RESUMO
With over 450 genes, solute carriers (SLCs) constitute the largest transporter superfamily responsible for the uptake and efflux of nutrients, metabolites, and xenobiotics in human cells. SLCs are associated with a wide variety of human diseases, including cancer, diabetes, and metabolic and neurological disorders. They represent an important therapeutic target class that remains only partly exploited as therapeutics that target SLCs are scarce. Additionally, many small molecules reported in the literature to target SLCs are poorly characterized. Both features may be due to the difficulty of developing SLC transport assays that fulfill the quality criteria for high-throughput screening. Here, we report one of the main limitations hampering assay development within the RESOLUTE consortium: the lack of a resource providing high-quality information on SLC tool compounds. To address this, we provide a systematic annotation of tool compounds targeting SLCs. We first provide an overview on RESOLUTE assays. Next, we present a list of SLC-targeting compounds collected from the literature and public databases; we found that most data sources lacked specificity data. Finally, we report on experimental tests of 19 selected compounds against a panel of 13 SLCs from seven different families. Except for a few inhibitors, which were active on unrelated SLCs, the tested inhibitors demonstrated high selectivity for their reported targets. To make this knowledge easily accessible to the scientific community, we created an interactive dashboard displaying the collected data in the RESOLUTE web portal (https://re-solute.eu). We anticipate that our open-access resources on assays and compounds will support the development of future drug discovery campaigns for SLCs.
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Aspartate is crucial for nucleotide synthesis, ammonia detoxification, and maintaining redox balance via the malate-aspartate-shuttle (MAS). To disentangle these multiple roles of aspartate metabolism, tools are required that measure aspartate concentrations in real time and in live cells. We introduce AspSnFR, a genetically encoded green fluorescent biosensor for intracellular aspartate, engineered through displaying and screening biosensor libraries on mammalian cells. In live cells, AspSnFR is able to precisely and quantitatively measure cytosolic aspartate concentrations and dissect its production from glutamine. Combining high-content imaging of AspSnFR with pharmacological perturbations exposes differences in metabolic vulnerabilities of aspartate levels based on nutrient availability. Further, AspSnFR facilitates tracking of aspartate export from mitochondria through SLC25A12, the MAS' key transporter. We show that SLC25A12 is a rapidly responding and direct route to couple Ca2+ signaling with mitochondrial aspartate export. This establishes SLC25A12 as a crucial link between cellular signaling, mitochondrial respiration, and metabolism.
Assuntos
Ácido Aspártico , Técnicas Biossensoriais , Ácido Aspártico/metabolismo , Humanos , Mitocôndrias/metabolismo , Células HEK293 , Células HeLa , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Fluorescência Verde/genéticaRESUMO
Pharmacological modulation of cannabinoid receptor type 2 (CB2R) holds promise for the treatment of neuroinflammatory disorders, such as Alzheimer's disease. Despite the importance of CB2R, its expression and downstream signaling are insufficiently understood in disease- and tissue-specific contexts. Herein, we report the first ligand-directed covalent (LDC) labeling of CB2R enabled by a novel synthetic strategy and application of platform reagents. The LDC modification allows visualization and study of CB2R while maintaining its ability to bind other ligands at the orthosteric site. We employed in silico docking and molecular dynamics simulations to guide probe design and assess the feasibility of LDC labeling of CB2R. We demonstrate selective, covalent labeling of a peripheral lysine residue of CB2R by exploiting fluorogenic O-nitrobenzoxadiazole (O-NBD)-functionalized probes in a TR-FRET assay. The rapid proof-of-concept validation with O-NBD probes inspired incorporation of advanced electrophiles suitable for experiments in live cells. To this end, novel synthetic strategies toward N-sulfonyl pyridone (N-SP) and N-acyl-N-alkyl sulfonamide (NASA) LDC probes were developed, which allowed covalent delivery of fluorophores suitable for cellular studies. The LDC probes were characterized by a radioligand binding assay and TR-FRET experiments. Additionally, the probes were applied to specifically visualize CB2R in conventional and imaging flow cytometry as well as in confocal fluorescence microscopy using overexpressing and endogenously expressing microglial live cells.
