroxytryptamine (serotonin) type 7 (5-HT 7 ) receptors, and its analog N-benzyl-4-(2-diphenyl)-1-piperazinehexanamide (MEL-9). Both ligands exhibited competitive displacement of ]-SB-269970) radioligand binding and insurmountable antagonism of 5-carboxamidotryptamine (5-CT)-stimulated cyclic adenosine monophosphate (cAMP) signaling in human embryonic kidney (HEK293) cells stably expressing human 5-HT 7 receptors. They also inhibited forskolin-stimulated adenylate cyclase activity in 5-HT 7 -expressing HEK293 cells but not in the parental cell line. The compounds elicited long-lasting (at least 24 h) concentration-dependent inhibition of radioligand binding at 5-HT 7 -binding sites in whole-cell radioligand binding assays, after pretreatment of the cells with the compounds and subsequent compound removal. In cAMP assays, pretreatment of cells with the compounds rendered 5-HT 7 receptors unresponsive to 5-CT and also rendered 5-HT 7 -expressing HEK293 cells unresponsive to forskolin. Compound 1-(2-biphenyl)piperazine (RA-7), a known active metabolite of LP-211 present in vivo, was able to partially inhibit 5-HT 7 radioligand binding in a long-lasting irreversible manner. Hence, LP-211 and MEL-9 were identified as high-affinity long-acting inhibitors of human 5-HT 7 receptor binding and function in cell lines. The detailed in vitro characterization of these two pharmacological tools targeting 5-HT 7 receptors may benefit the study of 5-HT 7 receptor function and it may lead to the development of novel selective pharmacological tools with defined functional properties at 5-HT 7 receptors. Abbreviations 5-CT, 5-carboxamidotryptamine; 5-HT, 5-hydroxytryptamine (serotonin); ANOVA, analysis of variance; E max , maximal response; Fmr1, fragile X mental retardation 1; Fos, FBJ murine osteosarcoma viral oncogene homolog; GPCR, G protein-coupled receptors; HEK293, human embryonic kidney cells; HTRF, homogeneous timeresolved fluorescence; IBMX, 3-isobutyl-1-methylxanthine; IC 50, irrev , potency of compounds inhibiting 5-HT 7 -stimulated cAMP signaling or whole-cell 5-HT

Selective Agents for Serotonin2C (5-HT2C) Receptor

The serotonin2C (5-HT2C) receptor has attracted a lot of attention owing to its role in appetite regulation, depression, obsessive-compulsive disorder (OCD), panic disorders, and substance abuse. This review summarizes nonpatent and patent literature up to November 2005 that deals with the synthesis and characterization of selective 5-HT2C receptor agonists and antagonists. Highlights on structure-activity relationships have been included, when possible

Increase of Capsaicin-Induced Trigeminal Fos-Like Immunoreactivity by 5-HT(7) Receptors.

OBJECTIVE: To explore whether pharmacological stimulation of the 5-hydroxytryptamine(7) (5-HT(7) ) receptor modulates Fos-like immunoreactivity in the trigeminal nucleus caudalis of rats. BACKGROUND: The serotonin 5-HT(7) receptor was proposed to be involved in migraine pathogenesis and evidence suggests it plays a role in peripheral nociception and hyperalgesia through an action on sensory afferent neurons. METHODS: The potential activating or sensitizing role of 5-HT(7) receptors on trigeminal sensory neurons, as visualized by Fos-like immunoreactivity in the superficial layers of the trigeminal nucleus caudalis in rats, was investigated using the 5-HT(7) receptor agonist, LP-211, in the absence and the presence of intracisternal capsaicin, respectively. The agonist effect was characterized with the 5-HT(7) receptor antagonist, SB-656104. Male Wistar rats received a subcutaneous injection of LP-211, SB-656104, and SB-656104 + LP-211. They were then anesthetized and prepared to receive an intracisternal injection of capsaicin or its vehicle. Animals were perfused and brains removed; sections of the brain stem from the area postrema to the CI level were obtained and processed for Fos immunohistochemistry. RESULTS: Capsaicin but not its vehicle induced Fos-like immunoreactivity within laminae I and II of trigeminal nucleus caudalis. Pretreatment with LP-211 had no effect on Fos-like immunoreactivity but strongly increased the response produced by capsaicin; this effect was abolished by SB-656104. Interestingly, capsaicin-induced Fos-like immunoreactivity was abolished by SB-656104 pretreatment thus suggesting involvement of endogenous 5-HT. CONCLUSIONS: Data suggest that 5-HT(7) receptors increase activation of meningeal trigeminovascular afferents and/or transmission of nociceptive information in the brain stem. This mechanism could be relevant in migraine and its prophylactic treatment

