The carrier of carbonic anhydrase (CA) activity was detected in gel among low molecular mass proteins from pea, spinach and Arabidopsis, after nondenaturing electrophoresis in PAAG of the dodecyl-β-d-maltoside treated PSII membranes (the fragments of thylakoid membrane containing PSII complexes). The elimination of Mn-stabilizing protein PsbO by treatment of PSII membranes with salts, did not lead to a decrease in CA activity observed in the gel although it reduced the amount of this protein down to 25% compared to the original sample. The isolated protein PsbO did not demonstrated CA activity. The distinguished features of CA activity of PSII membranes were as follows: 1) resistance to heating, 2) high sensitivity to ethoxyzolamide, the specific inhibitor of CA, and 3) stimulation of this activity by acetazolamide, another specific inhibitor of CA at low concentration of the latter. CA activity was not stimulated by acetazolamide in the PSII membranes samples from Arabidopsis thaliana mutants with knocked out gene At4g20990 encoding αCA4 (according to the nomenclature by Fabre et al., 2007). Taking into account the above data and our previous findings that the energy-dependent part of nonphotochemical quenching of chlorophyll a fluorescence is highly suppressed in that mutant, we suppose that thylakoid membranes of higher plants contain in the vicinity of PSII complex a true CA belonging to the α family of CAs.

In rational drug design, it is important to determine accurately and with high precision the binding constant (the affinity or the change in Gibbs energy, ∆G), the change in enthalpy (ΔH), and the entropy change upon small molecule drug binding to a disease-related target protein. These thermodynamic parameters of the protein-ligand association reaction are usually determined by isothermal titration calorimetry (ITC). Here, the repeatability, precision, and accuracy of the measurement of the affinity and the change in enthalpy upon acetazolamide (AZM) interaction with human carbonic anhydrase II (CA II) are discussed based on the measurements using several ITC instruments. The AZM-CA II reaction was performed at decreasing protein-ligand concentrations until the determination of ∆G and ΔH was not possible, indicating a lower limit for accuracy. To obtain the confidence intervals (CI) of the ∆G and ΔH of AZM binding to CA II, the binding reaction was repeated numerous times at the optimal concentration of 10 µM and 25 °C temperature. The CI (at a confidence level α = 0.95) for ΔH = - 51.2 ± 1.0 kJ/mol and ∆G = - 45.4 ± 0.5 kJ/mol was determined by averaging the results of multiple repeats.

Traditional chemotherapeutics used in cancer therapy do not preferentially accumulate in tumor tissues. The conjugation to delivery vehicles like antibodies or small molecules has been proposed as a strategy to increase the tumor uptake and improve the therapeutic window of these drugs. Here, we report the synthesis and the biological evaluation of a novel small molecule-drug conjugate (SMDC) comprising a high-affinity bidentate acetazolamide derivative, targeting carbonic anhydrase IX (CAIX), and cryptophycin, a potent microtubule destabilizer. The biological activity of the novel SMDC was evaluated in vitro, measuring binding to the CAIX antigen by surface plasmon resonance and cytotoxicity against SKRC-52 cells. In vivo studies showed a delayed growth of tumors in nude mice bearing SKRC-52 renal cell carcinomas.

Carbonic anhydrase IX (CA IX), over-expressed on cancer cells, catalyzes CO2 to bicarbonate and protons, contributing to the acidic extracellular pH (pHe), which enhances the multidrug resistance of tumor cells. Therefore, alleviating tumor acidosis would greatly improve the outcome of chemotherapy. This work fabricates acetazolamide (ACE)-loaded pH-responsive nanoparticles (ACE-NPs), which are quickly disintegrated in an acidic solution (pH 6.8), resulting in a quick release of ACE from these NPs to inhibit the expression of CA IX, thus up-regulating the pHe value. These ACE-NPs have no obvious in vitro cytotoxicity and in vivo studies confirm the accumulation of ACE-NPs in tumor tissue. In addition, mice treated with ACE and paclitaxel (PTX) co-loaded NPs show a smaller tumor size and a higher survival rate when compared to that of mice treated with ACE- or PTX-loaded NPs. This work reveals that simultaneous delivery of ACE and chemotherapy agents to tumor tissue can up-regulate the acidic pHe value, consequently enhancing the anti-tumor ability of chemotherapy medicine. These findings open a new window for enhancing the anti-tumor ability of traditional chemotherapy in clinic.

A novel series of silyl-yne containing chalcone derivatives 5a-5j was synthesized by exploiting Sonogashira coupling reaction and Claisen-Schimdt condensation reaction. The synthesized derivative were characterized by spectroscopic and elemental analysis. The selective inhibition of carbonic anhydrases is considered critical in the field of medicinal chemistry because carbonic anhydrases exits in several isoforms. Synthesized compounds were subjected to carbonic anhydrase -II assay. Except 5j, the other derivatives exhibited better potential than standard acetazolamide. Compound 5e was found to be potent derivative in the series with IC50 value 0.054 ± 0.001 µM. Binding analysis revealed that most potent derivative 5e binds in the active site of CA-II and single π-π stacking interaction was observed between ring structure of ligand and Phe129 having bond length 4.90 Å. Pharmacokinetics elicited that compounds obey Lipinski's rule and show significant drug score.

Special high K diets have cardio-protective effects and are often warranted in conjunction with diuretics such as furosemide for treating hypertension. However, it is not understood how a high K diet (HK) influences the actions of diuretics on renal K+ handling. Furosemide acidifies the urine by increasing acid secretion via the Na+-H+ exchanger 3 (NHE3) in TAL and vacuolar H+-ATPase (V-ATPase) in the distal nephron. We previously found that an alkaline urine is required for BK-αβ4-mediated K+ secretion in mice on HK. We therefore hypothesized that furosemide could reduce BK-αβ4-mediated K+ secretion by acidifying the urine. Treating with furosemide (drinking water) for 11 days led to decreased urine pH in both WT and BK-β4 knockout mice (BK-β4 KO) with increased V-ATPase expression and elevated plasma aldosterone levels. However, furosemide decreased renal K+ clearance and elevated plasma [K+] in WT but not BK-β4 KO. Western blotting and immunofluorescence staining showed that furosemide treatment decreased cortical expression of BK-β4 and reduced apical localization of BK-α in connecting tubules (CNT). Addition of the carbonic anhydrase inhibitor, acetazolamide, to furosemide water restored urine pH along with renal K+ clearance and plasma [K+] to control levels. Acetazolamide plus furosemide also restored the cortical expression of BK-β4 and BK-α in connecting tubules (CNT). These results indicate that in mice adapted to HK, furosemide reduces BK-αβ4-mediated K+ secretion by acidifying the urine.

Precise regulation of vas deferens fluid volume which is important for sperm survival might be influenced by testosterone. In order to investigate changes in vas deferens fluid volume and aquoporins (AQP) isoforms expression under testosterone influence, orchidectomized Sprague-Dawley rats were given 125 and 250 µg/kg/day testosterone with or without flutamide, an androgen receptor blocker or finasteride, a 5alpha-reductase inhibitor for seven consecutive days. Following treatment completion, vas deferens was perfused and changes in the fluid secretion rate and osmolality were determined in the presence of acetazolamide. Rats were then sacrificed and vas deferens was harvested for histology, tissue expression and distribution analyses of AQP-1, AQP-2, AQP-5, AQP-7 and AQP-9 proteins by Western blotting and immunohistochemistry, respectively. Our findings indicate that testosterone causes vas deferens fluid secretion rate to increase, which was antagonized by acetazolamide. Fluid osmolality increased following testosterone treatment and further increased when acetazolamide was given. Co-administration of flutamide or finasteride with testosterone causing both fluid secretion rate and osmolality to decrease. Histology revealed increased size of vas deferens lumen with increased thickness of vas deferens stroma. Expression of AQP-1, AQP-2 and AQP-9 were detected in vas deferens but not AQP-5 and AQP-7, and the levels of these proteins were increased by testosterone treatment mainly at the apical membrane of vas deferens epithelium. In conclusion, increased in vas deferens fluid secretion rate under testosterone influence mediated via the up-regulation of AQP-1, 2 and 9 might be important for vas deferens fluid homeostasis in order to ensure normal male fertility.

Thirty novel sulfonamide derivatives incorporating dipeptide were synthesized by facile acylation through benzotriazole mediated reactions and their structures were identified by 1H NMR, 13C NMR, MS and FT-IR spectroscopic techniques and elemental analysis. The carbonic anhydrase (CA, EC 4.2.1.1) inhibitory activity of the new compounds was assessed against four human (h) isoforms, hCA I, hCA II, hCA IV and hCA XII. Most of the synthesized compounds showed excellent in vitro carbonic anhydrase inhibitory properties comparable to those of the clinically used drug acetazolamide (AAZ). The new unprotected dipeptide-sulfonamide conjugates showed very effective inhibitory activity, in the low nanomolar range against II and XII, being less effective as hCA I and IV inhibitors. Four of the thirty compounds also showed strong inhibitory activity against hCA XII compared to AAZ.