Assuntos
Corantes Fluorescentes , Transdução de Sinais , Ligantes , Ligação Proteica , Corantes Fluorescentes/química , Receptores de CanabinoidesRESUMO
The solute carrier (SLC) superfamily represents the biggest family of transporters with important roles in health and disease. Despite being attractive and druggable targets, the majority of SLCs remains understudied. One major hurdle in research on SLCs is the lack of tools, such as cell-based assays to investigate their biological role and for drug discovery. Another challenge is the disperse and anecdotal information on assay strategies that are suitable for SLCs. This review provides a comprehensive overview of state-of-the-art cellular assay technologies for SLC research and discusses relevant SLC characteristics enabling the choice of an optimal assay technology. The Innovative Medicines Initiative consortium RESOLUTE intends to accelerate research on SLCs by providing the scientific community with high-quality reagents, assay technologies and data sets, and to ultimately unlock SLCs for drug discovery.
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Photopharmacology aims at the optical control of protein activity using synthetic photoswitches. This approach has been recently expanded to nuclear hormone receptors with the introduction of "photohormones" for the retinoic acid receptor, farnesoid X receptor, and estrogen receptor. Herein, we report the development and profiling of photoswitchable agonists for peroxisome proliferator-activated receptor γ (PPARγ). Based on known PPARγ ligands (MDG548, GW1929, and rosiglitazone), we have designed and synthesized azobenzene derivatives, termed AzoGW1929 and AzoRosi, which were confirmed to be active in cell-based assays. Subsequent computer-aided optimization of AzoRosi resulted in the photohormone AzoRosi-4, which bound and activated PPARγ preferentially in its light-activated cis-configuration.
Assuntos
Luz , PPAR gama/agonistas , Animais , Humanos , Simulação de Acoplamento Molecular , PPAR gama/química , PPAR gama/metabolismo , Conformação Proteica , Receptores Citoplasmáticos e Nucleares/agonistas , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores de Estrogênio/efeitos dos fármacos , Receptores de Estrogênio/metabolismo , Receptores do Ácido Retinoico/agonistas , Receptores do Ácido Retinoico/metabolismoRESUMO
Receptor tyrosine kinases (RTKs) are key regulators of cellular functions in metazoans. In vertebrates, RTKs are mostly activated by polypeptides but are not naturally sensitive to amino acids or light. Taking inspiration from Venus kinase receptors (VKRs), an atypical family of RTKs found in nature, we have transformed the human insulin (hIR) and hepatocyte growth factor receptor (hMET) into glutamate receptors by replacing their extracellular binding domains with the ligand-binding domain of metabotropic glutamate receptor typeâ 2 (mGluR2). We then imparted light sensitivity through covalent attachment of a synthetic glutamate-based photoswitch via a self-labelling SNAP tag. By employing a Xenopus laevis oocyte kinase activity assay, we demonstrate how these chimeric RTKs, termed light-controlled human insulin receptor (LihIR) and light-controlled human MET receptor (LihMET), can be used to exert optical control over the insulin or MET signaling pathways. Our results outline a potentially general strategy to convert RTKs into photoreceptors.
Assuntos
Luz , Proteínas Proto-Oncogênicas c-met/metabolismo , Receptor de Insulina/metabolismo , Receptores de Glutamato/metabolismo , Animais , Biotransformação , Humanos , Transdução de Sinais , Xenopus laevisRESUMO
Azobenzenes are versatile photoswitches that have found widespread use in a variety of fields, ranging from photopharmacology to the material sciences. In addition to regular azobenzenes, the cyclic diazocines have recently emerged. Although diazocines have fascinating conformational and photophysical properties, their use has been limited by their synthetic accessibility. Herein, we present a general, high-yielding protocol that relies on the oxidative cyclization of dianilines. In combination with a modular substrate synthesis, it allows for rapid access to diversely functionalized diazocines on gram scales. Our work systematically explores substituent effects on the photoisomerization and thermal relaxation of diazocines. It will enable their incorporation into a wide variety of functional molecules, unlocking the full potential of these emerging photoswitches. The method can be applied to the synthesis of a new cyclic azobenzene with a nine-membered central ring and distinct properties.
