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Search for New KCa3.1‐Targeting Small Molecules and Monoclonal Antibodies
Author(s) -
Shim Hesung,
Nguyen Hai,
Cui Yanjun,
Colussi Paul,
Bednenko Janna,
Wulff Heike
Publication year - 2020
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2020.34.s1.03756
Subject(s) - monoclonal antibody , chemistry , jurkat cells , small molecule , microbiology and biotechnology , ion channel , phage display , recombinant dna , antibody , biology , biochemistry , t cell , receptor , immunology , immune system , gene
The intermediate‐conductance Ca 2+ ‐activated K + channel KCa3.1 (also known as SK4 or the Gárdos channel) is encoded by the KCNN4 gene and plays an important role in the activation of T‐ and B‐cells, mast cells, macrophages and microglia by regulating membrane potential, cellular volume and calcium signaling. KCa3.1 is further involved in the proliferation of dedifferentiated vascular smooth muscle cells and fibroblast. Accordingly, KCa3.1 inhibitors are therapeutically relevant as immunosuppressants and for the treatment of a wide range of fibroproliferative disorders. Using the recently solved cryo‐EM structures of the full length KCa3.1 channel in the closed and two open states we virtually screened a small library of clinically used drugs with two software packages (Glide and AutoDock Vina). The top 50 scoring compounds for the triarylmethane binding site in the inner pore varied significantly between the two programs and the three states. We are currently in the process of functionally confirming the more interesting hits using a thallium flux assay and whole‐cell patch‐clamp. In parallel we are also searching for KCa3.1 blocking monoclonal antibodies, since antibodies constitute attractive alternatives to small molecules because of their exquisite specificity and long half‐lives. Human KCa3.1 was recombinantly expressed in Tetrahymena thermophila , a unicellular eukaryote that has shown a remarkable ability to produce correctly folded human ion channels at high yield. The recombinant KCa3.1 was purified and adhered to magnetic beads that were subsequently used to pan two human phage display libraries. Library screening identified 34 unique anti‐KCa3.1 antibodies, out of which two were found to functionally block KCa3.1 current in whole‐cell patch‐clamp experiments. The selected clone, TTG‐252, inhibits human KCa3.1 current with an IC 50 of 1.8 nM while showing no effect on human KCa1.1, KCa2.2, Kv11.1 (hERG) or Nav1.5. The antibody does not cross‐react to mouse or rat KCa3.1. Support or Funding Information Funded by R21NS101876.