The Glitazone Class of Drugs as Carbonic Anhydrase Inhibitors-A Spin-Off Discovery from Fragment Screening.

The approved drugs that target carbonic anhydrases (CA, EC 4.2.1.1), a family of zinc metalloenzymes, comprise almost exclusively of primary sulfonamides (R-SO2NH2) as the zinc binding chemotype. New clinical applications for CA inhibitors, particularly for hard-to-treat cancers, has driven a growing interest in the development of novel CA inhibitors. We recently discovered that the thiazolidinedione heterocycle, where the ring nitrogen carries no substituent, is a new zinc binding group and an alternate CA inhibitor chemotype. This heterocycle is curiously also a substructure of the glitazone class of drugs used in the treatment options for type 2 diabetes. Herein, we investigate and characterise three glitazone drugs (troglitazone 11, rosiglitazone 12 and pioglitazone 13) for binding to CA using native mass spectrometry, protein X-ray crystallography and hydrogen-deuterium exchange (HDX) mass spectrometry, followed by CA enzyme inhibition studies. The glitazone drugs all displayed appreciable binding to and inhibition of CA isozymes. Given that thiazolidinediones are not credited as a zinc binding group nor known as CA inhibitors, our findings indicate that CA may be an off-target of these compounds when used clinically. Furthermore, thiazolidinediones may represent a new opportunity for the development of novel CA inhibitors as future drugs.

Perfluoroalkyl Substances of Significant Environmental Concern Can Strongly Inhibit Human Carbonic Anhydrase Isozymes.

Perfluoroalkyl substances (PFASs) persist and are ubiquitous in the environment. The origins of PFAS toxicity and how they specifically affect the functions of proteins remain unclear. Herein, we report that PFASs can strongly inhibit the activity of human carbonic anhydrases (hCAs), which are ubiquitous enzymes that catalyze the hydration of CO2, are abundant in the blood and organs of mammals, and involved in pH regulation, ion homeostasis, and biosynthesis. The interactions between PFASs and hCAs were investigated using stopped-flow kinetic enzyme-inhibition measurements, native mass spectrometry (MS), and ligand-docking simulations. Narrow-bore emitters in native MS with inner diameters of ∼300 nm were used to directly and simultaneously measure the dissociation constants of 11 PFASs to an enzyme, which was not possible using conventional emitters. The data from native MS and stopped-flow measurements were in excellent agreement. Of 15 PFASs investigated, eight can inhibit at least one of four hCA isozymes (I, II, IX, and XII) with submicromolar inhibition constants, including perfluorooctanoic acid, perfluorooctanesulfonamide, and perfluorooctanesulfonic acid. Some PFASs, including those with both short and long perfluoromethylene chains, can effectively inhibit at least one hCA isozyme with low nanomolar inhibition constants.

Development of a cheminformatics platform for selectivity analyses of carbonic anhydrase inhibitors.

The selectivity for a specific human Carbonic Anhydrase (hCA) isoform is an important property a hCA inhibitor (CAI) should be endowed with, in order to constitute a valuable therapeutic tool for the treatment of a desired pathology. In this context, we developed a chemoinformatic platform that allows the analysis of the structure and selectivity profile of known CAIs reported in literature, with the aim of identifying structural motifs connected to ligand selectivity, thus providing useful guidelines for the design of novel ligands selective for the desired hCA isoform. The platform is able to perform ultrafast structure and selectivity analyses through ligand fingerprint similarity, with no need of structural information about the target receptor and ligands' binding mode. It is easily accessible to the non-expert user through the implementation of a KNIME Analytic Platform workflow and could be extended to analyze the selectivity profile of known ligands of different target proteins.

UPLC-QTOF-MS analysis of a carbonic anhydrase-inhibiting extract and fractions of Luffa acutangula (L.) Roxb (ridge gourd).

