Ntε-LCY genes from allotetraploid tobacco (Nicotiana tabacum), Ntε-LCY2 and Ntε-LCY1, and demonstrated the overall effect of Ntε-LCY genes on carotenoid biosynthesis and stress resistance. However, their genetic and functional characteristics require further research in polyploid plants.Lycopene epsilon-cyclase (ε-LCY) is a key enzyme in the carotenoid biosynthetic pathway (CBP) of higher plants. In previous work, we cloned two Ntε-LCY2 and Ntε-LCY1 mutants in allotetraploid N.tabacum K326. Ntε-LCY2 and Ntε-LCY1 had similar promoter cis-acting elements, including light-responsive elements. The Ntε-LCY genes were expressed in roots, stems, leaves, flowers, and young fruit, and their highest expression levels were found in leaves. Ntε-LCY2 and Ntε-LCY1 genes responded differently to normal light and high light stress. Both the Ntε-LCY2 and the Ntε-LCY1 mutants had a more rapid leaf growth rate, especially ntε-lcy2-1. The expression levels of CBP genes were increased in the ntε-lcy mutants, and their total carotenoid content was higher. Under both normal light and high light stress, the ntε-lcy mutants had higher photosynthetic capacities and heat dissipation levels than the wild type, and this was especially true of ntε-lcy2-1. The reactive oxygen species content was lower in leaves of the ntε-lcy mutants.Here, we used CRISPR/Cas9 to obtain Ntε-LCY genes Ntε-LCY1 and Ntε-LCY2 differed in several respects. The mutation of Ntε-LCY2 was associated with a greater increase in the content of chlorophyll and various carotenoid components, and it enhanced the stress resistance of tobacco plants under high light.In summary, the expression patterns and biological functions of the The online version contains supplementary material available at 10.1186/s12870-022-03634-5. Phothibition , 6. Phothibition . Therefohibition . Recent hibition . Therefohibition . Carotenhibition . The carhibition ; it can hibition . The methibition .40 hydrocarbon chain is an important branch point in the carotenoid biosynthetic pathway (CBP). The lycopene cyclase family is composed of lycopene epsilon-cyclase (ε-LCY) and lycopene beta-cyclase (β-LCY). The ε-LCY enzyme catalyzes only the formation of a δ ring at one end of the lycopene molecule to generate δ-carotene, and a β ring can then be formed at the other end of δ-carotene through the catalysis of β-LCY. δ-carotene finally forms α-carotene, and α-carotene undergoes hydroxylation and other modifications to form lutein in the α branch of the CBP. The β-LCY enzyme can also catalyze the formation of two β rings at both ends of lycopene to generate β-carotene, which is then hydroxylated and epoxidized to generate β-cryptoxanthin, zeaxanthin, antheraxanthin, violaxanthin, neoxanthin, and other carotenoids in the β branch of the CBP. Therefore, the relative activities of ε-LCY and β-LCY directly determine the ratio of α-carotene to β-carotene substrates, and relative flux through the two branches of the CBP has an important influence on the carotenoid composition of higher plants [ε-LCY gene has been cloned from a variety of plants and algae, including Arabidopsis, potato, tomato, maize, Chlorella, olive, and others [Cyclization of the end of the carotenoid Cr plants –17. To dd others , 19.ε-LCY gene exists as a single-copy gene in Arabidopsis and maize. It was reported that mutation of ε-LCY in Arabidopsis increased the contents of β-carotene, violaxanthin, zeaxanthin and other substances, but contents of lutein and other products of the α branch decreased [ε-LCY gene not only increased the carotenoid content of Brassica napus seeds [ε-LCY can significantly affect carotenoid content and composition in higher plants [LCY expression determined the ratio of α/β carotene and the content of carotenoids in banana [ε-LCY in banana increased the β-carotene content approximatelty six-fold in the fruit pulp; the content of α-carotene and lutein were significantly reduced, but agronomic traits were not significantly affected [ε-LCY plays an important role in the regulation of carotenoid composition and stress resistance in plants, although its specific mechanism of action may differ among plant species.The ecreased . Downregus seeds  but alsous seeds . The expr plants . Under hn banana . Mutatioaffected . These rN. tabacum is derived from the hybridization, doubling, and duplication of the ancestral species Nicotiana sylvestris and Nicotiana tomentosiformis. In Nicotiana tabacum, Ntε-LCY is present as two highly homologous copies, Ntε-LCY1 and Ntε-LCY2. The overall effect of Ntε-LCY gene silencing in tobacco was to improve the total carotenoid and chlorophyll contents while increasing the photosynthetic efficiency [Ntε-LCY gene increased abscisic acid (ABA) levels and enhanced the ability of tobacco to tolerate salt and drought stresses, however, overexpression of Ntε-LCY gene reduced the ability of tobacco to tolerate salt and drought stresses [Ntβ-LCY overexpression mimicked the phenotype of Ntε-LCY gene silencing [ficiency . Down-restresses . Ntβ-LCYilencing .N. tabacum K326 to investigate the functions of two Ntε-LCY homologs. We characterized the phenotypes and relevant secondary metabolites of ntε-lcy mutants created with CRISPR/Cas9, documented the expression patterns of Ntε-LCY1 and Ntε-LCY2, and analyzed cis-elements in their promoters. Finally, we compared the functional characteristics of Ntε-LCY1 and Ntε-LCY2 under high light stress.The existence of highly homologous genes is a challenging problem for gene functional characterization in polyploid plants. The homologous genes may or may not show strong/weak functional differentiation or temporal and spatial expression differences. Here, we used the model tetraploid plant ntε-lcy2-1, ntε-lcy2-2, ntε-lcy1-1, and ntε-lcy1-2) and wild-type (WT) plants were used as the experimental materials. Plants were grown in a greenhouse with a photosynthetic photon flux density (PAR) of 80–250 μmol m−2 s−1 and a 16-h light/8-h dark photoperiod. The temperature was 25 ± 2 °C, and the relative humidity was 60 ± 2%. Samples used for tissue expression analysis were obtained from 5-month-old wild-type tobacco plants. In other experiments, tobacco plants grown for 45 days were exposed to normal light (PAR = 80–250 μmol m−2 s−1) or high light (PAR = 440–1000 μmol m−2 s−1) for 2 h. Tobacco leaf samples in the same part were frozen in liquid nitrogen immediately before use, and additional samples were stored at − 80℃.Seeds of allotetraploid tobacco K326 were provided by the Zhengzhou Tobacco Research Institute. CRISPR/Cas9 mutants of K326 (Ntε-LCY2 and Ntε-LCY1) and of homologous genes from Nicotiana sylvestris and Nicotiana tomentosiformis were downloaded from the China Tobacco Genome Database v4.0 (http://10.6.0.76/). The CDSs of Ntε-LCY1 and Ntε-LCY2 were used as blastn queries to search for Solanum tuberosum and Solanum lycopersicum homologs in the website (https://phytozome.jgi.doe.gov/). These ε-LCY protein sequences were aligned by ClustalW, the neighbor-joining trees were constructed by using MEGA7 with 1000 replicates of bootstrap [ε-LCY proteins using MEME 4.12.0 software (parameters: -mod anr -nmotifs 12 -minw 6 -maxw 30) (http://meme-suite.org/tools/meme) [cis-acting elements were identified in the 3000-bp promoter regions upstream of Ntε-LCY1 and Ntε-LCY2 using PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) [https://zhanglab.ccmb.med.umich.edu/cgi-bin/itasser_submit.cgi) [The coding sequences (CDSs) of the target genes (ootstrap . Conservls/meme) . The home/html/) . The I-TNtε-LCY2 and Ntε-LCY1 were carried out according to the Mutation Sites Based Specific Primers Polymerase Chain Reaction (MSBSP-PCR) method [Ntε-LCY2 and Ntε-LCY1 genes was downloaded from the China Tobacco Genome Database, and target sites were identified using CRISPR Multi Targeter (http://www.multicrispr.net/index.html) based on the multiple PAM (NGG or CCN) sites. We selected optimal sgRNA sequences close to the 5' end of the Ntε-LCY2 and Ntε-LCY1 CDSs. Appropriate primers were designed ~ 150 bp upstream and downstream of the target site to detect the position and sequence of gene editing in Ntε-LCY2 and Ntε-LCY1 transgenic lines (Table SThe design of target sites and detection primers and the construction of CRISPR/Cas9 vectors for ) method . Exon anNtε-LCY2 and Ntε-LCY1 [Ntε-LCY2 and Ntε-LCY1 recombinant vectors were transformed individually into tobacco callus, and T0 transgenic-positive plants were identified based on the presence of the kanamycin resistance gene sequence using kanamycin gene primers (Table S1 generation plants were obtained. The mutation sites of the gene-editing positive plants were identified. using the MSBSP-PCR method [Ntε-LCY1/2-F + Ntε-LCY1/2-R), and the second round of PCR reaction produced no amplified products (Primers used: Ntε-LCY1/2-Target + Ntε-LCY1/2-R)  from the top of WT and ntε-lcy2-1, ntε-lcy2-2, ntε-lcy1-1, and ntε-lcy1-2 mutant plants were measured with a ruler (12 plants per genotype). The phenotypes of WT, ntε-lcy2-1, and ntε-lcy1-1 plants were photographed.To minimize the influence of off-target gene editing effects, two separate TChlorophyll a and chlorophyll b contents were measured as described in .Fresh, freeze-dried tobacco leaves were ground into powder with a ball mill . An appropriate amount of internal standard was added to 50 mg of the ground sample, and carotenoids were extracted with a mixture  of n-hexane, acetone, and ethanol that contained 0.01% BHT (g/mL). The extract was vortexed for 20 min at room temperature. After repeating the extraction, the supernatant was collected by centrifugation, then evaporated to dryness under nitrogen flow and reconstituted in a 3:1 mixture (v/v) of methanol and MTBE. Finally, the solution was filtered through a 0.22-μm filter and analyzed by high performance liquid chromatography-photo diode array detection-mass spectrometry (HPLC–DAD-MS) method with atmospheric pressure chemical ionization (APCI) mode for qualitative . Analystntε-lcy2-1, ntε-lcy1-1, and WT plants using the Spin Column Plant Total RNA Purification Kit , and cDNA was synthesized from the extracted RNA using TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix , and the RNA/cDNA quality was checked using a ultra-versus spectrophotometer. qRT-PCR was performed using Super Real PreMix Plus  on a Light Cycler 480 II system . The 2−∆∆Ct method was used to calculate relative gene expression [Ntε-LCY genes and related genes in the CBP were designed with Primer-BLAST software [Total RNA was extracted from leaf tissue of pression , using tsoftware  and are Chlorophyll fluorescence parameters were measured using an Imaging-PAM-MAXI chlorophyll fluorescence meter  between 9:00 a.m. and 11:00 a.m. The chlorophyll fluorometer was connected to a computer, and ImagingWin software  was used for data acquisition. The experimental protocol followed that described in .