RESUMEN
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.
RESUMEN
Transporters of the solute carrier superfamily (SLCs) are responsible for the transmembrane traffic of the majority of chemical substances in cells and tissues and are therefore of fundamental biological importance. As is often the case with membrane proteins that can be heavily glycosylated, a lack of reliable high-affinity binders hinders their functional analysis. Purifying and reconstituting transmembrane proteins in their lipidic environments remains challenging and standard approaches to generate binders for multi-transmembrane proteins, such as SLCs, channels or G protein-coupled receptors (GPCRs) are lacking. While generating protein binders to 27 SLCs, we produced full length protein or cell lines as input material for binder generation by selected binder generation platforms. As a result, we obtained 525 binders for 22 SLCs. We validated the binders with a cell-based validation workflow using immunofluorescent and immunoprecipitation methods to process all obtained binders. Finally, we demonstrated the potential applications of the binders that passed our validation pipeline in structural, biochemical, and biological applications using the exemplary protein SLC12A6, an ion transporter relevant in human disease. With this work, we were able to generate easily renewable and highly specific binders against SLCs, which will greatly facilitate the study of this neglected protein family. We hope that the process will serve as blueprint for the generation of binders against the entire superfamily of SLC transporters.
Asunto(s)
Unión Proteica , Proteínas Transportadoras de Solutos , Humanos , Proteínas Transportadoras de Solutos/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/química , Células HEK293RESUMEN
Zinc is an essential trace element that is involved in many biological processes and in cellular homeostasis. In pancreatic ß-cells, zinc is crucial for the synthesis, processing, and secretion of insulin, which plays a key role in glucose homeostasis and which deficiency is the cause of diabetes. The accumulation of zinc in pancreatic cells is regulated by the solute carrier transporter SLC30A8 (or Zinc Transporter 8, ZnT8), which transports zinc from cytoplasm in intracellular vesicles. Allelic variants of SLC30A8 gene have been linked to diabetes. Given the physiological intracellular localization of SLC30A8 in pancreatic ß-cells and the ubiquitous endogenous expression of other Zinc transporters in different cell lines that could be used as cellular model for SLC30A8 recombinant over-expression, it is challenging to develop a functional assay to measure SLC30A8 activity. To achieve this goal, we have firstly generated a HEK293 cell line stably overexpressing SLC30A8, where the over-expression favors the partial localization of SLC30A8 on the plasma membrane. Then, we used the combination of this cell model, commercial FluoZin-3 cell permeant zinc dye and live cell imaging approach to follow zinc flux across SLC30A8 over-expressed on plasma membrane, thus developing a novel functional imaging- based assay specific for SLC30A8. Our novel approach can be further explored and optimized, paving the way for future small molecule medium-throughput screening.
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Transportador 8 de Zinc , Zinc , Humanos , Transportador 8 de Zinc/genética , Transportador 8 de Zinc/metabolismo , Células HEK293 , Zinc/metabolismo , Membrana Celular/metabolismo , Células Secretoras de Insulina/metabolismo , Transporte Biológico , Proteínas de Transporte de Catión/genética , Proteínas de Transporte de Catión/metabolismoRESUMEN
The solute carrier transporter family 6 (SLC6) is of key interest for their critical role in the transport of small amino acids or amino acid-like molecules. Their dysfunction is strongly associated with human diseases such as including schizophrenia, depression, and Parkinson's disease. Linking single point mutations to disease may support insights into the structure-function relationship of these transporters. This work aimed to develop a computational model for predicting the potential pathogenic effect of single point mutations in the SLC6 family. Missense mutation data was retrieved from UniProt, LitVar, and ClinVar, covering multiple protein-coding transcripts. As encoding approach, amino acid descriptors were used to calculate the average sequence properties for both original and mutated sequences. In addition to the full-sequence calculation, the sequences were cut into twelve domains. The domains are defined according to the transmembrane domains of the SLC6 transporters to analyse the regions' contributions to the pathogenicity prediction. Subsequently, several classification models, namely Support Vector Machine (SVM), Logistic Regression (LR), Random Forest (RF), and Extreme Gradient Boosting (XGBoost) with the hyperparameters optimized through grid search were built. For estimation of model performance, repeated stratified k-fold cross-validation was used. The accuracy values of the generated models are in the range of 0.72 to 0.80. Analysis of feature importance indicates that mutations in distinct regions of SLC6 transporters are associated with an increased risk for pathogenicity. When applying the model on an independent validation set, the performance in accuracy dropped to averagely 0.6 with high precision but low sensitivity scores.