INTRODUCTION: Luffa acutangula (L.) Roxb, commonly known as ridge gourd (cucurbitaceae), is a common vegetable cultivated in India. It is also a well-used medicinal plant in Indian traditional medicine. OBJECTIVES: To analyse the phenolics content of the most potent carbonic anhydrase-inhibiting fraction from an extract of L. acutangula. MATERIALS AND METHODS: An aqueous ethanol extract of dried fruits of L. acutangula was successively fractionated into petroleum ether, dichloromethane and ethyl acetate. The extract and subsequent fractions were assessed for carbonic anhydrase-inhibitory activity and the enzyme inhibition kinetics were determined for the most active fraction. Total phenolic and flavonoid content of the extract and subsequent fractions were determined spectrophotometrically. Ultra-performance liquid chromatography-quadrupole/time-of-flight mass spectrometry (UPLC-QTOF-MS) analysis was used to tentatively identify the major phenolics in the most active fraction. RESULTS: The concentration of total phenolics and total flavonoids in the extract and each fraction thereof correlated with the level of carbonic anhydrase inhibition activity. The ethyl acetate fraction of the aqueous ethanol extract of L. acutangula had the highest carbonic anhydrase inhibition activity. The enzyme kinetics analysis indicated a mixed mode of inhibition. UPLC-QTOF-MS analysis of the ethyl acetate fraction indicated a number of phenolic acids, hydroxycoumarins, flavones, flavanones, and flavonoids. CONCLUSION: The correlation of total phenolic content with carbonic anhydrase inhibition suggested further research that might confirm that phenolic compounds of L. acutangula offer potential therapeutic benefits against carbonic anhydrase-related disorders.

Design, synthesis and evaluation of 18F-labeled cationic carbonic anhydrase IX inhibitors for PET imaging.

Carbonic anhydrase IX (CA-IX) is a marker for tumor hypoxia, and its expression is negatively correlated with patient survival. CA-IX represents a potential target for eliminating hypoxic cancers. We synthesized fluorinated cationic sulfonamide inhibitors 1-3 designed to target CA-IX. The binding affinity for CA-IX ranged from 0.22 to 0.96 µM. We evaluated compound 2 as a diagnostic PET imaging agent. Compound 2 was radiolabeled with 18F in 10 ± 4% decay-corrected radiochemical yield with 85.1 ± 70.3 GBq/µmol specific activity and >98% radiochemical purity. 18F-labeled 2 was stable in mouse plasma at 37 °C after 1 h incubation. PET/CT imaging was conducted at 1 h post-injection in a human colorectal cancer xenograft model. 18F-labeled 2 cleared through hepatobiliary and renal pathways. Tumor uptake was approximately 0.41 ± 0.06% ID/g, with a tumor-to-muscle ratio of 1.99 ± 0.25. Subsequently, tumor xenografts were visualized with moderate contrast. This study demonstrates the use of a cationic motif for conferring isoform selectively for CA-IX imaging agents.

Phenols and Polyphenols as Carbonic Anhydrase Inhibitors.

Phenols are among the largest and most widely distributed groups of secondary metabolites within the plant kingdom. They are implicated in multiple and essential physiological functions. In humans they play an important role as microconstituents of the daily diet, their consumption being considered healthy. The physical and chemical properties of phenolic compounds make these molecules versatile ligands, capable of interacting with a wide range of targets, such as the Carbonic Anhydrases (CAs, EC 4.2.1.1). CAs reversibly catalyze the fundamental reaction of CO2 hydration to bicarbonate and protons in all living organisms, being actively involved in the regulation of a plethora of patho/physiological processes. This review will discuss the most recent advances in the search of naturally occurring phenols and their synthetic derivatives that inhibit the CAs and their mechanisms of action at molecular level. Plant extracts or mixtures are not considered in the present review.

Synthesis and evaluation of (18)F-labeled tertiary benzenesulfonamides for imaging carbonic anhydrase IX expression in tumours with positron emission tomography.