−2 s−1), one exposed to high light (PAR = 440 μmol m−2 s−1) for 2 h. ImagingWin software was used to measure six areas of individual leaves, and five sets of leaves were measured for each genotype. Chlorophyll fluorescence parameters were calculated as follows: maximum photosynthetic efficiency of PSII (Fv/Fm) = (Fm − F0)/Fm, and non-photochemical quenching (NPQ) = (Fm-Fm')/Fm' [There were two different groups of tobacco seedlings, one exposed to normal light (PAR = 80 μmol mFm')/Fm' .2− content using the nitroblue tetrazolium (NBT) staining method. First, we added 5 mg of NBT to 50 mL 25 mM Hepes buffer solution and then added 50 μL 0.1% Triton X-100 to make a 0.1 mg/mL NBT solution. We used a punch to obtain tobacco leaf discs having a diameter of 0.5 cm from the same part of each plant. We immersed all the samples in the NBT dye solution, stained them for 24 h at 28℃ in the dark, removed the dye solution, added 80% ethanol, and placed the samples in a boiling water bath for 3 min. We removed the 80% ethanol, added absolute ethanol, and kept the samples in the boiling water bath for another 3 min. If necessary, these steps were repeated until no green color remained. We added absolute ethanol to the completely decolorized samples and photographed them under a microscope . H2O2 content was measured following the manufacturer’s instructions of a hydrogen peroxide kit  based on absorbance at 415 nm measured with a microplate reader . We confirm that all materials and methods were performed in accordance with the relevant guidelines/regulations/legislation in China.We analyzed the Ontε-lcy2-1, and ntε-lcy1-1 lines using one- or two-way ANOVA and Tukey’s post-hoc test .All experiments included at least three independent technical and biological replicates, and data are expressed as the mean ± standard error of the mean (SEM). All data analyses and processing were performed using Microsoft Excel and GraphPad Prism 5 software . Data for each experiment were compared among WT, Ntε-LCY2 and Ntε-LCY1, we analyzed their phylogenetic relationships, conserved domains, and homology to related genes in Solanaceae species. MEME analysis of their predicted amino acid sequences showed that Ntε-LCY2 and Ntε-LCY1 were highly conserved and had similarities and differences in their conserved motifs. Both Ntε-LCY2 and Ntε-LCY1 contained motifs 9, 5, 1, 8, 11, 10, 12, 2, 4, and 6, but Ntε-LCY2 contained motif 3 and Ntε-LCY1 contained motif 7. The CDS of Ntε-LCY2 is 1497 bp in length and encodes 498 amino acids, whereas that of Ntε-LCY1 was 1431 bp in length and encoded 476 amino acids. The coding sequence of the ε-LCY gene in both ancestral species was 1431 bp in length and encoded 476 amino acids. One ε-LCY gene was identified in tomato  and potato . In both cases, its coding sequence was 1584 bp in length and encoded 528 amino acids. The homology of the Ntε-LCY2 and Ntε-LCY1 CDSs was 87.95%. The phylogenetic relationships, conserved domains, and percent homologies of Ntε-LCY2, Ntε-LCY1, and related Solanaceae genes are presented in Fig. S. Although Ntε-LCY2 and Ntε-LCY1 proteins had very similar predicted structures, ligand binding sites, and enzyme active sites, there were some differences. For example, Ntε-LCY2 had a predicted F280 active site in motif 3 and a Y465 active site in motif 6, but these were absent in Ntε-LCY1. These differences may be related to the functional characteristics of Ntε-LCY2 and Ntε-LCY1 proteins  produced amplification products of ~ 300 bp in the first round of PCR and produced no amplification products in the second round, indicating that Ntε-LCY2 and Ntε-LCY1 homozygous mutants had been generated successfully . L3 leaf length and width were largest in the ntε-lcy2 lines, somewhat lower in the ntε-lcy1 lines, and smallest in the WT plants  method with atmospheric pressure chemical ionization (APCI) mode [ntε-lcy2-1 mutants showed the highest carotenoid levels, followed by the ntε-lcy1-1 mutants and the WT plants, consistent with the leaf phenotype data. Among the main types of carotenoids, α-carotene and β-cryptoxanthin contents were significantly higher in the Ntε-LCY mutants, especially in the leaves of ntε-lcy2-1  mode . The ntεNtε-LCY mutants. Chlorophyll content showed the same trend as leaf growth and carotenoid content: highest in ntε-lcy2-1, followed by ntε-lcy1-1 and the WT. Together, these data suggested that mutation of Ntε-LCY genes indeed enhanced the accumulation of carotenoids and chlorophylls. Ntε-LCY2 mutation promoted carotenoid and chlorophyll accumulation to a greater extent than Ntε-LCY1 mutation, suggesting that the homologs exhibited strong and weak functional differentiation.Chlorophyll a (Chl a) and Chlorophyll b (Chl b) contents were also much higher in the ntε-lcy2-1 and ntε-lcy1-1 mutants. The first CBP product, phytoene, is crucial for the regulation of the entire pathway. However, the contents of lutein and β-carotene, the main carotenoid components in N. tabacum, were also clearly increased in the mutants. We next examined the expression levels of genes that encoded CBP enzymes. qRT-PCR results showed that the expression levels of phytoene synthase (PSY), phytoene desaturase (PDS), ζ-carotene desaturase (ZDS), carotenoid isomerase (CRTISO), β-lycopene cyclase (β-LCY), β-carotene hydroxylase (β-OHase), violaxanthin deepoxidase (VDE), zeaxanthin epoxidase (ZE), and neoxanthin synthase (NXS) genes were upregulated in ntε-lcy plants under normal growing conditions. Their expression patterns showed the same trend: highest in ntε-lcy2-1, followed by ntε-lcy1-1, and lowest in the WT. Notably, the expression levels of β-LCY, ZE, and NXS were significantly higher in ntε-lcy plants than in the WT. However, the expression level of VDE was lower in Ntε-LCY mutants than in the WT under normal conditions  to low (WT). By contrast, the relative expression of CRTISO was significantly upregulated in ntε-lcy2-1 but downregulated in ntε-lcy1-1 and the WT. The relative expression of β-LCY was significantly downregulated in ntε-lcy1-1 and WT plants but only slightly affected by high light stress in ntε-lcy2-1. Finally, the relative expression of ZE was downregulated in the Ntε-LCY mutants and the WT under high light stress. Together, these results demonstrate that Ntε-LCY2 mutation produces stronger effects than Ntε-LCY1 mutation on carotenoid biosynthesis and the photosynthetic system.Carotenoids can protect the photosystems from photodamage and photoinhibition under high light stress. To gain insights into the biological effects of Ntε-LCY mutation could strongly induce carotenoid-related gene expression and increase carotenoid and chlorophyll levels, we further analyzed the expression of the two Ntε-LCY genes under normal light and high light stress using qRT-PCR. Under normal light conditions, the relative expression of Ntε-LCY2 was significantly higher in WT than in ntε-lcy1-1. In fact, the relative expression of Ntε-LCY2 in ntε-lcy1-1 was almost zero. After 2 h of high light exposure, the expression of Ntε-LCY2 was upregulated in both WT and ntε-lcy1-1, but its expression was still significantly higher in WT. This result indicated that the expression of Ntε-LCY2 is inhibited in the ntε-lcy1-1 mutant and that high light induces Ntε-LCY2 expression  was significantly higher in Ntε-LCY mutant plants than in the WT. However, there was no clear difference in Fv/Fm between ntε-lcy2-1 and ntε-lcy1-1. After a 2 h exposure to high light stress, Fv/Fm was lower in all genotypes, but it was higher in the Ntε-LCY mutant plants than in the WT plants, especially in the ntε-lcy2-1 mutant  in 45-day-old WT, ntε-lcy mutants, we next examined their O2− and hydrogen peroxide (H2O2) contents after a 2 h exposure to high light stress. Both ntε-lcy2-1 and ntε-lcy1-1 showed lighter nitroblue tetrazolium staining than the WT under normal light conditions, and the degree of staining did not differ significantly between ntε-lcy2-1 and ntε-lcy1-1. After a 2 h exposure to high light, the depth of blue staining was highest in the WT, followed by ntε-lcy1-1 and ntε-lcy2-1 , N. tabacum (Ntε-LCY2), Nicotianasylvestris (Nsyε-LCY), Nicotiana tomentosiformis (Ntomε-LCY), Solanum tuberosum (Stuε-LCY),and Solanum lycopersicum (Slyε-LCY) sequences are shown. b Spatial structures, ligand binding sites, andenzyme active sites of Ntε-LCY2 (497 amino acids) and Ntε-LCY1 (475 amino acids) from N.tobacumpredicted with the I-TASSER program. Figure S2. Screening gelelectrophoresis of homozygous mutants of Ntε-LCY2and Ntε-LCY1. antε-lcy2-1 homozygousmutant line with two rounds of PCR verification. bntε-lcy2-2 homozygousmutant line with two rounds of PCR verification. cntε-lcy1-1 homozygousmutant line with two rounds of PCR verification. dntε-lcy1-2 homozygousmutant line with two rounds of PCR verification. M, DL2000 DNA marker. FigureS3. Sequencing verification results of bacterial liquidcorresponding to two rounds of PCR reactions for Ntε-LCY2 and Ntε-LCY1homozygous mutant lines. a Partialpeak diagram of WT and two homozygous mutant linesof Ntε-LCY2  b Partial peak diagram of WT and twohomozygous mutant lines of Ntε-LCY1 . Table S1.cis-acting elementsin Ntε-LCY gene promoters (3000 bpupstream of the start codon) identified using PlantCARE online software.Important motifs and their functions are shown to highlight similarities anddifferences among the Ntε-LCYhomologs. Table S2. The primers and targetsequences used to construct the CRISPR/Cas9 vectors for Ntε-LCY2 and Ntε-LCY1 and identify the resulting mutants. Table S3. The primers used for quantitative real-time PCR(qRT-PCR) analysis.


