Synthesis of Small‐Molecule Fluorescent Probes for the In Vitro Imaging of Calcium‐Activated Potassium Channel K_Ca3.1

Abstract

Small-molecule probes for the in vitro imaging of K Ca 3.1 channel-expressing cells were developed. Senicapoc, showing high affinity and selectivity for the K Ca 3.1 channels, was chosen as the targeting component. BODIPY dyes 15-20 were synthesized and connected by aC u I -catalyzed azidealkyne [3+ +2]cycloaddition with propargyl ether senicapoc derivative 8,yielding fluorescently labeled ligands 21-26.The dimethylpyrrole-based imaging probes 25 and 26 allow staining of K Ca 3.1 channels in NSCLC cells.T he specificity was shown by removing the punctate staining pattern by preincubation with senicapoc.T he density of K Ca 3.1 channels detected with 25 and by immunostaining was identical. The punctate structure of the labeled channels could also be observed in living cells.M olecular modeling showed binding of the senicapoc-targeting component towards the binding site within the ion channel and orientation of the linker with the dye along the inner surface of the ion channel.Supportinginformation and the ORCID identification number(s) for the author(s) of this article can be found under: https://doi.

Co‐staining of KCa3.1 Channels in NSCLC Cells with a Small‐Molecule Fluorescent Probe and Antibody‐Based Indirect Immunofluorescence

“…The typical punctate staining pattern of the K Ca 3.1 channel was observed after incubation of the A549-3R cells with a 10 μM staining solution of 1 for 10 min. [7,12,14] (Figure 2A,B) The same staining pattern was obtained after incubation of the cells with the BODIPY-labeled senicapoc derivative 2 which has a longer linker (four OCH 2 CH 2 units) between the targeting component and the BODIPY dye. (Figure 2C,D) The most likely explanation for the strong perinuclear staining is the labeling of intracellularly located channels (e. g. channels in the ER, Golgi and/or mitochondria).…”

“…[15] As described, pre-incubation with senicapoc eliminated the punctate staining pattern of 1 and 2 confirming that the BODIPYlabeled senicapoc derivatives 1 and 2 label selectively K Ca 3.1 channels. [12] (Figure 2…”

“…[a] Antibody-based indirect immunofluorescence. [12] Additionally, patch-clamp experiments were performed to investigate the inhibitory activity of the BODIPY-labeled senicapoc derivatives 1 and 2 towards the K Ca 3.1 channel ( Figure 7).…”

“…[10,11] Recently we published the development and synthesis of novel fluorescently labeled small-molecule probes as tools for fast and efficient visualization of K Ca 3.1 channels. [12] Best results were achieved with compounds 1 and 2 (corresponding to the fluorescently labeled ligands previously reported), [12] which are dimethylpyrrole-based BODIPY-labeled derivatives of the K Ca 3.1 channel inhibitor senicapoc (3). (Figure 1).…”

“…The densities of K Ca 3.1 channel-related dots resulting from staining with 1 and from an antibody-based indirect immunofluorescence assay were identical. [12] However, co-staining was not successful when both staining protocols were performed sequentially. Therefore, a new protocol was developed that allowed co-staining using both protocols.…”