Assuntos
Compostos Azo/química , Azocinas/química , Compostos Azo/síntese química , Azocinas/síntese química , Cobre/química , Ciclização , Espectroscopia de Ressonância Magnética , Oxirredução , Processos Fotoquímicos , Espectrofotometria UltravioletaRESUMO
Chemical and electrical signaling at the synapse is a dynamic process that is crucial to neurotransmission and pathology. Traditional pharmacotherapy has found countless applications in both academic labs and the clinic; however, diffusible drugs lack spatial and temporal precision when employed in heterogeneous tissues such as the brain. In the field of photopharmacology, chemical attachment of a synthetic photoswitch to a bioactive ligand allows cellular signaling to be controlled with light. Azobenzenes have remained the go-to photoswitch for biological applications due to their tunable photophysical properties, and can be leveraged to achieve reversible optical control of numerous receptors and ion channels. Here, we discuss the most recent advances in photopharmacology which will improve the use of azobenzene-based probes for neuroscience applications.
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Compostos Azo , Desenho de Fármacos , Sondas Moleculares , Transmissão Sináptica , Compostos Azo/metabolismo , Luz , Sondas Moleculares/metabolismo , Transdução de Sinais/efeitos da radiação , Transmissão Sináptica/efeitos da radiaçãoRESUMO
Photoswitchable blockers of potassium channels can be used to optically control neuronal excitability and hold great promise for vision restoration. Here, we report a series of improved photoswitchable blockers that are furnished with a new pharmacophore based on the local anesthetic bupivacaine. These azobupivacaines (ABs) enable optical control over the delayed rectifier channel Kv2.1. and target the two-pore domain potassium channel TREK-1. For the first time, we have identified a compound that blocks conductance in the dark and potentiates it upon illumination. Using light as a trigger, ABs efficiently and reversibly silence action potential firing of hippocampal neurons in acute mouse brain slices.
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Potenciais de Ação/efeitos dos fármacos , Compostos Azo/farmacologia , Bupivacaína/análogos & derivados , Luz , Neurônios/efeitos dos fármacos , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio de Domínios Poros em Tandem/efeitos dos fármacos , Canais de Potássio Shab/efeitos dos fármacos , Animais , Compostos Azo/síntese química , Células HEK293 , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Humanos , Camundongos , Neurônios/metabolismo , Fenômenos Ópticos , Bloqueadores dos Canais de Potássio/síntese química , Canais de Potássio de Domínios Poros em Tandem/antagonistas & inibidores , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Canais de Potássio Shab/antagonistas & inibidores , Canais de Potássio Shab/metabolismoRESUMO
Understanding the activation and internalization of G protein-coupled receptors (GPCRs) using conditional approaches is paramount to developing new therapeutic strategies. Here, we describe the design, synthesis, and testing of ExONatide, a benzylguanine-linked peptide agonist of the glucagon-like peptide-1 receptor (GLP-1R), a class B GPCR required for maintenance of glucose levels in humans. ExONatide covalently binds to SNAP-tagged GLP-1R-expressing cells, leading to prolonged cAMP generation, Ca2+ rises, and intracellular retention of the receptor. These effects were readily switched OFF following cleavage of the introduced disulfide bridge using the cell-permeable reducing agent beta-mercaptoethanol (BME). A similar approach could be extended to a class A GPCR using GhrelON, a benzylguanine-linked peptide agonist of the growth hormone secretagogue receptor 1a (GHS-R1a), which is involved in food intake and growth. Thus, ExONatide and GhrelON allow SNAP-tag-directed activation of class A and B GPCRs involved in gut hormone signaling in a reversible manner. This tactic, termed reductively cleavable agONist (RECON), may be useful for understanding GLP-1R and GHS-R1a function both in vitro and in vivo, with applicability across GPCRs.
