Picomolar fluorescent probes for compound affinity determination to carbonic anhydrase IX expressed in live cancer cells

Abstract

Numerous human cancers, especially hypoxic solid tumors, express carbonic anhydrase IX (CAIX), a transmembrane protein with its catalytic domain located in the extracellular space. CAIX acidifies the tumor microenvironment, promotes metastases and invasiveness, and is therefore considered a promising anticancer target. We have designed a series of high affinity and high selectivity fluorescein-labeled compounds targeting CAIX to visualize and quantify CAIX expression in cancer cells. The competitive binding model enabled the determination of common CA inhibitors’ dissociation constants for CAIX expressed in exponentially growing cancer cells. All tested sulfonamide compounds bound the proliferating cells with similar affinity as to recombinantly purified CAIX. The probes are applicable for the design of selective drug-like compounds for CAIX and the competition strategy could be applied to other drug targets.

High-Affinity NIR-Fluorescent Inhibitors for Tumor Imaging via Carbonic Anhydrase IX

“…Contrary to our previously designed fluorescein-labeled compounds that nicely stained hypoxia-grown cell cultures and could be used to quantify the expression of the CAIX protein, the same cells stained with the NIR fluorescent compounds did not show any detectable signal. This was due to limited capabilities of detection in the infrared wave region.…”

“…The synthesis of NIR-fluorescent compounds was carried out as shown in Scheme . The inhibitor GZ18-23 and compounds leading to it were prepared by previously described procedures . The 3-mer and 11-mer PEG linkers were introduced via amide coupling.…”

“…To measure NIR-fluorescent compound binding to CAIX expressed in cancer cells, the GZ22-4 compound was applied in different concentrations to live HeLa cells grown under hypoxia and normoxia, as described in the Materials and Methods section, and was previously performed for the fluorescein-conjugated CAIX inhibitor GZ19-32 . In contrast to GZ19-32 , GZ22-4 compound showed no difference in the fluorescence signal in hypoxia vs normoxia, most likely due to the lower fluorescence quantum yield of NIR dye as compared to fluorescein.…”

“…Human cervical adenocarcinoma cells (HeLa) were kindly provided by Dr. A. Kanopka (Vilnius University). The CAIX-knockout cell line (HeLaCAIX KO ) was developed using the CRISPR-Cas9 knockout system as described previously . HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with GlutaMAX (Gibco, Thermo Fisher), supplemented with 10% of heat-inactivated fetal bovine serum, 100 units/mL penicillin, and 100 mg/mL streptomycin in a humidified atmosphere at 37 °C and 5% CO 2 .…”

“…The synthesis of compounds GZ18-20, GZ18-21, GZ18-22, and AZ21-1B has been previously described in the literature. 16 4.2. 3-((2-(Cyclooctylamino)-3,5,6-trifluoro-4sulfamoylphenyl)sulfonyl)propanoic Acid (GZ18-23).…”