Positional isomers of bispyridine benzene derivatives induce efficacy changes on mGlu5 negative allosteric modulation

Modulation of metabotropic glutamate receptor 5 (mGlu5) with partial allosteric antagonists has received increased interest due to their favourable in vivo activity profiles compared to the unfavourable side-effects of full inverse agonists. Here we report on a series of bispyridine benzene derivatives with a functional molecular switch affecting antagonistic efficacy, shifting from inverse agonism to partial antagonism with only a single change in the substitution pattern of the benzene ring. These efficacy changes are explained through computational docking, revealing two different receptor conformations of different energetic stability and different positional isomer binding preferences

Shining light on an mGlu5 photoswitchable NAM: A theoretical perspective

Metabotropic glutamate receptors (mGluRs) are important drug targets because of their involvement in several neurological diseases. Among mGluRs, mGlu5 is a particularly high-profile target because its positive or negative allosteric modulation can potentially treat schizophrenia or anxiety and chronic pain, respectively. Here, we computationally and experimentally probe the functional binding of a novel photoswitchable mGlu5 NAM, termed alloswitch-1, which loses its NAM functionality under violet light. We show alloswitch-1 binds deep in the allosteric pocket in a similar fashion to mavoglurant, the co-crystallized NAM in the mGlu5 transmembrane domain crystal structure. Alloswitch-1, like NAM 2-Methyl-6-(phenylethynyl)pyridine (MPEP), is significantly affected by P655M mutation deep in the allosteric pocket, eradicating its functionality. In MD simulations, we show alloswitch-1 and MPEP stabilize the co-crystallized water molecule located at the bottom of the allosteric site that is seemingly characteristic of the inactive receptor state. Furthermore, both NAMs form H-bonds with S809 on helix 7, which may constitute an important stabilizing interaction for NAM-induced mGlu5 inactivation. Alloswitch-1, through isomerization of its amide group from trans to cis is able to form an additional interaction with N747 on helix 5. This may be an important interaction for amide-containing mGlu5 NAMs, helping to stabilize their binding in a potentially unusual cis-amide state. Simulated conformational switching of alloswitch-1 in silico suggests photoisomerization of its azo group from trans to cis may be possible within the allosteric pocket. However, photoexcited alloswitch-1 binds in an unstable fashion, breaking H-bonds with the protein and destabilizing the co-crystallized water molecule. This suggests photoswitching may have destabilizing effects on mGlu5 binding and functionality

Illuminating phenylazopyridines to photoswitch metabotropic glutamate receptors: from the flask to the animals

Phenylazopyridines are photoisomerizable compounds with high potential to control biological functions with light. We have obtained a series of phenylazopyridines with light dependent activity as negative allosteric modulators (NAM) of metabotropic glutamate receptor subtype 5 (mGlu5). Here we describe the factors needed to achieve an operational molecular photoisomerization and its effective translation into in vitro and in vivo receptor photoswitching, which includes zebrafish larva motility and the regulation of the antinociceptive effects in mice. The combination of light and some specific phenylazopyridine ligands displays atypical pharmacological profiles, including light-dependent receptor overactivation, which can be observed both in vitro and in vivo. Remarkably, the localized administration of light and a photoswitchable compound in the peripheral tissues of rodents or in the brain amygdalae results in an illumination-dependent analgesic effect. The results reveal a robust translation of the phenylazopyridine photoisomerization to a precise photoregulation of biological activity

BRAFV600E Mutant Allele Frequency (MAF) Influences Melanoma Clinicopathologic Characteristics

Background: Cutaneous melanoma shows high variability regarding clinicopathological presentation, evolution and prognosis. Methods: Next generation sequencing was performed to analyze hotspot mutations in different areas of primary melanomas (MMp) and their paired metastases. Clinicopathological features were evaluated depending on the degree of variation of the BRAFV600E mutant allele frequency (MAF) in MMp. Results: In our cohort of 14 superficial spreading, 10 nodular melanomas and 52 metastases, 17/24 (71%) melanomas had a BRAFV600E mutation and 5/24 (21%) had a NRASQ61 mutation. We observed a high variation of BRAFV600E MAF (H-BRAFV600E) in 7/17 (41%) MMp. The H-BRAFV600E MMp were all located on the trunk, had lower Breslow and mitotic indexes and predominantly, a first nodal metastasis. Regions with spindled tumor cells (Spin) and high lymphocytic infiltrate (HInf) were more frequent in the H-BRAFV600E patients (4/7; 57%), whereas regions with epithelial tumor cells (Epit) and low lymphocytic infiltrate (LInf) were predominant (6/10; 60%) and exclusive in the low BRAFV600E MAF variation tumors (L-BRAFV600E). The H-BRAFV600E/Spin/HInf MMp patients had better prognostic features and nodal first metastasis. Conclusions: The H-BRAFV600E MMp were located on the trunk, had better prognostic characteristics, such as lower Breslow and mitotic indexes as well as high lymphocytic infiltrate.This work was supported by grants from ISCIII and FEDER (“una manera de hacerEuropa”) PI15/00711 to RMM and PI18/00573 to RMM & AM. CIBERONC (CB16/12/00231) to XMG.Postdoctoral AECC (POSTD004MACI - POSTD16) to AM. Predoctoral UdL to PS and predoctoralAECC fellowship to IR. The work was also supported by the Xarxa de Bancs de Tumors de Catalunyasponsored by “Pla Director d’Oncologia de Catalunya (XBTC)”and IRBLleida Biobank (B.0000682)and PLATAFORMA BIOBANCOS (PT17/0015/0027; PT20/00021

Development of subtype-selective photoswitchable positive allosteric modulators for mGlu receptors

Positive allosteric modulators (PAMs) for metabotropic glutamate receptors have been postulated to treat neuropsychiatric diseases. Besides, obtaining a reversible and efficient spatiotemporal control of mGlu activity would be therapeutically advantageous. Photopharmacology may provide a solution on this topic, since it is based on the use of light and photoswitchable ligands to modulate a protein activity. This approach offers new perspectives for drug discovery and promises a better drug action control reducing side effects to unattained levels.Peer reviewe

A modular ligand-directed approach to label endogenous aminergic GPCRs in live cells

In the last two decades, new technologies based on luminescence have been developed to monitor the organization, signalling or ligand binding of G Protein-Coupled Receptors. These technologies rely on the overexpression of genetically modified (and/or fluorescently tagged) receptors of interest. However, there is an increasing interest in developping approaches to conjugate chemical labels to specific residues of native GPCRs, despite of the low reactivity and the high abundance of such residues.Peer reviewe

A modular click ligand-directed approach to label endogenous aminergic GPCRs in live cells

New technologies based on luminescence have been essential to monitor the organization, signaling, trafficking or ligand binding of G Protein-Coupled Receptors (GPCRs), but they rely on the overexpression of genetically modified receptors. As more and more studies indicate the importance of studying native receptors in their natural environment, it is essential to develop approaches allowing the specific labeling of native receptors. Here we report an innovative ligand directed approach to specifically label residues of native GPCRs upon ligand binding. To this end, we developed a ligand-directed toolbox based on a novel approach that uses molecular modules to build fluorescent ligand-directed probes that can label an archetypical aminergic GPCR (D1R). Our probes can be readily prepared before the labeling reaction from two molecular modules: an activated electrophilic linker which includes a fluorescent dye and a GPCR ligand that may include nucleophilic groups. Thanks to a fast and specific click reaction, the nucleophilic ligand can barely react with the activated linker before it is bound to the native target GPCR and the labeling reaction occurs. Subsequently, the ligand unbinds the GPCR pocket, leaving the receptor fluorescently labeled and fully functional. This novel labeling approach allowed us to label both D1 receptor in transfected cells and native receptors in neuronal cell lines. This approach will pave the way to develop new reagents and assays to monitor endogenous GPCRs distribution, trafficking, activity or binding properties in their native environment.Funding Agence Nationale de la Recherche ANR-17-CE11-0046 Ministerio de Ciencia e Innovación, Agencia Estatal de Investigación and ERDF - A way of making Europe CTQ2017-89222-R Ministerio de Ciencia e Innovación, Agencia Estatal de Investigación and ERDF - A way of making Europe PCI2018-093047 Ministerio de Ciencia e Innovación, Agencia Estatal de Investigación and ERDF - A way of making Europe PID2020-120499RB-I00 Catalan government 2017 SGR 1604 the European Union's Horizon 2020 research and innovation program under Marie Skłodowska-Curie grant agreement No. 801342 (TecniospringINDUSTRY) and the Government of Catalonia's Agency for Business Competitive-ness (ACCIÓ). TECSPR19-1-0062Peer reviewe

A modular ligand-directed approach to label endogenous aminergic GPCRs in live cells

Peer reviewe

A MODULAR LIGAND-DIRECTED APPROACH TO LABEL ENDOGENOUS AMINERGIC GPCRS IN LIVE CELLS

In the last two decades, new technologies based on luminescence have been developed to monitor the organization, signalling or ligand binding of G Protein-Coupled Receptors. These technologies rely on the overexpression of genetically modified (and/or fluorescently tagged) receptors of interest. However, there is an increasing interest in developping approaches to conjugate chemical labels to specific residues of native GPCRs, despite of the low reactivity and the high abundance of such residues. Ligand directed approaches, may offer a solution to this problem. Such approaches consist in molecular probes that include a selective ligand moiety and a reactive moiety. Upon ligand binding, the labelling is directed to a nucleophilic amino acid in the vicinity of the binding pocket. However, this requires the use of non-nucleophilic ligand moieties, which is particularly difficult since many GPCR ligands contain amines or other nucleophilic functional groups. In the present work, we developed an innovative ligand-directed toolbox based on a novel approach. The later uses molecular modules to build fluorescent ligand-directed probes to label an archetypical aminergic GPCR (D1R). Our molecular probes can be readily prepared before the labelling reaction from two molecular modules: an activated electrophilic linker which includes a fluorescent dye and a GPCR ligand that may include nucleophilic groups. Thanks to a fast and specific chemical reaction, the nucleophilic ligand can barely react with the activated linker before it is bound to the native target GPCR and the labelling reaction occurs. Subsequently, the ligand will unbind the GPCR pocket, leaving the receptor fluorescently labelled and fully functional. This novel labelling approach allowed us to label endogenous D1 receptor both in transfected cells and primary cultures of neurons and will pave the way to develop new reagents and assays to monitor endogenous GPCRs distribution and activity in their native environment.Peer reviewe