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SLAS Discov ; 26(6): 783-797, 2021 07.
Article in English | MEDLINE | ID: covidwho-1293079


Classical high-throughput screening (HTS) technologies for the analysis of ionic currents across biological membranes can be performed using fluorescence-based, radioactive, and mass spectrometry (MS)-based uptake assays. These assays provide rapid results for pharmacological HTS, but the underlying, indirect analytical character of these assays can be linked to high false-positive hit rates. Thus, orthogonal and secondary assays using more biological target-based technologies are indispensable for further compound validation and optimization. Direct assay technologies for transporter proteins are electrophysiology-based, but are also complex, time-consuming, and not well applicable for automated profiling purposes. In contrast to conventional patch clamp systems, solid supported membrane (SSM)-based electrophysiology is a sensitive, membrane-based method for transporter analysis, and current technical developments target the demand for automated, accelerated, and sensitive assays for transporter-directed compound screening. In this study, the suitability of the SSM-based technique for pharmacological compound identification and optimization was evaluated performing cell-free SSM-based measurements with the electrogenic amino acid transporter B0AT1 (SLC6A19). Electrophysiological characterization of leucine-induced currents demonstrated that the observed signals were specific to B0AT1. Moreover, B0AT1-dependent responses were successfully inhibited using an established in-house tool compound. Evaluation of current stability and data reproducibility verified the robustness and reliability of the applied assay. Active compounds from primary screens of large compound libraries were validated, and false-positive hits were identified. These results clearly demonstrate the suitability of the SSM-based technique as a direct electrophysiological method for rapid and automated identification of small molecules that can inhibit B0AT1 activity.

Amino Acid Transport Systems, Neutral/metabolism , Electrophysiological Phenomena , High-Throughput Screening Assays/methods , Amino Acid Transport Systems, Neutral/agonists , Amino Acid Transport Systems, Neutral/antagonists & inhibitors , Animals , Biological Assay/methods , Biological Transport/drug effects , CHO Cells , Cell Membrane/metabolism , Cricetulus , Humans , Mice , Patch-Clamp Techniques/methods , Reproducibility of Results , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacology
Int J Mol Sci ; 21(17)2020 Aug 27.
Article in English | MEDLINE | ID: covidwho-831264


Outside of Mycobacterium tuberculosis and Mycobacterium leprae, nontuberculous mycobacteria (NTM) are environmental mycobacteria (>190 species) and are classified as slow- or rapid-growing mycobacteria. Infections caused by NTM show an increased incidence in immunocompromised patients and patients with underlying structural lung disease. The true global prevalence of NTM infections remains unknown because many countries do not require mandatory reporting of the infection. This is coupled with a challenging diagnosis and identification of the species. Current therapies for treatment of NTM infections require multidrug regimens for a minimum of 18 months and are associated with serious adverse reactions, infection relapse, and high reinfection rates, necessitating discovery of novel antimycobacterial agents. Robust drug discovery processes have discovered inhibitors targeting mycobacterial membrane protein large 3 (MmpL3), a protein responsible for translocating mycolic acids from the inner membrane to periplasm in the biosynthesis of the mycobacterial cell membrane. This review focuses on promising new chemical scaffolds that inhibit MmpL3 function and represent interesting and promising putative drug candidates for the treatment of NTM infections. Additionally, agents (FS-1, SMARt-420, C10) that promote reversion of drug resistance are also reviewed.

Anti-Bacterial Agents/pharmacology , Membrane Transport Proteins/metabolism , Mycobacterium Infections, Nontuberculous/drug therapy , Nontuberculous Mycobacteria/metabolism , Anti-Bacterial Agents/therapeutic use , Bacterial Proteins/metabolism , Biological Transport/drug effects , Drug Discovery , Drug Resistance, Multiple, Bacterial/drug effects , Gene Expression Regulation, Bacterial/drug effects , Humans , Iodophors/pharmacology , Iodophors/therapeutic use , Isoxazoles/pharmacology , Isoxazoles/therapeutic use , Mycobacterium Infections, Nontuberculous/metabolism , Mycolic Acids/metabolism , Nontuberculous Mycobacteria/drug effects , Polysaccharides/pharmacology , Polysaccharides/therapeutic use , Spiro Compounds/pharmacology , Spiro Compounds/therapeutic use