Visualization of K_Ca3.1 Channels in Tumor Cells by Optimized Senicapoc-Bodipy Conjugates

Upregulation of K_Ca3.1 channels was observed in highly aggressive tumor cells, such as non small cell lung cancer cells of the A549 line. In order to visualize K_Ca3.1 channels in these cells, novel fluorescent probes with increased polarity were designed. Key step of the synthesis was a 1,3-dipolar cycloaddition of senicapoc propargyl ether <b>4</b> with various azide substituted bodipy dyes. Due to their reduced lipophilicity and promising photophysical properties, the senicapoc-bodipy conjugates <b>7a</b> (log<i>P</i> = 4.3) and <b>16</b> (log<i>P</i> = 4.4) were able to stain K_Ca3.1 ion channels in fixed, living, and permeabilized A549-3R tumor cells. The apparent size of the observed fluorescent dots indicates labeling of single K_Ca3.1 channels. The recorded density is in good accordance with literature values. The specificity of K_Ca3.1 labeling by the senicapoc-bodipy conjugates <b>7a</b> and <b>16</b> was shown with HEK293 cells, blocking experiments and azide precursors. Subsequent staining of K_Ca3.1 ion channels with hydroxyphenyl derivative <b>16</b> and antibodies did not lead to overlapping (yellow) dots, as different states of the ion channel were stained by <b>16</b> (open state) and antibody (closed state). In patch clamp experiments, both senicapoc-bodipy conjugates <b>7a</b> and <b>16</b> reduced the current density, although less efficiently than senicapoc. MD simulations showed weaker interactions of the amide moiety of <b>16</b> with Thr250, explaining the lower channel inhibition of the open-pore blocker <b>16</b> compared to senicapoc (<b>1</b>). Due to their optimal imaging properties, high specificity, balanced lipophilicity/hydrophilicity, and sufficient water solubility, senicapoc-bodipy conjugates <b>7a</b> and <b>16</b> represent innovative diagnostic tools to image K_Ca3.1 channels.