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The cannabinoid receptor 1 (CB1) is an inhibitory G protein-coupled receptor abundantly expressed in the central nervous system. It has rich pharmacology and largely accounts for the recreational use of cannabis. We describe efficient asymmetric syntheses of four photoswitchable Δ9-tetrahydrocannabinol derivatives (azo-THCs) from a central building block 3-Br-THC. Using electrophysiology and a FRET-based cAMP assay, two compounds are identified as potent CB1 agonists that change their effect upon illumination. As such, azo-THCs enable CB1-mediated optical control of inwardly rectifying potassium channels, as well as adenylyl cyclase.
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Canabinoides/química , Dronabinol/química , Fármacos Fotossensibilizantes/química , Animais , Sítios de Ligação , Bioensaio , Encéfalo/efeitos dos fármacos , Desenho de Fármacos , Fenômenos Eletrofisiológicos , Óptica e Fotônica , Ratos , Receptor CB1 de Canabinoide , Transdução de SinaisRESUMO
Genetics and pharmacology are often seen as two distinct approaches to interrogating, elucidating, and manipulating biological systems. The former is renowned for its precision whereas the latter for its fast kinetics, reversibility, and practicality. Here, we show that both can be joined as "tethered pharmacology", wherein a genetically programmed bioconjugation site provides selectivity and a tethered pharmacophore provides function. The speed of onset, and especially cessation, of pharmacological activity can be greatly enhanced by incorporating photoswitches and using light as the trigger ("tethered photopharmacology"). Genetically encoded, tethered photopharmacology is a variant of optogenetics and could even play a role in medicine wherever gene therapy is viable. However, gene therapy may not be necessary if sufficiently selective tethering strategies that operate on wild-type receptors can be developed.
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Optogenética/métodos , Farmacologia/métodos , Humanos , Fatores de TempoRESUMO
G protein-coupled receptor (GPCR) signaling occurs in complex spatiotemporal patterns that are difficult to probe using standard pharmacological and genetic approaches. A powerful approach for dissecting GPCRs is to use light-controlled pharmacological agents that are tethered covalently and specifically to genetically engineered receptors. However, deficits in our understanding of the mechanism of such photoswitches have limited application of this approach and its extension to other GPCRs. In this study, we have harnessed the power of bioorthogonal tethering to SNAP and CLIP protein tags to create a family of light-gated metabotropic glutamate receptors (mGluRs). We define the mechanistic determinants of photoswitch efficacy, including labeling efficiency, dependence on photoswitch structure, length dependence of the linker between the protein tag and the glutamate ligand, effective local concentration of the glutamate moiety, and affinity of the receptor for the ligand. We improve the scheme for photoswitch synthesis as well as photoswitch efficiency, and generate seven light-gated group II/III mGluRs, including variants of mGluR2, 3, 6, 7, and 8. Members of this family of light-controlled receptors can be used singly or in specifically labeled, independently light-controlled pairs for multiplexed control of receptor populations.
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Ativação do Canal Iônico , Luz , Receptores de Glutamato Metabotrópico , Transdução de Sinais , Animais , Células HEK293 , Humanos , Ativação do Canal Iônico/genética , Ativação do Canal Iônico/efeitos da radiação , Ratos , Receptores de Glutamato Metabotrópico/genética , Receptores de Glutamato Metabotrópico/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/efeitos da radiaçãoRESUMO
The covalent attachment of synthetic photoswitches is a general approach to impart light sensitivity onto native receptors. It mimics the logic of natural photoreceptors and significantly expands the reach of optogenetics. Here we describe a novel photoswitch design-the photoswitchable orthogonal remotely tethered ligand (PORTL)-that combines the genetically encoded SNAP-tag with photochromic ligands connected to a benzylguanine via a long flexible linker. We use the method to convert the G protein-coupled receptor mGluR2, a metabotropic glutamate receptor, into a photoreceptor (SNAG-mGluR2) that provides efficient optical control over the neuronal functions of mGluR2: presynaptic inhibition and control of excitability. The PORTL approach enables multiplexed optical control of different native receptors using distinct bioconjugation methods. It should be broadly applicable since SNAP-tags have proven to be reliable, many SNAP-tagged receptors are already available, and photochromic ligands on a long leash are readily designed and synthesized.