ProteoMutaMetrics: machine learning approaches for solute carrier family 6 mutation pathogenicity prediction.

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.

Experimental and Computational Analysis of Newly Identified Pathogenic Mutations in the Creatine Transporter SLC6A8.

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.

An Overview of Cell-Based Assay Platforms for the Solute Carrier Family of Transporters.

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.

Label-free high-throughput screening assay for the identification of norepinephrine transporter (NET/SLC6A2) inhibitors.

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.

Parallel All-Optical Assay to Study Use-Dependent Functioning of Voltage-Gated Ion Channels in a Miniaturized Format.

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.

Three-Dimensional Control of Ion Channel Function through Optogenetics and Co-Culture.

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.

Satiety Innovations: Food Products to Assist Consumers with Weight Loss, Evidence on the Role of Satiety in Healthy Eating: Overview and In Vitro Approximation.

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.

An innovative cell-based assay for the detection of modulators of soluble guanylate cyclase.

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.