Three tertiary benzenesulfonamide inhibitors 4a-c were radiolabeled with (18)F and evaluated for imaging carbonic anhydrase IX (CA IX) expression with positron emission tomography. All three inhibitors exhibit <10 nM affinity for CA IX with no measurable affinity for CA II. Despite good affinity/selectivity to CA IX and excellent stability in plasma, uptake of [(18)F]4a-c in CA IX-expressing HT-29 tumours was low without significant contrast. [(18)F]4a,b were excreted rapidly, while [(18)F]4c exhibited significant in vivo defluorination leading to high bone uptake. Due to minimal uptake in HT-29 tumours compared to normal organs/tissues, (18)F-labeled benzenesulfonamides [(18)F]4a-c are not suitable as CA IX imaging agents.

Quantitative Characterization of Three Carbonic Anhydrase Inhibitors by LESA Mass Spectrometry.

Liquid extraction surface analysis (LESA) coupled to native mass spectrometry (MS) presents unique analytical opportunities due to its sensitivity, speed, and automation. Here, we examine whether this tool can be used to quantitatively probe protein-ligand interactions through calculation of equilibrium dissociation constants (Kd values). We performed native LESA MS analyses for a well-characterized system comprising bovine carbonic anhydrase II and the ligands chlorothiazide, dansylamide, and sulfanilamide, and compared the results with those obtained from direct infusion mass spectrometry and surface plasmon resonance measurements. Two LESA approaches were considered: In one approach, the protein and ligand were premixed in solution before being deposited and dried onto a solid substrate for LESA sampling, and in the second, the protein alone was dried onto the substrate and the ligand was included in the LESA sampling solvent. Good agreement was found between the Kd values derived from direct infusion MS and LESA MS when the protein and ligand were premixed; however, Kd values determined from LESA MS measurements where the ligand was in the sampling solvent were inconsistent. Our results suggest that LESA MS is a suitable tool for quantitative analysis of protein-ligand interactions when the dried sample comprises both protein and ligand.

Design of a carbonic anhydrase IX active-site mimic to screen inhibitors for possible anticancer properties.

Recently, a convincing body of evidence has accumulated suggesting that the overexpression of carbonic anhydrase isozyme IX (CA IX) in some cancers contributes to the acidification of the extracellular matrix, which in turn promotes the growth and metastasis of the tumor. These observations have made CA IX an attractive drug target for the selective treatment of certain cancers. Currently, there is no available X-ray crystal structure of CA IX, and this lack of availability has hampered the rational design of selective CA IX inhibitors. In light of these observations and on the basis of structural alignment homology, using the crystal structure of carbonic anhydrase II (CA II) and the sequence of CA IX, a double mutant of CA II with Ala65 replaced by Ser and Asn67 replaced by Gln has been constructed to resemble the active site of CA IX. This CA IX mimic has been characterized kinetically using (18)O-exchange and structurally using X-ray crystallography, alone and in complex with five CA sulfonamide-based inhibitors (acetazolamide, benzolamide, chlorzolamide, ethoxzolamide, and methazolamide), and compared to CA II. This structural information has been evaluated by both inhibition studies and in vitro cytotoxicity assays and shows a correlated structure-activity relationship. Kinetic and structural studies of CA II and CA IX mimic reveal chlorzolamide to be a more potent inhibitor of CA IX, inducing an active-site conformational change upon binding. Additionally, chlorzolamide appears to be cytotoxic to prostate cancer cells. This preliminary study demonstrates that the CA IX mimic may provide a useful model to design more isozyme-specific CA IX inhibitors, which may lead to development of new therapeutic treatments of some cancers.

An integrated microfluidic device for large-scale in situ click chemistry screening.

An integrated microfluidic device has been developed to perform 1024 in situ click chemistry reactions in parallel using the bovine carbonic anhydrous II (bCAII) click chemistry system as a proof-of-concept study and a rapid hit identification approach using SPE purification and electrospray-ionization mass spectrometry, multiple reaction monitoring (MRM) analysis, all of which improves the sensitivity and throughput of the downstream analysis.

Validated HPLC-DAD Method for the Simultaneous Determination of Six Selected Drugs Used in the Treatment of Glaucoma.