In the crystal of the title compound, molecules are linked by C—H⋯O hydrogen bonds and a C—H⋯π interaction, forming a supramolecular framework. 12H10O6, an intramolecular O—H⋯O hydrogen bond forms an S(6) ring motif. The molecule is essentially planar with an r.m.s. deviation of 0.051 Å for all non-H atoms. In the crystal molecules are linked by C—H⋯O hydrogen bonds and a C—H⋯π interaction, forming a supramolecular framework.In the title compound, C I, is an intermediate in the synthesis of cytogenin, a naturally occurring isocoumarin that was first isolated from a cultured broth of Streptoverticillium eurocidicum . The details of the syntheses of the precursors A and B and cytogenin have been described elsewhere  ring motif , 3-(bromomethyl)-6-methoxy-1-oxo-1H-isochromen-8-yl acetate (B), and cytogenin, are described in the PhD thesis of Tiouabi  was dissolved with stirring in 5 ml of anhydrous DMF in a 50 ml flask equipped with a magnetic stirrer and under an atmosphere of argon. HCO2Na  was added and the mixture was stirred overnight at room temperature. The evolution of the reaction was monitored by thin-layer chromatography, using dimethylchloride as eluent. On completion of the reaction, the mixture was diluted with ethyl acetate and then washed with an aqueous saturated solution of NaCl. The organic phase was dried using anhydrous Na2SO4, then filtered and concentrated using a rotary evaporator, yielding compound I in the form of a white solid .Method 2: The acetoxybromoisocoumarin B  was dissolved with stirring in 3 ml of anhydrous DMF in a 50 ml flask equipped with a magnetic stirrer and under an atmosphere of argon. HCO2Na  was added, the temperature was raised to 80°C and the mixture stirred for 4 h. The evolution of the reaction was monitored by thin-layer chromatography, using dimethylchloride as eluent. On completion of the reaction, the mixture was diluted with ethyl acetate and then washed with an aqueous saturated solution of NaCl. The organic phase was dried using anhydrous Na2SO4, then purified by column chromatography . Compound I was obtained in the form of a white solid .IAnalytical data for : Rf  0.44. 1H NMR : 3.90 , 4.99 , 6.42 , 6.53 , 6.55 , 8.17 , 11.0 . 13C NMR : 56.19 C(OCH3), 61.61 C(3a), 100.67 C(9), 101.80 C(5), 103.13 C(7), 107.82 C(4), 138.21 C(10), 150.27 C(3), 160.37 C(3 b), 164.23 C(1), 165.75 C(8), 167.35 C(6). HR–MS [ESI(+)]: ms 273.03634 [M + Na]+. IR : 3129 br, 1728 s, 1690 vs, 1622 m, 1400 vs, 1164 vs, 1064 w.I were obtained by slow evaporation of a solution in chloroform.Colourless block-like crystals of diffrn_reflns_laue_measured_fraction_full value (0.947) below minimum (0.95) is given. This involves 29 random reflections out of the expected 1034 for the IUCr cut-off limit of (sin θ)/λ = 0.6 Å−1; viz. 2.8%.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2414314620013917/bh4057sup1.cifCrystal structure: contains datablock(s) I, Global. DOI: 10.1107/S2414314620013917/bh4057Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2414314620013917/bh4057Isup3.cmlSupporting information file. DOI: 2039333CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


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. CAIX is a transmembrane protein that catalyzes the reversible hydration of CO2 into a bicarbonate anion and an acid proton, acidifies tumor cell microenvironment, and promotes metastasis and invasiveness6. Humans have 15 CA isozymes, where CAI, CAII, CAIII and CAVII are cytosolic, CAIX, CAXII and CAXIV—transmembrane, CAIV—attached to membrane via a lipid residue, CAVI—secreted, CAVA and CAVB—mitochondrial. CAVIII, CAX, and CAXI are catalytically inactive isozymes because the Zn(II) is absent in their active site7.Since the discovery that carbonic anhydrase isozyme IX (CAIX) is highly overexpressed in numerous cancers9. Compounds that would exhibit high affinity and selectivity for CAIX would have a promising therapeutic potential. Furthermore, the CAIX-selective fluorescent11 or PET12 probes could visualize the tumor for optically guided surgery or patient stratification.In healthy humans, CAIX is primarily expressed in the stomach and testes. However, its expression increases dramatically in numerous cancers, including cervical carcinoma, esophageal carcinoma, pancreatic tumor, kidney carcinoma, endometrial adenocarcinoma, ovarian tumor, urinary bladder carcinoma, colon adenocarcinoma, lung tumor, liver carcinoma, breast adenocarcinoma, squamous cell carcinoma of the head and neck, and others18. To possess high affinity, fluorines had to be introduced into the benzene ring directly attached to the sulfonamide head-group that binds the Zn(II) in the active site of CA. These fluorines withdraw electrons from the sulfonamide group and diminish its pKa leading to an increase in affinity. The compound selectivity for CAIX is increased by introducing a bulky hydrophobic group as a meta substituent in the benzene ring relative to the sulfonamide group. However, these compounds were only shown to bind the purified CAIX selectively but have not been demonstrated to recognize CAIX in the cellular environment in the presence of thousands of other competing proteins.We have designed and evaluated a series of compounds that bind CAIX with 10 pM to 50 pM affinities and showed 30 to 100,000-fold selectivities over other CA isozymes19, various fluorescent probe-labeled sulfonamides have been synthesized. Naphthalene sulfonamide was used as a fluorogenic group20, fluorescein was attached to para- or meta- substituted benzene sulfonamide or acetazolamide23. Rhodamine24 and infrared dyes were attached to acetazolamide27 to image carbonic anhydrases, especially to follow CAIX expression in cell cultures and mice tumors. Fluorescent dye displacement assays have been used to determine inhibitor efficacy for bovine CAII28.Since the demonstration by Whitesides and coworkers that various substituents can be introduced in CA sulfonamide inhibitors, reviewed inHere we have designed a series of compounds by conjugating fluorescein to CAIX-selective high-affinity inhibitors. Fluorescein was attached to VD11-4-2 with high affinity for CAIX and also other compounds that possess average or low affinities for CAIX as controls to account for non-specific binding. These fluorescent compounds not only helped to visualize CAIX expression in cancer cells but also became the basis of a technique to determine dissociation constants of common non-fluorescent CAIX inhibitors via competition with fluorescein-labeled compounds. We demonstrated that the tested inhibitors exhibited comparable affinities between recombinantly produced CAIX and the CAIX on the cell surface of live cancer cells. The dynamics of CAIX appearance on cancer cells was visualized and quantified. Super-resolution microscopy demonstrated the co-localization of fluorescently-labeled compounds with the CAIX antibodies.Kd ~ 30 pM) and high selectivity over the remaining 11 catalytically active human CA isozymes14, were labeled with fluorescein as described in the methods part. The chemical structures of all compounds used in this study are shown in Fig. 16.A series of our previously designed compounds, which have been demonstrated to bind human CAIX with high affinity . In addition, we used the stopped-flow assay (SFA) to determine whether the compounds inhibited the catalytic activity of CA isozymes. However, the SFA technique cannot determine the affinities lower than the enzyme concentration used in the assay. Since we used 10–50 nM concentrations of CA isozymes, the SFA could not determine the affinities in the pM range. In addition, we used isothermal titration calorimetry (ITC), the standard assay to determine affinity and enthalpy of protein–ligand binding. However, direct ITC has a limitation that it can determine affinities up to approximately 10 nM Ki = 1.5 µM). Compound affinities for the recombinantly purified catalytic domain of human CAIX and other isoforms are listed in Table According to our TSA results, GZ19-32 possessed high affinity for CAIX (pM range). The CAI-selective GZ19-31 had a medium affinity (nM range) for CAIX . The first column shows the staining of DNA in the nuclei. This was a direct visual determination of the specific compound binding and staining of CAIX expressed on cancer cell membrane under hypoxia.To visualize the binding of fluorescein-labeled compounds to live cells, we performed microscopy of cells incubated with GZ19-32 Fig. . The CAIIn the individual cells, the fluorescein-labeled GZ19-32 was seen to localize to philopodia and threads connecting the cells and also other places on the cell membrane indicating localization of CAIX Fig. . Super-R32, was used , or when the primary antibody was omitted in the staining procedure .The colocalization between GZ19-32 and CAIX was also demonstrated with another CAIX-specific monoclonal antibody M75 . In contrast, no yellow colocalization spots were seen when the antibody 1B10, specific for another membrane-associated isozyme CAXII To further study the binding and selectivity of designed fluorescent compounds to the cellular CAIX, we decided to knock-out CAIX in cancer cells. The CRISPR-Cas9 methodology was applied to generate the CAIX-knockout HeLa cells (CAIX-KO) as described in the methods part. The CAIX knockout in HeLa cells was confirmed by sequencing and immunofluorescence cell staining, Western blot (WB), and flow cytometry using antibodies for CAIX Fig. .Figure 6CAIX-KO was also validated by measuring the inhibition of CA activity in live cells by an inhibitor of CAIX, the GZ18-23. The compound showed strong inhibition of the CA activity by the gas-analysis mass spectrometry in hypoxic HeLa WT cells, but there was no effect in hypoxic HeLa CAIX-KO cells Fig. .Figure 7The compound GZ18-23 was designed to retain the affinity towards CAIX of our previously discovered VD11-4-2. The new inhibitor had the same chemical structure as VD11-4–2 except that the hydroxyl group was replaced with the carboxylic group that improved its aqueous solubility and pharmacokinetic properties Fig. A.Under hypoxia, HeLa cells express numerous CA isozymes. Since the GZ18-23 inhibitor