From X-ray crystallographic structure to intrinsic thermodynamics of protein-ligand binding using carbonic anhydrase isozymes as a model system.

Carbonic anhydrase (CA) was among the first proteins whose X-ray crystal structure was solved to atomic resolution. CA proteins have essentially the same fold and similar active centers that differ in only several amino acids. Primary sulfonamides are well defined, strong and specific binders of CA. However, minor variations in chemical structure can significantly alter their binding properties. Over 1000 sulfonamides have been designed, synthesized and evaluated to understand the correlations between the structure and thermodynamics of their binding to the human CA isozyme family. Compound binding was determined by several binding assays: fluorescence-based thermal shift assay, stopped-flow enzyme activity inhibition assay, isothermal titration calorimetry and competition assay for enzyme expressed on cancer cell surfaces. All assays have advantages and limitations but are necessary for deeper characterization of these protein-ligand interactions. Here, the concept and importance of intrinsic binding thermodynamics is emphasized and the role of structure-thermodynamics correlations for the novel inhibitors of CA IX is discussed - an isozyme that is overexpressed in solid hypoxic tumors, and thus these inhibitors may serve as anticancer drugs. The abundant structural and thermodynamic data are assembled into the Protein-Ligand Binding Database to understand general protein-ligand recognition principles that could be used in drug discovery.

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

Tumor imaging and delivery of therapeutic agents may be achieved by designing high-affinity and high-selectivity compounds recognizing a tumor cell-expressing biomarker, such as carbonic anhydrase IX (CA IX). The CAIX, overexpressed in many hypoxic solid tumors, helps adjust to the energy requirements of the hypoxic environment, reduces intracellular acidification, and participates in the metastatic invasion of adjacent tissues. Here, we designed a series of sulfonamide compounds bearing CAIX-recognizing, high-affinity, and high-selectivity groups conjugated via a PEG linker to near-infrared (NIR) fluorescent probes used in the clinic for optically guided cancer surgery. We determined compound affinities for CAIX and other 11 catalytically active CA isozymes by the thermal shift assay and showed that the affinity Kd value of CAIX was in the subnanomolar range, hundred to thousand-fold higher than those of other CA isozymes. Similar affinities were also observed for CAIX expressed on the cancer cell surface in live HeLa cell cultures, as determined by the competition assay. The NIR-fluorescent compounds showed excellent properties in visualizing CAIX-positive tumors but not CAIX-negative knockout tumors in a nude mice xenograft model. These compounds would therefore be helpful in optically guided cancer surgery and could potentially be developed for anticancer treatment by radiotherapy.

Aerobic vaginitis is associated with carbonic anhydrase IX in cervical intraepithelial neoplasia.

The aim of this study was to analyze the association between vaginal microbiota, carbonic anhydrase IX (CAIX) and histological findings of cervical intraepithelial neoplasia (CIN). The study included 132 females, among them 66 were diagnosed with high-grade intraepithelial lesion (CIN2, CIN3, and cancer), 14 with low-grade disease, and 52 assigned to the control group. An interview focused on the behavior risk factors, together with vaginal fluid pH measurement, wet mount microscopy, detection of Chlamydia trachomatis, and Trichomonas vaginalis were performed. After colposcopy, high-grade abnormalities were detected via direct biopsies and treated with conization procedure. Conuses were immuno-stained with CAIX antibody. The histological findings were CIN1 (n = 14), and CIN2+ (included CIN2 (n = 10), CIN3 (n = 49), and cancer (n = 7; squamous cell carcinomas)). Prevalence of bacterial vaginosis (BV) was similar between the groups. Moderate or severe aerobic vaginitis (msAV) was diagnosed more often among CIN2+ (53.0%) than CIN1 (21.4%). Moderate or strong immunostaining of CAIX (msCAIX) was not detected among CIN1 cases. Thus, msAV was prevalent in CAIX non-stained group (p = 0.049) among CIN2 patients. Co-location of msAV and msCAIX was found in CIN3. Regression model revealed that msAV associated with high-grade cervical intraepithelial neoplasia independently from smoking and the number of partners.