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

Abstract

The Ca²⁺ activated K⁺ channel K_Ca 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 K_Ca 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 K_Ca 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 K_Ca 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 K_Ca 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 K_Ca 3.1 channel confirmed the interaction of 9 a and 9 b with a binding pocket in the channel pore.

Keywords: BODIPY; KCa3.1 channel; fluorescent probes; non-small cell lung cancer; positron emission tomography; senicapoc.

Figure 1

Prominent inhibitors of the K_Ca3.1 channel: the antifungal drug clotrimazole (1) represents the first member of this class of K_Ca3.1 channel blockers, which was developed into TRAM‐34 (2) and senicapoc (3).

Scheme 1

Senicapoc (3) and senicapoc derived fluorinated PET tracers. Reactions and reaction conditions: (a) [¹⁸F]KF, K222, CH₃CN, 90 °C, 15 min, radiochemical yield (decay corrected) 4±1.5 %. (b) 1. N₃CH₂CH₂OTs+[¹⁸F]KF, K222, CH₃CN, 110 °C, 3 min, distillation under helium flow; 2. prepared N₃CH₂CH₂ ¹⁸F+6, CuSO₄, sodium ascorbate, H₂O, DMF 60 °C, 30 min. radiochemical yield (decay corrected) 13±4.6,%, over two steps.

Figure 2

The fluorescent probe 8 consists of a senicapoc warhead (targeting unit) connected via a linker with a fluorescent label.

Figure 3

Design of novel fluorescent dye labelled senicapoc derivatives 9. In order to increase the polarity, the linker will be extended by one oxyethylene unit and one F‐atom at the B‐atom will be replaced by a methoxy moiety.

Figure 4

Binding of compound 9b (magenta surface) in K_Ca3.1 (PDB 6cno,^[43] light grey: K_Ca3.1 channel as ribbons, blue: calmodulin as surface representation). a) Compound 9b binding in the full homotetrameric K_Ca3.1 channel. b) 9b bound to the channel, with one monomer removed. c) A detailed view on 9b in the channel pore. The figures were created using UCSF Chimera.

Scheme 2

Synthesis of senicapoc BODIPY conjugates. Reagents and reaction conditions: (a) F₃CCO₂H, CH₂Cl₂, rt, 24 h. (b) DDQ, CH₂Cl₂, rt, 30 min. (c) BF₃ ⋅ OEt₂, NEt₃, rt, 16 h, 13 % (over three steps). (d) TMSOTf, CH₂Cl₂, 0 °C, 2.5 min; then CH₃OH, DIPEA, 0 °C, 5 min, 39 %. (e) CuSO₄, Na‐ascorbate, DMF, H₂O, rt, 16 h, 43 % (9a), 25 % (9b). Details for the synthesis of aldehyde 10 are given in the Supporting Information.

Figure 5

Staining of NSCLC tumor cells A549‐3R with senicapoc BODIPY conjugates. 5 A,B; Staining with difluoro derivative 9a; 5 C,D: Staining with methoxy‐fluoro derivative 9b.

Figure 6

Staining of NSCLC tumor cells A549‐3R after preincubation with senicapoc. A549‐3R cells were preincubated with senicapoc (30 μM, 100 μL) and incubated with 9a (Figure 6A) and 9b (Figure 6B). The staining solutions contained senicapoc (100 μL) as well.