This work presents a simple, sensitive, and generic HPLC-diode-array detection method for the simultaneous determination of six drugs prescribed for the treatment of open-angle glaucoma and ocular hypertension. The investigated drugs include brimonidine tartarate (BMN), acetazolamide (AZA), brinzomaide (BZA), dorzolamide HCl (DZA), levobunolol HCl (LVB), and timolol maleate (TIM). Efficient chromatographic separation was achieved using a Thermo Hypersil BDS C18 column (4.6 × 250 mm, 5 µm) with a mobile phase consisting of phosphate buffer pH 5 and acetonitrile in a ratio of 78 + 22. The flow rate was 1 mL/min, and quantification was based on measuring peak areas at 298 nm for TIM and 254 nm for the other drugs. Peaks were perfectly resolved, with retention times at 3.06, 3.87, 4.53, 5.78, 7.31, and 10.78 min for BMN, AZA, DZA, TIM, LVB, and BZA respectively. The developed method was validated according to International Conference on Harmonization guidelines with respect to system suitability, linearity, ranges, accuracy, precision, robustness, and LODs and LOQs. The proposed method showed good linearity in the ranges of 2-80, 2.5-100, 2.5-100, 5-200, 3.75-150, and 1.75-70 µg/mL for BMN, AZA, DZA, TIM, LVB, and BZA, respectively. LODs were 0.20-1.01 µg/mL for the analyzed compounds. Applicability of the proposed method to real-life situations was assessed through the analysis of five different pharmaceutical formulations, and satisfactory results were obtained.

Geographical characterization by MAE-HPLC and NIR methodologies and carbonic anhydrase inhibition of Saffron components.

A microwave-assisted extraction method was optimised for the recovery of bioactive compounds from Crocus sativus L. stigmas with the use of water/ethanol mixture. HPLC-DAD was employed to evaluate the extraction parameters, in particular, solvent type and volume, and the duration of the procedure. Microwave-assisted extraction enhanced the recovery of the active principles, limiting extraction time and solvent waste. Moreover, NIR experiments were performed in order to compare spectra in pseudo-absorbance of Saffron samples with different geographical origins through the application of the chemometric techniques. Moreover, the biological evaluation of crocin 1, safranal and its semisynthetic derivatives as selective inhibitors of five isoforms of human carbonic anhydrase was also explored.

Investigation of sulfonamides inhibition of carbonic anhydrase enzyme using multiphotometric and electrochemical techniques.

Two approaches for sulfonamides (SA's) determination, based on carbonic anhydrase enzyme inhibition have been investigated in this work. In the first method, nine different SA's have been screened using simultaneous multiphotometric measurement in multiwell plates. The sulfanilamide (SAD) showed significant inhibition compared to other sulfonamides. The carbonic anhydrase (CA) kinetic interactions reveal noncompetitive binding of SAD. Interferences from other inhibitors with enzyme were studied and the results showed very good selectivity toward SAD. In the second approach, an electrochemical enzyme inhibition biosensor, based on CA entrapped in a carbon paste electrode using carbon black nanoparticles and solid paraffin, was successfully applied to SAD measurements. Results from the quantitative analysis of SAD are discussed in terms of detection limit, linear range and sensitivity using multiphotometric and biosensor-based methods The biosensor developed was successfully applied to the determination of SAD at submicromolar levels and it is recommended for application for in situ analysis.

Inhibition profiling of human carbonic anhydrase II by high-throughput screening of structurally diverse, biologically active compounds.

Human carbonic anhydrase II (CA II), a zinc metalloenzyme, was screened against 960 structurally diverse, biologically active small molecules. The assay monitored CA II esterase activity against the substrate 4-nitrophenyl acetate in a format allowing high-throughput screening. The assay proved to be robust and reproducible with a hit rate of approximately 2%. Potential hits were further characterized by determining their IC(50) and K(d) values and tested for nonspecific, promiscuous inhibition. Three known sulfonamide CA inhibitors were identified: acetazolamide, methazolamide, and celecoxib. Other hits were also found, including diuretics and antibiotics not previously identified as CA inhibitors, for example, furosemide and halazone. These results confirm that many sulfonamide drugs have CA inhibitory properties but also that not all sulfonamides are CA inhibitors. Thus many, but not all, sulfonamide drugs appear to interact with CA II and may target other CA isozymes. The screen also yielded several novel classes of nonsulfonamide inhibitors, including merbromin, thioxolone, and tannic acid. Although these compounds may function by some nonspecific mechanism (merbromin and tannic acid), at least 1 (thioxolone) appears to represent a genuine CA inhibitor. Thus, this study yielded a number of potentially new classes of CA inhibitors and preliminary experiments to characterize their mechanism of action.