possesses high selectivity only for CAIX, the application of the inhibitor did not inhibit all CA activity. As seen in Fig. After the confirmation of compound binding by microscopy, we attempted to determine the quantitative strength of interaction between the fluorescein-labeled compounds and CAIX expressed on cells. We have performed a series of experiments with various concentrations of the compounds applied to live cells grown in 12-well plates for 72 h and incubated for various time periods and oxygen tensions. These experiments yielded the quantitative affinities of the compounds in addition to the visualization by microscopy.Application of the GZ19-32 compound at various concentrations yielded a concentration-dependent increase in fluorescence Fig. . In the Kd values for compounds GZ19-32 and GZ20-40 that are selective for CAIX were similar and equal to 0.15 nM (see below). The position of the curves did not indicate the Kd but instead showed the concentration of the interacting protein (CAIX) equal to approximately 5 nM in this figure, as obtained by the fit to Morrison equation. This value depends on the time used to grow the HeLa cells under hypoxia.The Application of small concentrations of the fluorescently labeled ligand GZ19-32 caused all of it to bind to CAIX on HeLa’s cell surface. Saturation was reached at a concentration of approximately 10 nM. The cells bound only GZ19-32 and GZ20-40 that were specific for CAIX and differed only in the length of the linker, and both contained the same CAIX-specific head-group. However, the GZ19-31 ligand, which contained the head-group specific for CAI isozyme, did not bind to the cells under the same conditions. Similarly, the compound GZ20-37, where sulfonamide amino group was substituted with the methyl group, did not bind CAIX in this concentration region. The binding was also abolished for the CAIX-KO HeLa cells Fig. .However, when micromolar concentrations were applied, all above listed compounds bound to all cells indicating that a low-affinity or non-specific binding occurred in the micromolar concentration range Fig. . At concThe GZ19-32 compound dosing profiles were also determined for the WT HeLa cells grown under hypoxia for 1 to 4 days Fig. . There wFigure Kd of 0.1 nM, but since the calculated concentration of CAIX was 10 nM, it would be necessary to add 10 nM to achieve saturation and full inhibition of the CAIX enzymatic activity  of EA3-2 equal to 5.5 nM. This value matched the Kd observed for EA3-2 binding to recombinant CAIX  bound with the KdA = 5.5 nM. The 22-point EA3-2 compound dilution was prepared by two 12-step serial dilutions where the first started from 80 µM and the second started from 80 nM in twofold steps. The 11th point of the 80 µM series coincided with the first point of the 80 nM series and there were two control points at 0 nM from each dilution. Such 22-concentration dilutions covered an entire region from micromolar to sub-nanomolar and yielded the most accurate Kd values  bound with the A series of compounds were competitively dosed in a mixture with the constant concentration of GZ19-32 (10 nM) to determine their affinities for the cell-expressed CAIX. The competition curves showed well-defined profiles where the more potent binders out-competed GZ19-32 at lower concentrations than the weak ones Fig. . The comKd values around 0.2 nM, which matched the values observed for compound binding to recombinantly purified CAIX as determined by TSA. The other four inhibitors exhibited much weaker affinity than the two most selective CAIX inhibitors. Acetazolamide, a widely used CA inhibitor, yielded the Kd of 15 nM, which was close to the value of 21 nM, obtained by TSA for its binding to recombinant CAIX. The SLC-0111 compound exhibited the Kd of 50 nM, close to the value of 100 nM, observed for its binding to recombinant CAIX .For the comparison, we performed VD11-4-2 competition experiment in hypoxic chamber without cell exposure to normoxia. The competitive curves were indistinguishable indicating that it was suitable to grow the cells for 72 h under hypoxia, while performing the competition experiment under normoxia for 1.5 h .Kd values for compound binding to recombinant CAIX and CAIX on the cell surface. There was a relatively good agreement between the two methods that determines compound affinity for CAIX. However, the compounds VD11-4-2 and GZ18-23 had a lower affinity to the cell-expressed CAIX than to the purified CAIX. This difference was likely due to a rather long residence time, approximately 5 h for VD11-4-234, and thus incomplete achievement of the equilibrium. However, an overall good match validated this approach to determine compound affinities for cell-expressed CAIX. We suggest that this method could also be used to determine affinities of any compounds for cellular CAIX that have not been previously tested for recombinant purified CAIX.Figure para-substituted tetrafluorobenzenesulfonamide that is a marginal binder of CAIX, but selectively and with high-affinity recognizes CAI. The GZ19-32 is a VD11-4-2-based fluorescein-labeled compound that selectively and with high affinity binds to CAIX. The GZ20-37 is a methyl-substituted sulfonamide-containing compound lacking the amino group and thus unable to strongly bind CAIX, nor other CA isoforms. This compound is a mixture of 1:1 ratio of para and meta-substituted isomers. The GZ20-40 is analogous to GZ19-32, also designed to recognize CAIX, but contains a longer linker between the head-group and fluorescein. Compound GZ19-32 and GZ19-33 1H and 19F NMR spectra are shown in figure The GZ19-31 contains a 13 or 1,2,3,4,5-pentafluoro-6-(methylsulfonyl)benzene35, triethylamine  and 3-mercaptopropanoic acid  were added to methanol (50 mL). The reaction was refluxed for 13 h, then evaporated under reduced pressure. The resultant precipitate was washed with water and purified by recrystallization.1,2,3,4,5-Pentafluorobenzenesulfonamide3-thio)propanoic acid (GZ18-21).13 168–169 °C).Recrystallization was accomplished from water. Yield: 1.41 g, 83%; mp: 167–168 °C phenyl)thio)propanoic acid (GZ19-28).3:AcOH, 45:45:1 v/v/v): Rf = 0.4; 1H NMR : δ 2.59 , 3.24 , 3.52 , 12.51 ; 13C NMR : δ 29.73  = 3.3 Hz, SCH2), 35.06 (CH2COOH), 45.96 (CH3SO2), 119.45  = 15.1 Hz), 121.26  = 20.2 Hz), 143.97  = 258.1 Hz, 2 J(19F–13C) = 17.1 Hz, 3 J(19F–13C) = 4.5 Hz), 146.93  = 245.7 Hz), 172.80 (COOH); 19F NMR : δ -132.51 ÷ − 132.67 , -138.65 ÷ − 138.81 . HRMS (m/z): [M]− calcd. for C10H7F4O4S2, 330.9727; found, 330.9730.Recrystallization was accomplished from methanol. Yield: 1.32 g, 84%; mp: 173–174 °C; TLC sulfonyl)propanoic acid (GZ18-22)36.2O2 was added by portions of 0.1 mL every 15 min . The progress of reaction was monitored by TLC. The solvent was then evaporated under reduced pressure and crude product was purified by recrystallization from water. Yield: 0.97 g, 69%; mp: 217–218 °C; TLC (EtOAc): Rf = 0.8; 1H NMR : δ 2.76 , 3.81 , 8.67 , 12.67 ; 13C NMR : δ 27.65 (CH2COOH), 53.19 (SO2CH2), 120.87  = 14.5 Hz), 128.26  = 15.1 Hz), 143.36  = 251.2 Hz), 145.10  = 255.9 Hz, 2 J(19F—13C) = 16.6 Hz), 171.55 (COOH); 19F NMR : δ -135.89 , -136.75 .Appropriate compound (GZ18-21)  was added to acetic acid (40 mL) and the mixture was heated for 13 h at 65–70 °C. H3-phenyl)sulfonyl)propanoic acid (GZ19-30).2O2 was added by portions of 0.1 mL every 15 min . The progress of reaction was monitored by TLC. The solvent was then evaporated under reduced pressure and crude product was purified by recrystallization from methanol and water (4:1 v/v). Yield: 0.96 g, 67%; mp: 230–231 °C; TLC : Rf = 0.51; 1H NMR : δ 2.76 , 3.57 , 3.83 , 12.68 ; 13C NMR : δ 27.66 (CH2COOH), 45.89 (CH3SO2), 53.21 (SO2CH2), 122.47  = 14.8 Hz), 124.76  = 15.0 Hz), 144.90  = 257.0 Hz), 171.47 (COOH); 19F NMR : δ -135.32 , − 135.77 . HRMS (m/z): [M]− calcd. for C10H8F4O6S2, 362.9620; found, 362.9622.Appropriate compound (GZ19-28)  was added to acetic acid (10 mL) and the mixture was heated for 13 h at 90 °C. HAppropriate compound   was dissolved in DMSO (1.7 mL), then cyclooctylamine  was added, and the reaction mixture was stirred for 5 h at room temperature, under argon atmosphere. Reaction progress was monitored using TLC. The reaction mixture was quenched with saturated sodium chloride solution. The resulting precipitate was extracted with ethyl acetate and dried over sodium sulfate. The extract was then evaporated under reduced pressure and purified with column chromatography.3-((2-(cyclooctylamino)-3,5,6-trifluoro-4-sulfamoylphenyl)sulfonyl)propanoic acid (GZ18-23)36.3:AcOH, 45:45:1 v/v/v). Yield: 0.17 g, 66%; mp: 143–144 °C; TLC : Rf = 0.61; 1H NMR : δ 1.47–1.59 , 1.8–1.89 , 2.72 , 3.75 , 3.79 , 6.64 , 8.36 , 12.03 ; 13C NMR : δ 23.27 (cyclooctane), 25.45 (cyclooctane), 27.19 (cyclooctane), 27.67 (CH2COO), 32.65 (cyclooctane), 52.90 (SO2CH2), 55.87  = 11.3 Hz), 114.75  = 12.8 Hz, 2 J(19F–13C) = 5.5 Hz), 128.08  = 18.2 Hz, 2 J(19F–13C) = 13.9 Hz), 135.34  = 14.6 Hz, 2 J(19F–13C) = 1.6 Hz), 137.25  = 245.7 Hz, 2 J(19F–13C) = 17.5 Hz, 3 J(19F–13C) = 5.0 Hz), 144.43  = 253.5 Hz), 146.28  = 250.5 Hz, 2 J(19F–13C) = 16.8 Hz, 3 J(19F–13C) = 3.9 Hz), 171.52 (COOH); 19F NMR : δ -124.87-(-125.02) , -133.76 , − 150.55 .Purified with column chromatography (EtOAc:CHCl3-((2-(cyclooctylamino)-3,5,6-trifluoro-4-(methylsulfonyl)phenyl)sulfonyl)propanoic acid and 3-((3-(cyclooctylamino)-2,5,6-trifluoro-4-(methylsulfonyl)phenyl)sulfonyl)propanoic acid (GZ19-36).-Meta (3-((3-(cyclooctylamino)-2,5,6-trifluoro-4-(methylsulfonyl)phenyl)sulfonyl)propanoic acid (GZ19-36)) and –orto (3-((2-(cyclooctylamino)-3,5,6-trifluoro-4-(methylsulfonyl)phenyl)sulfonyl)propanoic acid) isomers were not separated and obtained in 9:11 ratio (unspecified isomers). Purified with column chromatography . Yield: 0.11 g, 47%; TLC : Rf = 0.36; 1H NMR : δ isomer 1 (45%)–1.47–1.69 , 1.81–1.91 , 2.75 , 3.49 , 3.77 , 3.81 , 6.67 , 12.71 , isomer 2 (55%)–1.47–1.69 , 1.81–1.91 , 2.72 , 3.51 , 3.75 , 3.81 , 6.71 , 12.71 ; 13C NMR : δ 23.22; 23.25; 25.46 (2), 27.21; 27.25; 27.68; 27.70; 32.62; 32.65; 45.87; 45.93; 52.94 (2), 55.91 , 56.06 , 115.99  = 12.6 Hz, 2 J(19F–13C) = 5.9 Hz (2)), 124.81  = 18.1 Hz, 2 J(19F–13C) = 3.2 Hz (2)), 134.98  = 12.4 Hz), 135.61  = 13.7 Hz), 137.99  = 248.3 Hz (2)), 145.41  = 254.2 Hz (2)), 146.34  = 260.3 Hz, (2)), 171.48; 171.52; 19F NMR : δ isomer 1 (45%)–− 123.61–(− 123.73) , − 134.06 , − 150.16 , isomer 2 (55%)–− 124.87-(-125.01) , − 133.17 , -151.08 . HRMS (m/z): [M]+ calcd. for C18H24F3NO6S2, 472.1070; found, 472.1071.The mixture of appropriate starting material  , EDC , 5-aminofluoresceine  and 5 mL of dry pyridine was stirred in room temperature under argon atmosphere for 24 h. Reaction progress was monitored using TLC. The solvent was evaporated under reduced pressure, the crude product was poured into saturated sodium chloride solution (5 mL), acidified till neutral pH and extracted with ethyl acetate. The organic layer was washed with sat. sodium chloride solution and dried over sodium sulfate. The solvent was evaporated under reduced pressure, and residue was purified with column chromatography.N--3-sulfonyl)propanamide (GZ19-31).3, 1:1 v/v). Yield: 6.1 mg, 6%; mp: 175–176 °C; TLC : Rf = 0.26; 1H NMR : δ 2.97 , 3.95 , 6.54 , 6.59 , 6.67 , 7.22 , 7.74 , 8.22 , 8.66 , 10.13 , 10.64 ; 13C NMR : δ 30.07 (CH2CONH), 53.05 (SO2CH2), 83.57 (quaternary C), 102.65 (xanthene), 110.12 (xanthene), 113.04 (xanthene), 113.85 (Isobenzofuranone), 120.74  = 15.2 Hz), 125.05 (Isobenzofuranone), 126.70 (Isobenzofuranone), 127.52 (Isobenzofuranone), 128.34  = 15.3 Hz), 129.60 (xanthene), 140.73 (Isobenzofuranone), 143.45  = 262.3 Hz), 145.01  = 265.5 Hz), 147.44 (Isobenzofuranone), 152.36 (xanthene), 159.93 (C–OH), 168.19 (CON), 169.00 (COO); 19F NMR : δ − 135.64 , − 136.63 . HRMS (m/z): [M]− calcd. for C29H18F4N2O10S2, 693.0262; found, 693.0248.Purified with column chromatography (acetone:CHCl3-((2-(cyclooctylamino)-3,5,6-trifluoro-4-sulfamoylphenyl)sulfonyl)-N-propanamide (GZ19-32).3, 2:3 v/v). Yield: 52 mg, 30%; mp: 189–190 °C; TLC : Rf = 0.54; 1H NMR : δ 1.36–1.70 , 1.76–1.89 , 2.94 , 3.75 , 3.90 , 6.50 , 6.58 , 6.64 , 6.68 , 7.21 , 7.75 , 8.23 , 8.34 , 10.12 , 10.61 ; 13C NMR : δ 23.25 (cyclooctane), 25.42 (cyclooctane), 27.17 (cyclooctane), 29.77 (CH2CONH), 32.73 (cyclooctane), 52.97 (SO2CH2), 55.90  = 10.9 Hz), 83.57 (quaternary C), 102.68 (xanthene), 110.14 (xanthene), 112.99 (xanthene), 113.87 (isobenzofuranone), 114.71  = 12.6 Hz, 2 J(19F–13C) = 6.4 Hz), 124.99 (isobenzofuranone), 126.71 (isobenzofuranone), 127.49 (isobenzofuranone), 127.99  = 17.9 Hz, 2 J(19F–13C) = 3.2 Hz), 129.51 (xanthene), 135.34  = 13.9 Hz), 137.31  = 251.7 Hz), 140.79 (isobenzofuranone), 144.91  = 262.9 Hz), 146.27  = 255.1 Hz, 2J(19F–13C) = 18.6 Hz), 147.35 (isobenzofuranone), 152.38 (xanthene), 159.93 (C–OH), 168.14 (COO), 169.01 (CON); 19F NMR : δ − 125.05 , − 134.00 , − 150.65 .nHRMS (m/z): [M]- calcd. for C37H34F3N3O10S2, 802.1710; found, 802.1711.Purified with column chromatography -3-phenyl)thio)propanamide (GZ19-33).3, 2:3 v/v). Yield: 59 mg, 47%; mp: 181–182 °C; TLC : Rf = 0.34; 1H NMR : δ 2.77 , 3.40 , 3.51 , 6.55 , 6.61 , 6.68 , 7.22 , 7.76 , 8.27 , 10.13 , 10.51 ; 13C NMR : δ 30.07 (SCH2), 37.46 (CH2CON), 45.96 (CH3SO2), 83.54 (quaternary C), 102.66 (xanthene), 110.16 (xanthene), 113.03 (xanthene), 113.76 (isobenzofuranone), 119.54  = 15.0 Hz), 121.18  = 20.1 Hz), 124.98 (isobenzofuranone), 126.67 (isobenzofuranone), 127.47 (isobenzofuranone), 129.59 (xanthene), 140.82 (isobenzofuranone), 143.91  = 257.9 Hz, 2J(19F—13C) = 17.3 Hz, 3 J(19F–13C) = 4.6 Hz), 147.08  = 245.4 Hz, 2 J(19F–13C) = 14.7 Hz), 147.39 (isobenzofuranone), 152.37 (xanthene), 159.92 (C–OH), 169.04 (COO), 169.82 (CON); 19F NMR : δ -132.04 , − 138.74 . HRMS (m/z): [M]− calcd. for C30H19F4NO8S2, 660.0410; found, 660.0408.Purified with column chromatography (acetone:CHCl3-((2-(cyclooctylamino)-3,5,6-trifluoro-4-(methylsulfonyl)phenyl)sulfonyl)-N-propanamide and 3-((3-(cyclooctylamino)-2,5,6-trifluoro-4-(methylsulfonyl)phenyl)sulfonyl)-N-propanamide (GZ20-37).3, 1:1 v/v). Yield: 30 mg, 23%; TLC : Rf = 0.8; 1H NMR : δ 1.39–1.68 , 1.78–1.88 , 2.95 , 2.98 , 3.45 , 3.48 , 3.75 , 3.92 , 3.93 , 6.54 , 6.57 , 6.58 , 6.62 , 6.67 , 6.70 , 7.19 , 7.21 , 7.71 , 7.74 , 8.19  ir 8.23 , 10.13 , 10.64 ; 13C NMR : δ 23.16 (cyclooctane), 23.25 (cyclooctane), 25.38 (cyclooctane), 25.45 (cyclooctane), 27.15 (cyclooctane), 27.27 (cyclooctane), 29.86 , 32.55 (cyclooctane), 32.79 (cyclooctane), 45.84 , 52.72 (SO2CH2), 52.96 (SO2CH2), 55.90  = 11.1 Hz), 56.03  = 11.1 Hz), 83.50 (quaternary C), 83.52 (quaternary C), 102.66 , 110.14 , 112.99 , 113.76 , 115.05 , 124.97 , 126.62 , 127.49 , 128.17 , 129.49 (xanthene), 129.54 (xanthene), 134.97  = 15.1 Hz), 134.97  = 15.4 Hz), 137.65  = 267.0 Hz), 140.69 (isobenzofuranone), 140.77 (isobenzofuranone), 143.45  = 251.6 Hz), 146.17  = 262.3 Hz), 147.38 , 152.37 , 159.93 , 168.11 (COO), 168.15 (COO), 168.92 (CON), 168.96 (CON); 19F NMR : δ -123.63 , -125.32 , -133.28 , -134.15 , -150.08 , -151.23 . HRMS (m/z): [M]+ calcd. for C38H35F3N2O10S2, 801.1763; found, 801.1756.Purified with column chromatography (acetone:CHClTert-butyl 4-(3-((2-(cyclooctylamino)-3,5,6-trifluoro-4 sulfamoylphenyl)sulfonyl)propanamido) butanoate (GZ20-38).tert-butyl 4-aminobutanoate  and 12 mL of dry pyridine was stirred in room temperature under argon atmosphere for 24 h. Reaction progress was monitored using TLC : Rf = 0.4. Solvent was evaporated under reduced pressure, crude product was poured into saturated sodium chloride solution (10 mL), acidified till neutral pH and extracted with ethyl acetate. Organic layer was washed with sat. sodium cloride solution and dried over sodium sulfate. Solvent was evaporated under reduced pressure and residue was purified with column chromatography (same eluent as for TLC). Yield: 0.18 g, 55%; mp: 125–126 °C; 1H NMR : δ 1.39 , 1.43–1.70 , 1.8–1.89 , 2.19 , 2.57 , 2.97 , 3.74 , 3.79 , 6.6 , 8.05 , 8.36 ; 13C NMR : δ 23.27 (cyclooctane), 24.91 (NHCH2CH2), 25.46 (cyclooctane), 27.21 (cyclooctane), 28.21 ((CH3)3C), 28.29 (CH2CONH), 32.61 (CH2COO), 32.68 (cyclooctane), 38.54 (CONHCH2), 53.12 (SO2CH2), 55.87  = 11.4 Hz, CH cyclooctane), 79.99 ((CH3)3C), 114.85  = 12.4 Hz, 2 J(19F–13C) = 5.8 Hz), 128.01  = 18.4 Hz, 2 J(19F–13C) = 14.1 Hz), 135.29  = 13.7 Hz), 137.12  = 240.2 Hz), 144.46  = 252.9 Hz), 146.22  = 250.1 Hz, 2 J(19F–13C) = 16.1 Hz, 3 J(19F–13C) = 5.9 Hz), 168.20 (CON), 172.41 (COO); 19F NMR : δ -125.04 , -133.93 , -150.61 . HRMS (m/z): [M]+ calcd. for C25H39F3N3O7S2, 614.2176; found, 614.2170.The mixture of GZ18-23 , EDC , HOBT , 4-(3-((2-(cyclooctylamino)-3,5,6-trifluoro-4-sulfamoylphenyl)sulfonyl)propanamido)butanoic acid (GZ20-39).3:EtOAc, 1:1 v/v): Rf = 0.1. Solvent was evaporated under reduced pressure, crude product was dissolved in toluene and evaporated again. Yield: 42.4 mg, 95%; oily residue; 1H NMR : δ 1.41–1.70 , 1.79–1.89 , 2.2 , 2.57 , 2.98 , 3.74 , 3.79 , 6.59 , 8.1 , 8.33 , 10.3 (COOH); 19F NMR : δ − 125.01 , − 133.95 , -150.6 . HRMS (m/z): [M]+ calcd. for C21H31F3N3O7S2, 558.1550; found, 558.1559.The mixture of GZ20-38 , TFA  and 3 mL of DCM was stirred at room temperature under argon atmosphere for 20 h. Reaction progress was monitored using TLC (CHCl4-(3-((2-(cyclooctylamino)-3,5,6-trifluoro-4-sulfamoylphenyl)sulfonyl)propanamido)-N-butanamide (GZ20-40).3, 1:1 v/v). Yield: 17.1 mg, 11%; mp: 180–181 °C; TLC : Rf = 0.15; 1H NMR : δ 1.41–1.68 , 1.73 , 1.80–1.89 , 2.38 , 2.60 , 3.07 , 3.76 , 3.76–3.82 , 6.54 , 6.59 , 6.60 , 6.67 , 7.20 , 7.81 , 8.14 , 8.33 , 8.37 , 10.12 , 10.37 ; 13C NMR : δ 23.28 (cyclooctane), 25.42 (NHCH2CH2), 25.46 (cyclooctane), 27.19 (cyclooctane), 28.33 (CH2CONH), 32.57 (CH2COO), 32.71 (cyclooctane), 38.75 (CONHCH2), 53.17 (SO2CH2), 55.89  = 11.2 Hz), 83.48 (quaternary C), 102.67 (xanthene), 110.20 (xanthene), 113.01 (xanthene), 113.79 (isobenzofuranone), 114.86  = 13.1 Hz, 2 J(19F–13C) = 5.3 Hz), 124.85 (isobenzofuranone), 126.71 (isobenzofuranone), 127.39 (isobenzofuranone), 128.08  = 16.0 Hz), 129.52 (xanthene), 135.31  = 13.2 Hz), 137.31 , 141.26 (isobenzofuranone), 144.41  = 253.64 Hz), 146.16  = 251.9 Hz), 147.08 (isobenzofuranone), 152.36 (xanthene), 159.91 (C–OH), 168.31 (COO), 169.11 (CON), 171.96 (CH2CH2CH2CON); 19F NMR : δ -124.96 , -133.98 , -150.55 . HRMS (m/z): [M]+ calcd. for C41H42F3N4O11S2, 887.2238; found, 887.2244.Purified with column chromatography (acetone:CHCl3-(cyclooctylamino)-2,5,6-trifluoro-4-((2-hydroxyethyl)sulfonyl)benzenesulfonamide (VD11-4–2)14.2,3,5,6-tetrafluoro-4-((2-hydroxyethyl)sulfonyl)benzenesulfonamide (VD10-35)13.2,3,5,6-tetrafluoro-4-((2-hydroxyethyl)thio)benzenesulfonamide (VD10-13)13.4-chloro-N-(2-hydroxyethyl)-2-(phenylthio)-5-sulfamoylbenzamide (EA3-2)37.4-(3-(4-fluorophenyl)ureido)benzenesulfonamide (SLC-0111). The SLC-0111 compound has been synthesized as previously described30.N-acetamide (AZM). Acetazolamide has been purchased from Sigma Aldrich, Schnelldorf, Germany.38. All isozymes were expressed in E. coli except CAIX that has been prepared in mammalian cells. The proteins were chromatographically purified on ion-exchange or benzene sulfonamide containing affinity columns. Protein MW was confirmed by mass spectrometry with 1 Da precision as predicted from gene sequence.All twelve catalytically active human CA isozymes were prepared recombinantly as previously described. Isozymes CAIX, CAXII, and CAXIV were the catalytic domains of the proteins39. Protein solutions containing 1,8-anilinonaphthalenesulfonate fluorescent probe were heated from 25 to 100 °C at a rate of one degree per minute while following the fluorescence of the probe. The temperature scanning curves provided the melting temperature Tm of the protein. The Tm has a distinct dependence on the concentration of a compound. Stronger binders shift the Tm to a greater extent than the weak binders. The Tm dependencies on compound concentration were fit to obtain the affinity Kd40.Compound affinities for the twelve catalytically active CA isozymes were determined by the thermal shift assay as previously described2 to acid protons and bicarbonate) by selected compounds has been determined as previously described42. The CA enzymatic activity and its inhibition were followed by the rate of change in pH spectrophotometrically followed by the change in color of indicator phenol red. Addition of increased concentrations of an inhibitor slowed the rate of the change in