Developments in fluorescent probes for receptor research

Early reports on the identification of fluorescent probes for receptors date back to mid-1970s. Fluorescent probes were initially used to visualize molecular targets in an analogous way to the use of fluorescent antibodies but with the same resolution as isotopically labelled ligands. In parallel to the rapid development of techniques, such as fluorescence correlation spectroscopy, multi-photon excitation fluorescence microscopy, fluorescence polarization and in vivo fluorescence imaging, fluorescent probes are becoming multifaceted tools in life science. The present review will focus on how the design of fluorescent ligands for receptors has evolved to meet the needs of most recent fluorescence application

Serotonergic modulation by 5-HT7 receptors in mouse spinal cord dorsal horn

Background Serotonergic receptors of the 5-HT7 type (5-HT7Rs) are widely expressed in the central nervous system, where they modulate seve ral functions, such as sleep induction, learning, mood, and vegetative behaviours. Along the pain axis, 5-HT7Rs are expressed on nociceptive primary afferent fibers and in the dorsal horn, both on neurons and astrocytes. [1]. Behavioural experiments have produ ced controversial results about anti- and pro-nociceptive actions of 5-HT7Rs. The low agonist selectivity and the different pain animal models used have likely contributed to the heterogeneity of the results [2]. To investigate the effects of 5-HT7Rs on spinal pain, we have performed an electrophysiological study on mouse spinal cord slices, using the selective agonist LP-211 [3]. The recorded neurons have been functionally characterized, in order to identify the neural circuits involved in the serotonergic modulation. Methods Patch-clamp recording was performed on lamina II neurons in spinal cord slices obtained from postnatal CD1 mice (P15-P25) [4]. Excitatory postsynaptic currents (EPSCs) were recorded in voltage clamp; evoked EPSCs were elicited by stimulating the dorsal root with a suction electrode in the Aδ and C fibers range. Results Application of 1 μM LP-211 to the spinal cord slice induced a facilitation of glutamatergic transmission: the frequency of spontaneous EPSCs was significantly increased in a subpopulation of neurons (control: 0.9±0.2 Hz; LP-211: 1.8±0.6 Hz; 5 responsive neurons out of 8). The recorded neurons were characterized from their firing pattern: significant effects of LP-211 were observed in both tonic and de layed firing neurons, corresponding to inhibitory and excitatory interneurons, respectively. Application of 1 μM LP-211 in the presence of 10 μM SB269970 (a 5-HT7R antagonist) did not alter spontaneous EPSC frequency in 11 lamina II neurons, confirming the involvement of 5-HT7Rs in glutamate release facilitation. EPSCs evoked by dorsal root stimulation were also tested with LP-211. The currents, evoked by paired pulse protocol, were significantly potentiated by the compound (mean potentiation: 19±4.2%; 4 responsive neurons out 7). The second EPSC was less potentiated than the first and the paired pulse ratio decreased in 3 neurons. Conclusion The compound LP-211 is able to selectively activate 5-HT7Rs in the dorsal horn, causing a facilitatory effect of both spontaneous and evoked EPSCs. The decrease of paired-pulse ratio suggests that LP-211 activates presynaptic 5-HT7Rs, increasing glutamate release. The study of specific effects of these receptors on the different neuron populations will be critical to determine whether 5-HT7Rs exert anti- or pro-nociceptive effects at the spinal level. References 1. Cortes-Altamirano JL et al., Curr. Neuropharm, 2018, 16:210-221 2. Bardoni R, Curr. Neuropharm, 2019, 17:1133-1145 3. Hedlund PB et al, Neurosci Lett. 2010 481:12-6. 4. Betelli C et al., Mol. Pain, 2015, 11: