Imaging of KCa 3.1 Channels in Tumor Cells with PET and Small-Molecule Fluorescent Probes.

The Ca2+ activated K+ channel KCa 3.1 is overexpressed in several human tumor cell lines, e. g. clear cell renal carcinoma, prostate cancer, non-small cell lung cancer. Highly aggressive cancer cells use this ion channel for key processes of the metastatic cascade such as migration, extravasation and invasion. Therefore, small molecules, which are able to image this KCa 3.1 channel in vitro and in vivo represent valuable diagnostic and prognostic tool compounds. The [18 F]fluoroethyltriazolyl substituted senicapoc was used as positron emission tomography (PET) tracer and showed promising properties for imaging of KCa 3.1 channels in lung adenocarcinoma cells in mice. The novel senicapoc BODIPY conjugates with two F-atoms (9 a) and with a F-atom and a methoxy moiety (9 b) at the B-atom led to the characteristic punctate staining pattern resulting from labeling of single KCa 3.1 channels in A549-3R cells. This punctate pattern was completely removed by preincubation with an excess of senicapoc confirming the high specificity of KCa 3.1 labeling. Due to the methoxy moiety at the B-atom and the additional oxyethylene unit in the spacer, 9 b exhibits higher polarity, which improves solubility and handling without reduction of fluorescence quantum yield. Docking studies using a cryo-electron microscopy (EM) structure of the KCa 3.1 channel confirmed the interaction of 9 a and 9 b with a binding pocket in the channel pore.

Imaging of the calcium activated potassium channel 3.1 (KCa 3.1) in vivo using a senicapoc-derived positron emission tomography tracer.

The calcium-activated potassium channel 3.1 (KCa 3.1) is overexpressed in many tumor entities and has predictive power concerning disease progression and outcome. Imaging of the KCa 3.1 channel in vivo using a radiotracer for positron emission tomography (PET) could therefore establish a potentially powerful diagnostic tool. Senicapoc shows high affinity and excellent selectivity toward the KCa 3.1 channel. We have successfully pursued the synthesis of the 18 F-labeled derivative [18 F]3 of senicapoc using the prosthetic group approach with 1-azido-2-[18 F]fluoroethane ([18 F]6) in a "click" reaction. The biological activity of the new PET tracer was evaluated in vitro and in vivo. Inhibition of the KCa 3.1 channel by 3 was demonstrated by patch clamp experiments and the binding pose was analyzed by docking studies. In mouse and human serum, [18 F]3 was stable for at least one half-life of [18 F]fluorine. Biodistribution experiments in wild-type mice were promising, showing rapid and predominantly renal excretion. An in vivo study using A549-based tumor-bearing mice was performed. The tumor signal could be delineated and image analysis showed a tumor-to-muscle ratio of 1.47 ± 0.24. The approach using 1-azido-2-[18 F]fluoroethane seems to be a good general strategy to achieve triarylacetamide-based fluorinated PET tracers for imaging of the KCa 3.1 channel in vivo.

Targeting Kca3.1 Channels in Cancer.

The Kca3.1 channels, previously designated as IK1 or SK4 channels and encoded by the KCNN4 gene, are activated by a rise of the intracellular Ca2+ concentration. These K+ channels are widely expressed in many organs and involved in many pathologies. In particular, Kca3.1 channels have been studied intensively in the context of cancer. They are not only a marker and a valid prognostic tool for cancer patients, but have an important share in driving cancer progression. Their function is required for many characteristic features of the aggressive cancer cell behavior such as migration, invasion and metastasis as well as proliferation and therapy resistance. In the context of cancer, another property of Kca3.1 is now emerging. These channels can be a target for novel small molecule-based imaging probes, as it has been validated in case of fluorescently labeled senicapoc-derivatives. The aim of this review is (i) to give an overview on the role of Kca3.1 channels in cancer progression and in shaping the cancer microenvironment, (ii) discuss the potential of using Kca3.1 targeting drugs for cancer imaging, (iii) and highlight the possibility of combining molecular dynamics simulations to image inhibitor binding to Kca3.1 channels in order to provide a deeper understanding of Kca3.1 channel pharmacology. Alltogether, Kca3.1 is an attractive therapeutic target so that senicapoc, originally developed for the treatment of sickle cell anemia, should be repurposed for the treatment of cancer patients.

Co-staining of KCa 3.1 Channels in NSCLC Cells with a Small-Molecule Fluorescent Probe and Antibody-Based Indirect Immunofluorescence.

The Ca2+ activated potassium channel 3.1 (KCa 3.1) is involved in critical steps of the metastatic cascade, such as proliferation, migration, invasion and extravasation. Therefore, a fast and efficient protocol for imaging of KCa 3.1 channels was envisaged. The novel fluorescently labeled small molecule imaging probes 1 and 2 were synthesized by connecting a dimethylpyrrole-based BODIPY dye with a derivative of the KCa 3.1 channel inhibitor senicapoc via linkers of different length. Patch-clamp experiments revealed the inhibition of KCa 3.1 channels by the probes confirming interaction with the channel. Both probes 1 and 2 were able to stain KCa 3.1 channels in non-small-cell lung cancer (NSCLC) cells following a simple, fast and efficient protocol. Pre-incubation with unlabeled senicapoc removed the punctate staining pattern showing the specificity of the new probes 1 and 2. Staining of the channel with the fluorescently labeled senicapoc derivatives 1 or 2 or with antibody-based indirect immunofluorescence yielded identical or very similar densities of stained KCa 3.1 channels. However, co-staining using both methods did not lead to the expected overlapping punctate staining pattern. This observation was explained by docking studies showing that the antibody used for indirect immunofluorescence and the probes 1 and 2 label different channel populations. Whereas the antibody binds at the closed channel conformation, the probes 1 and 2 bind within the open channel.

Synthesis of Small-Molecule Fluorescent Probes for the In†Vitro Imaging of Calcium-Activated Potassium Channel KCa 3.1.

Small-molecule probes for the in vitro imaging of KCa 3.1 channel-expressing cells were developed. Senicapoc, showing high affinity and selectivity for the KCa 3.1 channels, was chosen as the targeting component. BODIPY dyes 15-20 were synthesized and connected by a CuI -catalyzed azide-alkyne [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 KCa 3.1 channels in NSCLC cells. The specificity was shown by removing the punctate staining pattern by pre-incubation with senicapoc. The density of KCa 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. Molecular 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.