Sample preparation and RPHPLC determination of diuretics in human body fluids.

This article describes reverse phase high-performance liquid chromatography (RPHPLC) methods for determination of diuretics in different human body fluids (whole blood, plasma, serum or urine). Sample preparation procedures, including solid-phase extraction, liquid-liquid extraction, dilution, precipitation as well as automated RPHPLC procedures, are discussed in order to present the advantages and disadvantages of each type of sample preparation. Also, values of analytical recovery of each procedure used for sample preparation are summarized. The most important RPHPLC parameters (detection mode, stationary phase, mobile phase, sensitivity, etc.) are also summarized and discussed.

Native State Mass Spectrometry, Surface Plasmon Resonance, and X-ray Crystallography Correlate Strongly as a Fragment Screening Combination.

Fragment-based drug discovery (FBDD) is contingent on the development of analytical methods to identify weak protein-fragment noncovalent interactions. Herein we have combined an underutilized fragment screening method, native state mass spectrometry, together with two proven and popular fragment screening methods, surface plasmon resonance and X-ray crystallography, in a fragment screening campaign against human carbonic anhydrase II (CA II). In an initial fragment screen against a 720-member fragment library (the "CSIRO Fragment Library") seven CA II binding fragments, including a selection of nonclassical CA II binding chemotypes, were identified. A further 70 compounds that comprised the initial hit chemotypes were subsequently sourced from the full CSIRO compound collection and screened. The fragment results were extremely well correlated across the three methods. Our findings demonstrate that there is a tremendous opportunity to apply native state mass spectrometry as a complementary fragment screening method to accelerate drug discovery.

Carbonic anhydrase isoenzymes CA I and CA II in the human eye.

The distribution of carbonic anhydrase was studied in human donor eyes by the cobalt-phosphate histochemical method of Hansson and by immunofluorescence and immunoperoxidase techniques using antisera specific against the human cytoplasmic isoenzymes CA I and CA II. Corneal endothelium displayed specific immunological staining for CA I and CA II. Distinct enzyme activity was observed histochemically in the plasma membranes and cytoplasm of the endothelium. In the ciliary processes immunological evidence for the presence of CA II was found both in pigmented (PE) and in nonpigmented (NPE) epithelium. Activity was observed in the cytoplasm and basolateral membranes of NPE, but only in the basal membranes of PE. In the lens the plasma membranes of both the epithelium and fibers displayed intense activity, whereas cytoplasmic enzyme activity was seen only in the epithelium. There was no activity in the lens capsule. Immunofluorescence studies were difficult because of autofluorescence, but the immunoperoxidase technique indicated the presence of both CA I and CA II in the lens. In the central retina, Müller cells stained for CA II. Histochemically, enzyme activity was seen in the cytoplasm and at the plasma membranes. Activity was also observed in some but not all cones. Electron microscopy revealed this to be located in the cristae and plasma membranes adjacent to the pigment epithelium. Activity was also found in PE. Neurons and rods lacked both immunological staining and activity. Endothelial cells of capillaries in ciliary processes and in the choroid stained for CA I and exhibited histochemical activity, particularly those which faced neighboring epithelial cells containing the enzyme. The isoenzyme CA III, which is resistant to inhibition by sulfonamides, did not appear to be present in these ocular tissues, since the histochemical staining of enzyme activity was completely abolished by 10(-6) M acetazolamide.

Determination of methazolamide concentrations in human biological fluids using high performance liquid chromatography.

Methazolamide is a carbonic anhydrase inhibitor used to treat glaucoma. In vivo, methazolamide readily distributes into red blood cells. Therefore, both blood and plasma concentration data are needed in order to characterize the pharmacokinetics of methazolamide. In the present study, an analytical method using high performance liquid chromatography was validated for determination of methazolamide concentrations in several biological fluids. Through slight modification of a previously reported method for acetazolamide, another carbonic anhydrase inhibitor, methazolamide was readily quantitated in whole blood, plasma and urine. Sample preparation involved liquid-liquid extraction with ethyl acetate followed by a washing step using phosphate buffer (pH 8.0). After back extraction into glycine buffer (pH 10.0), samples were then washed with ether and injected onto the chromatograph. Chromatography was performed using a C-18, 5 microns reverse-phase column with UV detection at a wavelength of 285 nm. Mobile phase consisted of 0.05 M sodium acetate (pH 4.0) and acetonitrile (20%). The assay was validated over two standard concentration ranges from 1 to 100 micrograms ml-1, concentrations reflective of those expected in vivo, Calibration curves were linear for all biological fluids and coefficients of variation for interday and intraday reproducibility studies were less than 8% (range 3.1-7.9%). The method was used to measure methazolamide concentrations in blood, plasma and urine following oral administration to five human subjects.

Quantitation of acetazolamide in rat plasma, brain tissue and cerebrospinal fluid by high-performance liquid chromatography.

A simple and sensitive high-performance liquid chromatographic method for the analysis of acetazolamide (AZ) in rat blood (plasma/serum, whole blood and serum ultrafiltrate), brain tissue and cerebrospinal fluid (CSF) was described. Quantitative extraction of AZ with ethyl acetate from both buffered plasma and brain tissue homogenate (pH 8.0) was achieved. Each extract was evaporated to dryness and the residue was chromatographed on a reversed-phase column. CSF was directly analysed without extraction step. The limits of detection were 0.05 microgram ml-1 for plasma, 0.02 microgram g-1 for brain tissue and 0.004 microgram ml-1 for CSF. Calibration curves were linear over the working ranges of 0.1-100 micrograms ml-1 for plasma, 0.05-50 micrograms g-1 for brain tissue and 0.025-50 micrograms ml-1 for CSF. The reproducibility of AZ assay in the rat biologic media indicated very low relative standard deviations (RSDs). The recoveries of AZ added to plasma and brain tissue were more than 96% with an RSD of less than 5%. The present method was applied to studies of plasma concentration profiles of the drug after administration and its distribution into central nervous system.

Application of TLC-densitometry, first-derivative UV-spectrophotometry and ratio derivative spectrophotometry for the determination of dorzolamide hydrochloride and timolol maleate.

Three methods are described for the simultaneous determination of dorzolamide hydrochloride (DORZ) and timolol maleate (TIM) in ophthalmic solutions. The first method is based on application of thin layer chromatographic separation of both drugs followed by the densitometric measurements of their spot areas. After separation on silica gel GF(254) plates, using methanol-ammonia 25% (100:1.5 v/v) as the mobile phase, the chromatographic zones corresponding to the spots were scanned at 253 and 297 nm, respectively. The calibration function was established in the ranges of 2-18 microg for DORZ and 0.5-4.5 microg for TIM. The second method depends on first derivative ultraviolet spectrophotometry, with zero-crossing measurement method. The first derivative values D(1) at 250.2 and 312.5 nm were selected for the assay of DORZ and TIM, respectively. Calibration graphs follow Beer's law in the range 10-64 and 2.5-16 microg ml(-1), respectively. The third method is based on ratio first derivative spectrophotometry. The signals in the first derivative of the ratio spectra at 244 and 306.2 nm were selected to determine DORZ and TIM in the mixture and calibration graphs are linear in the range of 5-40 and 5.0-17.5 microg ml(-1), respectively. The proposed methods were successfully applied to the determination of these compounds in synthetic mixtures and in pharmaceutical preparations. The proposed methods are simple, rapid and suitable for quality control application.