pH. The assay was performed using the stopped-flow equipment. The rate of spontaneous acidification due to CO2 hydration in water was subtracted from the enzymatically catalyzed rate.Inhibition of the CAIX catalytic activity  and cultivated in conditions of normoxia (21% O2 and 5% CO2 in cell incubator) or hypoxia ). To visualize CAIX, live cells were incubated with the H7 antibody (dilution 1:100 in FluoroBright DMEM (FB) (Thermo Fisher)) in CO2 incubator for 30 min. After washing 3 × 5 min with PBS at room temperature (gently rocking), the cells were incubated w/ secondary Texas-Red-conjugated anti-mouse antibodies (dilution 1:100 in FB) in CO2 incubator for 30 min. After washing 3 × 5 min with PBS at room temperature, the cells were incubated with GZ19-32 or GZ19-37 (2 or 10 nM in FB) for 5 min and washed 3 × 3 min with PBS. The PBS was replaced by FB supplemented with 10 µM nuclear stain Hoerchst 33,342 (Thermo Fisher). After 10 min, the cells were imaged by fluorescence microscopy.HeLa cells (ATCC) were cultured in DMEM medium (Thermo Fisher) supplemented with 10% FBS, 2 mM glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin at 37 °C in humidified atmosphere, containing 5% CO46. Briefly, live cell widefield epifluorescence and TIRF were conducted using an inverted microscope built around a Till iMIC body , equipped with UPLSAPO 20 × oil (NA 0.85) and TIRF APON 60 × oil (NA 1.49) objective lenses . The samples were sequentially excited with 405 nm (150 mW), 488 nm (100 mW) PhoxX laser diodes  or AOTF controlled 561 nM (106 mW) Cobolt Jive DPSS laser  combined in the SOLE-6 light engine (Omicron-Laserage). Excitation light was launched into Yanus scan head, which along with a Polytrope galvanometric mirror  was used to position the laser for widefield epifluorescence or azimuthal 360° TIRF (approx. 100 nm depth) illumination. Excitation and emission light was spectrally separated with the imaging filter cubes containing a 390/40 BrightLine exciter filter , a zt 405 rdc 2 mm beamsplitter , and a ET 460/50 emitter filter (Chroma) for Hoechst 33,342 staining or containing a zt 488/561 rpc 2 mm dual-line beamsplitter (Chroma) and a 524/628 BrightLine dual-band bandpass emission filter  for GZ compounds (excited with 488 nm laser) or Texas-Red-conjugated antibodies (excited with 561 nm laser). Additionally, the brightfield channel was used for the determination of cell borders. The electron-multiplying charge-coupled device Ultra 897 camera  was mounted to a microscope through a TuCam adapter with 2 × magnification (Andor Technology). The camera was cooled down to − 100 °C with the assistance of a liquid recirculating chiller Oasis 160 . All measurements were performed in the 8-well CG imaging chambers, and at least 5 selected areas per well were captured as 16-bit OME-TIFF multicolor Z-stacks , with 100 ms exposure time and EM gain 300 in the case of widefield epifluorescence imaging or two-color time-stacks (1000 frames at 15 Hz) in the case of TIRF/SRRF experiments. For different fluorescent ligands and stains, the laser powers were varied to enable optimal signal levels . The widefield epifluorescence Z-stacks were deconvolved with EpiDEMIC plugin47, the objects were segmented with Spot Detection plugin in an ICY platform48, and the degree of multichannel co-localizations were estimated by Mander’s coefficients using Colocalization Studio49. The SRRF images were reconstructed with GPU of NVIDIA Quadro P4000 video card, using Fiji ImageJ 1.53i with NanoJ-SRRF plugin 1.14 50 . For each fluorescent ligand at each concentration, at least two independent experiments were performed.Imaging was carried out with the microscopy setup as described in2. Cells were exposed to hypoxic conditions in the humidified CO2 incubator with oxygen control , and conditions were 1% O2, 5% CO2 and 94% N2 and 37 ℃. Both cell lines were regulary checked for mycoplasma contamination using SouthernBiotech™ Mycoplasma Detection Kit (# OB1310001) according to manufacturer’s directions.Human cervical adenocarcinoma cells (HeLa) were kindly provided by Dr. A. Kanopka (Vilnius University) and lung carcinoma cells (A549) were kindly provided by Dr. V. Petrikaitė (Vilnius University). Cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) with GlutaMAX™  supplemented with 10% fetal bovine serum (ThermoFisher) in a humidified atmosphere at 37 ℃ and 5% COStaining of fixed cells. HeLa WT and HeLa-CAIX-KO cells were cultured on the glass coverslips in 12-well plates under hypoxia for three days. Cells were fixed by submerging glass coverslips into 20 ℃ methanol for at least 5 min. To visualize CAIX, live cells were rehydrated and stained using H7 antibody31 diluted 1:200 in PBS, supplemented with 0.05% Tween-20 (PBS-Tw) in 37 ℃ incubator for 30 min. After washing 3 × 10 min with PBS-Tw at room temperature (RT), cells were incubated with secondary Alexa Fluor 488-conjugated anti-mouse antibodies , dilution 1:200 in PBS-Tw, in 37 ℃ incubator for 30 min. After washing 3 × 10 min with PBS-Tw at RT, glass coverslips were briefly submerged into distilled water and mounted on a glass slide cells down on a drop of ProLong™ Gold Antifade Mountant with DAPI. Cells were observed using an automated fluorescence microscope EVOS FL Auto (ThermoFisher).Staining of live cells. HeLa cells were cultured in 12-well plates under normoxia or hypoxia for three days. To visualize CAIX, live cells were incubated with purified H7 antibody31 in the CO2 incubator for 30 min. The 1.13 mg/ml antibody stock solution was used at a dilution 1:100 in FluoroBright (FB)(ThermoFisher) medium. After washing 3 × 5 min with PBS at RT (gently rocking), cells were incubated with secondary Alexa Fluor 594-conjugated anti-mouse antibodies , dilution 1:100 in FB, in CO2 incubator for 30 min. After washing 3 × 5 min with PBS at RT, cells were incubated with GZ19-32 (10 nM in FB) for 5 min at room temperature and washed 3 × 3 min with PBS. PBS was replaced by FB and a drop of NucBlue Live ready probe (ThermoFisher) was added into the wells. After 10 min cells were observed using an automated fluorescence microscope EVOS FL Auto (Thermo Fisher).GeneArt CRISPR Nuclease vector (Invitrogen) was used for the disruption of CAIX gene in Hela cells. Three pairs of gRNA oligomers: fwd. GATGCAGGAGGATTCCCCCTGTTTT, rev. AGGGGGAATCCTCCTGCATCCGGTG; fwd. ACCCCAGAATAATGCCCACAGTTTT, rev. TGTGGGCATTATTCTGGGGTCGGTG; fwd. TGAAGTGACTCCACAAGGGTGTTTT, rev. ACCCTTGTGGAGTCACTTCACGGTG, were annealed to make 3 double-stranded oligos which were subcloned into linearized GeneArt CRISPR Nuclease vector. The obtained contructs were used for co-transfection of HeLa cells using TurboFect transfection reagent (ThermoFisher) according to the manufacturer’s protocol. Transfection was performed for only 6 h to avoid cytotoxicity. Transfected cells were cultured in fresh medium for 48 h and then transferred to 96 well plates for single cell colony formation. No cell sorting was performed because thransfection efficiency of HeLa cells by TurboFect reaches 70–90%. After 3 weeks, colonies were checked for CAIX gene disruption. Genomic PCR was performed using Phire Tissue Direct PCR Master Mix (ThermoFisher) according to the manufacturer’s protocol. Sequencing of PCR products confirmed the presence of deletions in CAIX gene. A clone chosen for further work contained a large deletion (413–6941 nt.) in one of the alleles and 2 small deletions (163–192 nt. and 412–415 nt.) in the other allele. A 4 nt. deletion (412–415) caused a frame shift, leading to a meaningless gene translation and thus gene inactivation. The absence of CAIX protein in HeLa-KO cells was confirmed by Western blotting, flow cytometry, and immunofluorescence staining using three types of antibodies that recognize human CAIX.Hela WT and HeLa-CAIX-KO cells were cultured in hypoxia for 72 h, harvested by scraping, centrifuged and extracted in RIPA buffer with 1 mM PMSF on ice for 30 min. After high speed centrifugation, the supernatants were mixed with SDS sample buffer, separated by SDS–polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (Carl Roth). Membranes were stained with Ponceau S solution (0.1% w/v in 5% acetic acid) and cut into the strips, corresponding to each loading well. All incubations with antibodies were performed in 5 ml tubes, while washing was done in 10 cm Petri dishes. Nonspecific binding was blocked by 3% bovine serum albumin (BSA) in PBS, containing 0.05% Tween-20 (PBS-Tw) for 30 min at RT. Monoclonal M75 antibody  was used at the dilution 1:200 and β-tubulin Monoclonal Antibody  was used at the dilution 1:2000. Antibodies were diluted in PBS-Tw supplemented with 1% BSA and 0.02% sodium azide and membranes were incubated with slow shaking at RT overnight. After washing in PBS-Tw, goat anti-mouse IgG antibody conjugated with alkaline phoshpatase  were used at the dilution 1:1000 in PBS-Tw with 1% BSA for 1 h at RT. After washing with PBS-Tw and PBS , protein bands were visualized using 1-Step NBT/BCIP Substrate Solution (Thermo Fisher). Color development reaction was stopped by transferring membranes to cold distilled water.2 versus 1% O2 conditions for 48 h. Cells were harvested, washed, resuspended in FACS buffer and stained for 30 min using anti-human CAIX AF488 antibody  and Mouse IgG2A Alexa Fluor 488-conjugated Antibody  as an isotype control (ISO). At the end, 7AAD staining was added for live cell separation. After centrifugation, the cells were suspended in 200 μl of FACS buffer and live cells were analyzed by flow cytometry using BD FACS Canto II system. Data were analyzed using FlowJo. The mean fluorescence intensity (MFI) values are shown as averages of two experiments.Hela WT and CAIX-KO cells were cultured in 6-well plates in DMEM uder 21% O18O depletion of doubly labeled 13C18O2 through several hydration and dehydration steps with a quadrupole mass spectrometer  as previously described51. HeLa cells were cultured in Dulbecco’s Modified Eagle’s Medium–high glucose , supplemented with 10% fetal calf serum and 1% penicillin / streptomycin. The cell line was cultured under hypoxia (1% O2) for three days prior to measurements. Cells were detached from the plate with Accumax Cell Detachment Solution , washed and resuspended in HEPES-buffered saline . Approximately 3 × 106 cells, dissolved in 5 mL of HEPES-buffered saline , were used for every single measurement. To ensure an equal amount of cells within one set of measurements cells were pooled and then aliquoted according to the number of tested inhibitor concentrations. The cell suspensions were pre-incubated with the inhibitor for up to three hours. For every measurement, the non-catalyzed reaction was determined for 12 min in the presence of inhibitor, before cell suspension was added to the measuring cuvette and the catalyzed reaction was determined for 12 min. For every batch of cells, CA activity in the presence of inhibitor was normalized to the activity in the absence of inhibitor. IC50 values were determined by Hill1 fit using OriginPro 8.6 (OriginLab Corporation). CA enzymatic activity in units (U) was calculated as defined by Badger and Price52. From this definition, one unit corresponds to 100% stimulation of the non-catalyzed 18O depletion of doubly labeled 13C18O2.Catalytic activity of CA in hypoxic human cervical adenocarcinoma (HeLa) cells was determined by monitoring the 53. HeLa cells were cultured under hypoxia (1% O2) for three days prior to measurements. Cells were detached from the plate with Accumax Cell Detachment Solution , washed and resuspended in HEPES-buffered saline . For each measurement, 100,000 cells were incubated with 1 nM or 1 µM of GZ18-23 for 3 h prior to adding the XTT  / electron coupling reagent . XTT-specific absorbance was measured at 492 nm using a microplate reader . Background absorption was measured at 690 nm. Cells, which were not incubated in GZ18-23, were used as control (100%).Potential cytotoxic effects on HeLa cells were determined by XTT assay. The XTT was carried out as previously described by Scudiero et al.2) or under normoxia (21% O2), for 70 to 76 h. The medium was removed. Then 200 µL of serially diluted compound (twelve twofold steps) in the FB medium, usually starting at 80 nM , was added and incubated at 37 ℃ under normoxia in the CO2 incubator for 20 min (2 or 5 min in some experiments where specified). Then the compound solution was removed and the cells were washed 3 times for 1.5–2 min with 400 µL of PBS at RT. Then 180 µL of TrypLe express enzyme (ThermoFisher) was added to each well and incubated in CO2 incubator for 5 min to 10 min until the cells deattached from the surface. After addition of 20 µL of Defined Trypsin Inhibitor solution (ThermoFisher), cells were resuspended by pipetting and 150 µL of the suspension from each well were transferred to black Thermo Scientific™ Nunc MicroWell 96-Well Optical-Bottom Plates for fluorescence and absorbance measurements. The fluorescence was recorded at 485 nm excitation and 520 nM emission wavelengths in the plate reader . The absorbance was recorded at 650 nm to measure the optical density of the cell suspension.HeLa cell culture was grown in 12-well plates, either under hypoxia (1% OThe compound solution was prepared by weighing 3–5 mg of a purified compound and dissolving in DMSO at 10 mM concentration. The 1 mM compound stock in DMSO was prepared by taking 10 µL of 10 mM stock and adding to 90 µL DMSO. The 10 µM compound stock was prepared by taking 10 µL of 1 mM compound stock in DMSO and adding 990 µL of the FB medium (this stock contained 1% of DMSO). Then 80 nM compound solution was prepared by taking 10 µL of 10 µM solution and adding 1.24 mL of the FB medium and placed to the first Eppendorf tube. Then 300 µL of FB medium were added to the Eppendorf tubes 2 to 12. A serial dilution was performed by taking 300 µL of solution from tube 1 to 2, then from 2 to 3 and so on while mixing vigorously at each step. No solution was transferred to the last tube thus it contained only FB medium and no compound. Then 200 µL from 12 tubes were transferred to the 12 wells of cell culture-containing 12-well plate, starting from the lowest concentration. Several different dilution series were prepared in various experiments beginning with 40 nM, 80 nM, 160 nM, 80 µM, or 160 µM depending on compound affinity using the scheme described above.The same compound solutions and cells were used in the competition experiments as in the dosing experiments. The 20 nM solution of GZ19-32 was prepared by adding 6 µL of 10 µM solution of GZ19-32 in 1% DMSO and 3 mL of the FB medium. A serial twofold, twelve step dilution is prepared of the unlabeled compound. Same volumes of 20 nM GZ19-32 were added to the twelve serially diluted compound solutions and mixed. The obtained mixture is then applied to the cells grown in 12-well culture plates, starting from the lowest concentration of unlabeled compound. Incubation, washing and fluorescence measurements were performed the same way as in the compound dosing experiments. In the dosing experiments, the concentration of compound was as listed. However, in the competition experiments, the compound concentrations were lower by a factor of two because equal volumes of 20 nM GZ19-32 (in most experiments) and competing compound solutions were prepared and applied to cells as a mixture. The concentration of DMSO was constant in the serially diluted solutions.2 200 workstation to make sure that the experiment performed in normoxia and hypoxia yielded the same binding constants .Usually, the experiment was performed for 1.5 h at RT (20 ℃) under normoxic conditions. However, where specified, the experiment has been performed entirely under hypoxic conditions in Ruskin InvivOf) is equal to the molar concentration of bound GZ19-32 per molar concentration of total available CAIX, the so-called total protein Application of fluorescently labeled ligand GZ19-32 to cancer cells expressing CAIX will stain the cells in dose-dependent manner. The fraction of saturation increases from 0 to 1. The fraction of bound GZ19-32  yields the bound fraction as:This equation was fit to the dosing curves of several fluorescently-labeled ligands to both the cells grown in hypoxia and in normoxia, both to the WT and CRISPR-Cas9 CAIX knockout cells. The cells bound both the GZ19-32 and GZ20-40 ligands that differed only in the length of the linker and both contained the same head-group. However, the GZ19-31 ligand, that contained the CAI-selective head-group, did not bind to the cells. Furthermore, the ligands that had their sulfonamide amino group substituted with the methyl completely abandoned their capability of binding CAIX in this concentration region. The binding was also abolished for the CAIX-KO HeLa cells. However, at larger, micromolar, concentrations, all ligands bound to all cells indicating that a low-affinity binding occurred in this concentration region.54 and also has been applied to ITC55. According to it, the fraction of fluorescently labeled GZ19-32 (ligand B) bound to CAIX in the presence of any competing ligand A can be calculated using the equation:The model of ligand binding to a receptor in the presence of another competing ligand was derived in 1995We made a competition (displacement) experiment, where the concentration of the GZ19-32 fluorescently labeled ligand was kept constant at 10 nM and the concentration of the unlabeled ligand (VD11-4–2 or any other ligand whose affinity for CAIX may not be known) was varied from 0 to 80 nM , we obtained a competitive dosing curve, where the unlabeled ligand out-competed the fluorescently-labeled one at sufficiently high concentrations.1/2 residence time on CAIX34. Therefore, the equilibrium could not be achieved in 20 min when both ligands were present. However, if both ligands are applied as a mixture, an approximate equilibrium is achieved based on diffusion rates of labeled and unlabeled ligands.This result was obtained only if we applied the mixture of both ligands onto the cells. However, if the cells were pre-saturated with GZ19-32 fluorescently labeled ligand for 20 min and then the competing unlabeled VD11-4-2 was applied, there was essentially no decrease in fluorescence occurring, and the competition did not occur. The unlabeled ligand was unable to displace the labeled one. This is explained by the long residence time of both ligands. We have previously shown by SPR that VD11-4-2 had 5.5 h tThe role of CAIX in cancer is still not fully understood, despite numerous studies. As shown here, the CRISPR-Cas9 knockout of CAIX in HeLa cells led to a cell line that despite a slower growth under hypoxic conditions than the WT was still able to grow in the absence of CAIX. This result may indicate that even full inhibition of CAIX in human cancers may not completely stop cancer progression due to alternative growth pathways by potentially over-expressing other membrane CA isozymes such as CAXII or CAXIV. The CAIX-KO cell line should be further studied to understand the role of CAIX better.The inhibition of CAIX may be still beneficial in treating CAIX-expressing cancers because it would reduce the acidification in the tumor microenvironment that has been generated by the high amount of CAIX present under hypoxic conditions. The easiest way to reduce the acidification is to apply chemical compounds–inhibitors of CAIX. There are two difficulties in the design of the inhibitors. First, the inhibitors must possess a sufficiently high affinity for CAIX. Second, the inhibitors must possess a sufficiently low affinity for the remaining 11 isozymes of CA or any other proteins in the human body. In other words, there should be sufficient selectivity and specificity in binding CAIX over other CAs.14. To visualize the location of this compound in cancer cells, attachment of a fluorescent group to a lead compound that would both visualize and quantify the binding.In this work, we analyzed the binding of inhibitor GZ18-23 that has been demonstrated to reach pM affinity for recombinant CAIX and significant selectivity over the remaining 11 catalytically active human CA isozymes. The compound had significantly improved aqueous solubility and pharmacokinetic properties as compared to earlier lead VD11-4-2An important factor in the design of CAIX inhibitors is the concentration of CAIX molecules in the tumor. Our results here show that the concentration of CAIX in the