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Creatine is an essential metabolite for the storage and rapid supply of energy in muscle and nerve cells. In humans, impaired metabolism, transport, and distribution of creatine throughout tissues can cause varying forms of mental disability, also known as creatine deficiency syndrome (CDS). So far, 80 mutations in the creatine transporter (SLC6A8) have been associated to CDS. To better understand the effect of human genetic variants on the physiology of SLC6A8 and their possible impact on CDS, we studied 30 missense variants including 15 variants of unknown significance, two of which are reported here for the first time. We expressed these variants in HEK293 cells and explored their subcellular localization and transport activity. We also applied computational methods to predict variant effect and estimate site-specific changes in thermodynamic stability. To explore variants that might have a differential effect on the transporter's conformers along the transport cycle, we constructed homology models of the inward facing, and outward facing conformations. In addition, we used mass-spectrometry to study proteins that interact with wild type SLC6A8 and five selected variants in HEK293 cells. In silico models of the protein complexes revealed how two variants impact the interaction interface of SLC6A8 with other proteins and how pathogenic variants lead to an enrichment of ER protein partners. Overall, our integrated analysis disambiguates the pathogenicity of 15 variants of unknown significance revealing diverse mechanisms of pathogenicity, including two previously unreported variants obtained from patients suffering from the creatine deficiency syndrome.
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Encefalopatías Metabólicas Innatas , Creatina , Discapacidad Intelectual Ligada al Cromosoma X , Proteínas del Tejido Nervioso , Proteínas de Transporte de Neurotransmisores en la Membrana Plasmática , Humanos , Creatina/deficiencia , Células HEK293 , Discapacidad Intelectual Ligada al Cromosoma X/genética , Proteínas del Tejido Nervioso/deficiencia , Proteínas del Tejido Nervioso/genética , Proteínas de Transporte de Neurotransmisores en la Membrana Plasmática/deficiencia , Proteínas de Transporte de Neurotransmisores en la Membrana Plasmática/genética , Encefalopatías Metabólicas Innatas/genética , Análisis Mutacional de ADN/métodos , Mutación Missense , Biología Computacional/métodosRESUMEN
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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The human norepinephrine transporter (NET) is an established drug target for a wide range of psychiatric disorders. Conventional methods that are used to functionally characterize NET inhibitors are based on the use of radiolabeled or fluorescent substrates. These methods are highly informative, but pose limitations to either high-throughput screening (HTS) adaptation or physiologically accurate representation of the endogenous uptake events. Recently, we developed a label-free functional assay based on the activation of G protein-coupled receptors by a transported substrate, termed the TRACT assay. In this study, the TRACT assay technology was applied to NET expressed in a doxycycline-inducible HEK 293 JumpIn cell line. Three endogenous substrates of NET-norepinephrine (NE), dopamine (DA) and epinephrine (EP)-were compared in the characterization of the reference NET inhibitor nisoxetine. The resulting assay, using NE as a substrate, was validated in a manual HTS set-up with a Z' = 0.55. The inhibitory potencies of several reported NET inhibitors from the TRACT assay showed positive correlation with those from an established fluorescent substrate uptake assay. These findings demonstrate the suitability of the TRACT assay for HTS characterization and screening of NET inhibitors and provide a basis for investigation of other solute carrier transporters with label-free biosensors.
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Fluoxetina/análogos & derivados , Ensayos Analíticos de Alto Rendimiento , Proteínas de Transporte de Noradrenalina a través de la Membrana Plasmática/antagonistas & inhibidores , Técnicas Biosensibles , Dopamina/metabolismo , Doxiciclina/farmacología , Epinefrina/metabolismo , Fluoxetina/química , Fluoxetina/aislamiento & purificación , Humanos , Norepinefrina/metabolismo , Proteínas de Transporte de Noradrenalina a través de la Membrana Plasmática/metabolismo , Especificidad por SustratoRESUMEN
Voltage-gated ion channels produce rapid transmembrane currents responsible for action potential generation and propagation at the neuronal, muscular, and cardiac levels. They represent attractive clinical targets because their altered firing frequency is often the hallmark of pathological signaling leading to several neuromuscular disorders. Therefore, a method to study their functioning upon repeated triggers at different frequencies is desired to develop new drug molecules selectively targeting pathological phenotype. Optogenetics provides powerful tools for millisecond switch of cellular excitability in contactless, physiological, and low-cost settings. Nevertheless, its application to large-scale drug-screening operations is still limited by long processing time (due to sequential well read), rigid flashing pattern, lack of online compound addition, or high consumable costs of existing methods. Here, we developed a method that enables simultaneous analysis of 384-well plates with optical pacing, fluorescence recording, and liquid injection. We used our method to deliver programmable millisecond-switched depolarization through light-activated opsin in concomitance with continuous optical recording by a fluorescent indicator. We obtained 384-well pacing of recombinant voltage-activated sodium or calcium channels, as well as induced pluripotent stem cell (iPSC)-derived cardiomyocytes, in all-optical parallel settings. Furthermore, we demonstrated the use-dependent behavior of known ion channel blockers by optogenetic pacing at normal or pathological firing frequencies, obtaining very good signal reproducibility and accordance with electrophysiology data. Our method provides a novel physiological approach to study frequency-dependent drug behavior using reversible programmable triggers. The all-optical parallel settings combined with contained operational costs make our method particularly suited for large-scale drug-screening campaigns as well as cardiac liability studies.
