ABSTRACT
Despite significant advances in the development of therapeutic interventions targeting autoimmune diseases and chronic inflammatory conditions, lack of effective treatment still poses a high unmet need. Modulating chronically activated T cells through the blockade of the Kv1.3 potassium channel is a promising therapeutic approach; however, developing selective Kv1.3 inhibitors is still an arduous task. Phage display-based high throughput peptide library screening is a rapid and robust approach to develop promising drug candidates; however, it requires solid-phase immobilization of target proteins with their binding site preserved. Historically, the KcsA bacterial channel chimera harboring only the turret region of the human Kv1.3 channel was used for screening campaigns. Nevertheless, literature data suggest that binding to this type of chimera does not correlate well with blocking potency on the native Kv1.3 channels. Therefore, we designed and successfully produced advanced KcsA-Kv1.3, KcsA-Kv1.1, and KcsA-Kv1.2 chimeric proteins in which both the turret and part of the filter regions of the human Kv1.x channels were transferred. These T+F (turret-filter) chimeras showed superior peptide ligand-binding predictivity compared to their T-only versions in novel phage ELISA assays. Phage ELISA binding and competition results supported with electrophysiological data confirmed that the filter region of KcsA-Kv1.x is essential for establishing adequate relative affinity order among selected peptide toxins (Vm24 toxin, Hongotoxin-1, Kaliotoxin-1, Maurotoxin, Stichodactyla toxin) and consequently obtaining more reliable selectivity data. These new findings provide a better screening tool for future drug development efforts and offer insight into the target-ligand interactions of these therapeutically relevant ion channels.
Subject(s)
Kv1.3 Potassium Channel , Potassium Channel Blockers , Recombinant Fusion Proteins , Animals , Humans , Bacterial Proteins/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/chemistry , Bacterial Proteins/antagonists & inhibitors , Binding Sites , Kv1.3 Potassium Channel/metabolism , Kv1.3 Potassium Channel/antagonists & inhibitors , Kv1.3 Potassium Channel/genetics , Kv1.3 Potassium Channel/chemistry , Ligands , Peptide Library , Potassium Channel Blockers/chemistry , Potassium Channel Blockers/pharmacology , Potassium Channels/metabolism , Potassium Channels/chemistry , Potassium Channels/genetics , Recombinant Fusion Proteins/metabolism , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Cell LineABSTRACT
Chlorotoxin (CTX), a scorpion venom-derived 36-residue miniprotein, binds to and is taken up selectively by glioblastoma cells. Previous studies provided controversial results concerning target protein(s) of CTX. These included CLC3 chloride channel, matrix metalloproteinase 2 (MMP-2), regulators of MMP-2, annexin A2, and neuropilin 1 (NRP1). The present study aimed at clarifying which of the proposed binding partners can really interact with CTX using biochemical methods and recombinant proteins. For this purpose, we established two new binding assays based on anchoring the tested proteins to microbeads and quantifying the binding of CTX by flow cytometry. Screening of His-tagged proteins anchored to cobalt-coated beads indicated strong interaction of CTX with MMP-2 and NRP1, whereas binding to annexin A2 was not confirmed. Similar results were obtained with fluorophore-labeled CTX and CTX-displaying phages. Affinity of CTX to MMP-2 and NRP1 was assessed by the "immunoglobulin-coated bead" test, in which the proteins were anchored to beads by specific antibodies. This assay yielded highly reproducible data using both direct titration and displacement approach. The affinities of labeled and unlabeled CTX appeared to be similar for both MMP-2 and NRP1 with estimated KD values of 0.5 to 0.7 µM. Contrary to previous reports, we found that CTX does not inhibit the activity of MMP-2 and that CTX not only with free carboxyl end but also with carboxamide terminal end binds to NRP1. We conclude that the presented robust assays could also be applied for affinity-improving studies of CTX to its genuine targets using phage display libraries.
Subject(s)
Glioblastoma , Matrix Metalloproteinase 2 , Neuropilin-1 , Scorpion Venoms , Humans , Glioblastoma/metabolism , Matrix Metalloproteinase 2/metabolism , Neuropilin-1/metabolism , Scorpion Venoms/metabolism , Cell Line, Tumor , Protein BindingABSTRACT
Regular monitoring of arsenic concentrations in water sources is essential due to the severe health effects. Our goal was to develop a rapidly responding, sensitive and stable sensing layer for the detection of arsenic. We have designed flagellin-based arsenic binding proteins capable of forming stable filament structures with high surface binding site densities. The D3 domain of Salmonella typhimurium flagellin was replaced with an arsenic-binding peptide motif of different bacterial ArsR transcriptional repressor factors. We have shown that the fusion proteins developed retain their polymerization ability and have thermal stability similar to that of wild-type filament. The strong arsenic binding capacity of the monomeric proteins was confirmed by isothermal titration calorimetry (ITC), and dissociation constants (Kd) of a few hundred nM were obtained for all three variants. As-binding fibers were immobilized on the surface of a gold electrode and used as a working electrode in cyclic voltammetry (CV) experiments to detect inorganic arsenic near the maximum allowable concentration (MAC) level. Based on these results, it can be concluded that the stable arsenic-binding flagellin variant can be used as a rapidly responding, sensitive, but simple sensing layer in a field device for the MAC-level detection of arsenic in natural waters.