cell culture was between 1 and 20 nM depending on the growth stage. This means that no matter how strongly the drug binds the CAIX there would be a need to have 20 nM concentration of the inhibitor to saturate and fully inhibit the enzyme, whose concentration is 20 nM. At such a high concentration, the inhibitor does not necessarily need to possess picomolar affinity. However, greater affinity and slower off-rate would be highly beneficial in achieving as complete inhibition of CAIX as possible.Kd for CAIX and 1 nM Kd for CAXII. Thus there was 30-fold selectivity for CAIX over CAXII. Such inhibition of CAXII may be beneficial since CAXII has also been implicated in cancer progression58. Thus the compound may serve as a cancer related-CA specific inhibitor. Furthermore, GZ18-23 possessed 100-fold selectivity over CAXIV, 300 × over CAXIII, 1000 × over CAVII, and more than 3000 × over the remaining CA isozymes. Notably, the selectivity over the ubiquitous isozyme CAI reached 150-thousand-fold.The inhibitor GZ18-23 possessed 30 pM For comparison, acetazolamide possesses only 2 × selectivity over CAII, 3 × selectivity over CAVII and CAXIV, and 100 × selectivity over the ubiquitous CAI. Such selectivities are relatively low and lead to undesired inhibition of other CA isozymes in addition to CAIX and thus elevated toxicity. Furthermore, the relatively low affinity requires a high dose of the inhibitor to saturate the CAIX while also nearly saturating numerous other isozymes.23. Our results here indicate non-specific binding of various compounds to unknown targets other than CAIX when applying the fluorescein-labeled inhibitors at concentrations higher than approximately 200 nM to 2 µM. The fluorescent compounds bound to all cells, including the cells that have been grown under normoxic conditions and the cells that had CAIX knocked-out. Thus the compounds when applied at higher concentrations than 200 nM bound to targets other than CAIX. Still, these observations may not apply to previously used fluorescein-labeled compounds due to different chemical structures.Application of fluorescein-labeled compounds has previously been performed while administering rather high, micromolar concentrations of compoundsAt high concentrations, the fluorescein-labeled compounds likely bound not only to the other CA isozymes but also to other proteins because the compounds that had the sulfonamide amino group substituted with the methyl and thus did not possess the capability of recognizing the CA via the sulfonamide-Zn(II) coordination bond still bound to the cells grown both under hypoxic and normoxic conditions and also to the CAIX-KO cells Fig. . Thus, aKd for a series of compounds matched the affinities determined by the thermal shift assay (TSA) and other assays for the recombinantly prepared catalytic domain of CAIX. The competitive binding model has been applied for the first time for CAIX inhibitors in live cells to the best of our knowledge.One of the importances of this study is the determination of common CA inhibitor affinities for cellular CAIX. The technique that involves the competition of the studied compound with a high affinity for CAIX fluorescein-labeled compound GZ19-32 showed that the Kd of the fluorescein-labeled compound. It may seem that the curves shifted to the left indicate stronger binding relative to the unshifted ones. However, our results showed that the midpoints (around 10 nM) did not indicate the Kd. The actual Kd was around 100 pM. The position of the curve instead indicated the concentration of the CAIX enzyme in the system. A shift to the right in the position showed an increase or decrease of the produced CAIX concentration. In addition, the shift to the right was also associated with an increased plateau of the curve on the ordinate axis. The saturation occurred at higher concentrations of the fluorescein-labeled inhibitor as illustrated in Fig. Another important finding of this study is that the dosing curves as shown in Fig. s around  nM did nKd value. However, it is important to remember that the Kd is logarithmically related to the change in Gibbs energy upon binding. The Gibbs energy distributes almost normally while the Kd has a log-normal distribution and thus the error of Kd should not be expressed as ± value, but instead it should be stated that the error of Kd is ‘two-fold’ or some other value59. Thus if the Kd has been determined to be 10 nM, and if the error is ‘two-fold’ then the error should be expressed in the confidence intervals between 5 and 20 nM . In our experience, various techniques yield a precision of around twofold in best cases. Greater precisions are very hard to achieve and they require multiple repetitions of the experiments. Our measurements yielded the precision of 1.6 to threefold. Therefore, if the values differ by a factor of 2 they should be considered within an error margin.The precision of the dissociation constant is often overestimated while the error is usually underestimated. Quite often, scientists claim that the error is around 5 or 10% of the Kd obtained by TSA of compound binding to recombinantly prepared catalytic domain of CAIX with the KdA values . However, numerous inhibitors are described in the literature using the stopped-flow assay (SFA) measuring the inhibition of CA enzymatic activity60. We prefer TSA over SFA because of the wider dynamic range. The SFA cannot determine compound affinities in the pM range because in the SFA the concentration of CAIX is usually around 10 nM. Therefore, all inhibitors with the IC50 below this value will appear to have the IC50 of 5 nM, half of the concentration of the enzyme. Thus it is impossible to determine the inhibitor affinities that are stronger than 5 nM61. Application of the Cheng-Prusoff equation to obtain the Ki from IC50 depending on the KM of the isozyme and the CO2 concentration may reach a value of 1–2 nM, but the pM range is still not accessible29. Therefore, in our opinion, TSA is a preferred assay over SFA to be used in the inhibitor design.Here we have determined the Kd within the error margin41 under the condition that both assays are applied in the correct concentration range of approximately 10 nM to 1 mM. However, all assays have advantages and limitations. Here we have introduced an additional assay where the affinity of compounds for CAIX on the cell surface can be determined via competition with the fluorescein-labeled CAIX-specific ligand.On the other hand, the TSA does not have this limitation and can determine pM and greater affinities in the same assay with weak mM inhibitors. The enzyme concentration here is 10 µM, but this does not limit the determination of the melting temperature. However, we argue that it is important to use as many assays for affinity determination as possible. There are numerous assays available such as TSA, SFA, isothermal titration calorimetry (ITC), surface plasmon resonance (SPR), microscale thermophoresis (MST), and the mass-spectrometry-based assay. We have previously shown that TSA, SFA, and SPR assays yield the same 63. In addition, the SSRF/TIRF microscopy and deconvoluted Z stacks of widefield microscopy showed that the CAIX is expressed on the cell surface in a distinctly separated conglomerates consisting of at least 10 molecules of CAIX. These conglomerates are separated by 0.7 to 1.8 µm. The function of such agglomerate is not clear but the observation was confirmed by near-perfect colocalization of fluorescein-labeled compounds with the H7 antibody. Agglomerates may be related to the focal points because CAIX has been demonstrated to be associated with focal contacts and cell adhesion64. It was necessary to use super-resolution microscopy for the demonstration of the co-localization. Based on the estimate of bound GZ19-32 molecules and if the conglomerates are separated on an average 1 µm distances evenly distributed throughout the cell surface, the numbers indicate that there should be approximately 100 CAIX molecules in each agglomerate. One possible explanation could be that the places of high concentration of CAIX on cell membrane are related to protein clustering in lipid rafts65.The staining of live HeLa cells with the fluorescein-labeled compound provided a picture of CAIX distribution on the cell surface. We confirmed previous observations that the CAIX is preferably expressed on invadopodiaKd values determined by the competitive assay were the observed ones, and they were not intrinsic. When sulfonamide ligands bind to any CA isozymes containing the Zn(II), they undergo a binding-linked deprotonation of the sulfonamide group. The CA isozymes also may undergo binding-linked protonation of the hydroxide bound to the Zn(II) in the unliganded form of the enzyme. It is important to dissect these linked reactions in order to study the relationships between the compound structure and the affinity for the enzyme. We consider the concept of the intrinsic affinities to be an important part of the structure-based drug design.The calculation of the intrinsic binding constants has been previously described66 and this model fully applies to the binding of the fluorescein-labeled compounds described here. These compounds can be widely used for the discovery and design of novel CAIX inhibitors for anticancer therapy.It should be kept in mind that the A series of designed fluorescein-labeled compounds recognized the CAIX expressed on the surface of live cancer cells with the same affinity as the recombinant CAIX. Control compounds that bear methyl-displaced headgroup or a non-CAIX selective group did not bind cellular CAIX in the 10 nM concentration range and could be clearly distinguished from CAIX-selective probes. The CAIX-CRISPR-Cas9 knockout cells and the cells grown under normoxia did not express CAIX and thus did not bind the CAIX-selective probe. However, non-specific binding was observed above 1 µM concentration. The probes could be used for time-dependent quantification of CAIX expression, visualization of CAIX on the cell surface and also for the determination of commonly used CAIX inhibitor affinities for cellular CAIX via the competition model of the fluorescein-labeled compounds.Supplementary Information.