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Bioensayo , Bloqueadores de los Canales de Calcio/farmacología , Optogenética/métodos , Bloqueadores de los Canales de Potasio/farmacología , Proteínas Algáceas/antagonistas & inhibidores , Proteínas Algáceas/genética , Proteínas Algáceas/metabolismo , Canales de Calcio/genética , Canales de Calcio/metabolismo , Canales de Calcio Tipo L/genética , Canales de Calcio Tipo L/metabolismo , Línea Celular , Chlamydomonas reinhardtii , Colorantes Fluorescentes/química , Expresión Génica , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Activación del Canal Iónico/efectos de los fármacos , Miocitos Cardíacos/citología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Canal de Sodio Activado por Voltaje NAV1.5/genética , Canal de Sodio Activado por Voltaje NAV1.5/metabolismo , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Imagen Óptica/métodos , Canales de Potasio de Rectificación Interna/antagonistas & inhibidores , Canales de Potasio de Rectificación Interna/genética , Canales de Potasio de Rectificación Interna/metabolismo , Canales de Potasio de Dominio Poro en Tándem/antagonistas & inhibidores , Canales de Potasio de Dominio Poro en Tándem/genética , Canales de Potasio de Dominio Poro en Tándem/metabolismo , Rodopsina/antagonistas & inhibidores , Rodopsina/genética , Rodopsina/metabolismoRESUMEN
The lack of miniaturized and cost-effective methods to control cellular excitability with dosable and temporally precise electrical perturbations represents a long-lasting and unsolved bottleneck for ion channel drug discovery pipelines. Here we developed a high-throughput-compatible fluorescent-based cellular assay that combines optogenetics and co-culture approaches to obtain spatial, temporal, and quantitative control of ion channel activity. The modularity and increased flexibility of control of this light-tandem assay, combined with contained costs and compatibility with conventional drug-screening platforms, make this system suitable for temporally precise screening of ion channel function in controlled conformations and can also be used to recapitulate other complexly regulated biological processes.
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Descubrimiento de Drogas/métodos , Ensayos Analíticos de Alto Rendimiento , Canales Iónicos/química , Optogenética/métodos , Canales de Calcio Tipo L/química , Línea Celular , Técnicas de Cocultivo , Humanos , Canales Iónicos/agonistas , Canales Iónicos/antagonistas & inhibidores , Cinética , LigandosRESUMEN
The prevalence of overweight and obesity is increasing globally, driven by the availability of energy-dense palatable foods. Most dietary strategies fail because of hunger generated by calorie restriction, and interventions that specifically control hunger and/or promote fullness may aid success. Current consumers have a limited choice of satiety-enhancing products with proven health benefits, and innovative ways to produce new foods (as structural modification) to enhance satiety/satiation may provide new opportunities. However, this potential is hindered by the cost of product testing. Within the SATIN-SATiety INnovation project-an in vitro platform has been developed to offer a cost-effective means of assessing the potential satiation/satiety effect of novel foods. This combines in vitro technologies to assess changes in colonic bacteria metabolism, appetite hormone release and the stability and bioavailability of active compounds in the new products/ingredients. This article provides a brief review of nutrients for which an impact on short-term appetite regulation has been demonstrated, and a summary of the changes to food structure which can be used to produce a change in appetite expression. Furthermore, the SATIN in vitro platform is discussed as a means of assessing the impact of nutritional and structural manipulations on appetite.
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Alimentos , Saciedad , Pérdida de Peso , Animales , Dieta Saludable , Ingestión de Alimentos , Humanos , SeguridadRESUMEN
Guanylate cyclase (GC) catalyzes the biosynthesis of cyclic guanosine 3',5'- monophosphate (cGMP) from GTP. GC exists in two isoenzyme forms: soluble and membrane-bound. The soluble GC (sGC) is a heterodimer composed of an alpha and a beta subunit, and it contains heme as a prosthetic group. The most important physiological activator of sGC is nitric oxide, which activates the enzyme upon binding to the heme moiety. By producing the second messenger cGMP, which regulates various effector systems such as phosphodiesterases, ion channels, and protein kinases, sGC plays an important role in different physiological processes, thus representing a very attractive pharmacological target. In fact, the pathogenesis of several diseases, especially those of the cardiovascular system, has been linked to inappropriate regulation of sGC. In order to find new modulators for this important enzyme, an innovative cell-based assay has been developed and optimized for the use in high-throughput screening. This luminescent assay, which is suitable for both 96- and 384-well plate formats, has been achieved by stably expressing the alpha and beta subunits of a mutated form of sGC in Chinese hamster ovary cells. The mutated form synthesizes cyclic adenosine 3',5'-monophosphate instead of cGMP, allowing the detection of enzymatic activity by a reporter gene approach. We demonstrated that this cell line responds to compounds typically used in the field of sGC research and that it represents an innovative and robust assay to screen for sGC modulators with high efficiency and high sensitivity by means of standard luminescence readers.