Ion Channels, Transporters, and Sensors Interact with the Acidic Tumor Microenvironment to Modify Cancer Progression.

Solid tumors, including breast carcinomas, are heterogeneous but typically characterized by elevated cellular turnover and metabolism, diffusion limitations based on the complex tumor architecture, and abnormal intra- and extracellular ion compositions particularly as regards acid-base equivalents. Carcinogenesis-related alterations in expression and function of ion channels and transporters, cellular energy levels, and organellar H+ sequestration further modify the acid-base composition within tumors and influence cancer cell functions, including cell proliferation, migration, and survival. Cancer cells defend their cytosolic pH and HCO3- concentrations better than normal cells when challenged with the marked deviations in extracellular H+, HCO3-, and lactate concentrations typical of the tumor microenvironment. Ionic gradients determine the driving forces for ion transporters and channels and influence the membrane potential. Cancer and stromal cells also sense abnormal ion concentrations via intra- and extracellular receptors that modify cancer progression and prognosis. With emphasis on breast cancer, the current review first addresses the altered ion composition and the changes in expression and functional activity of ion channels and transporters in solid cancer tissue. It then discusses how ion channels, transporters, and cellular sensors under influence of the acidic tumor microenvironment shape cancer development and progression and affect the potential of cancer therapies.

Boron-containing carbonic anhydrases inhibitors.

Over the last decades, the medicinal chemistry of boron-based compounds has been extensively explored, designing valuable small molecule drugs to tackle diseases and conditions, such as cancer, infections, inflammatory and neurological disorders. Notably, boron has proven to also be a valuable element for the development of inhibitors of the metalloenzymes carbonic anhydrases (CAs), a class of drug targets with significant potential in medicinal chemistry. Incorporating boron into carbonic anhydrase inhibitors (CAIs) can modulate the ligand ability to recognize the target and/or influence selectivity towards different CA isoforms, using the tail approach and boron-based tails. The electron-deficient nature of boron and its associated properties have also led to the discovery of novel zinc-binding CAIs, such as boronic acids and the benzoxaboroles, capable of inhibiting the CAs upon a Lewis acid-base mechanism of action. The present manuscript reviews the state-of-the-art of boron-based CAIs. As research in the applications of boron compounds in medicinal chemistry continues, it is anticipated that new boron-based CAIs will soon expand the current array of such compounds. However, further research is imperative to fully unlock the potential of boron-based CAIs and to advance them towards clinical applications.

A CatSper-Uninvolved Mechanism to Induce Forward Sperm Motility in the Internal Fertilization.

Sperm-specific cation channel (CatSper), sperm-specific Na + /H + exchanger (sNHE), and soluble adenylyl cyclase (sAC) are necessary in the signaling pathways to control sperm motility in many animals, whereas some animals have lost some or all of them. In the present study, we examined CatSper-uninvolved signaling for vigorous undulation of the undulating membrane that is attached to the sperm tail and gives thrust for forward motility in the internally fertilizing newt Cynops pyrrhogaster. Reverse-transcription PCR failed to detect sNHE in the newt sperm. However, the pH of sperm cytoplasm was raised under a high extracellular pH equivalent to that of egg jelly, where sperm motility is initiated by sperm motility-initiating substance (SMIS). Carbonic anhydrase XII/ XVI and SLC4A4/8 were suggested to be present in the sperm, and transported bicarbonates raised the intracellular pH. In egg jelly extract that contained SMIS, the anion transporter inhibitor DIDS weakened the undulation of the undulating membrane, while bicarbonates enhanced it. The cyclic AMP concentration was found to increase in sperm cytoplasm in the egg-jelly extract. An inhibitor of sAC (KH7) weakened the undulation of the undulating membrane, and dibutyryl cyclic AMP blocked the inhibitory effect. Inhibitor of transmembrane AC (DDA) limitedly affected the undulation. The undulation was weakened by an inhibitor of protein kinase A (H89), and by an inhibitor of transient receptor potential (TRP) channels (RN1747). Our results support the conclusions that the high pH of the egg jelly triggers a signaling pathway through sAC, PKA, and TRP channels, and coacts with SMIS to induce forward sperm motility.

Acetazolamide and human carbonic anhydrases: retrospect, review and discussion of an intimate relationship.

Acetazolamide (AZM) is a strong pharmacological sulphonamide-type (R-SO2-NH2, pKa 7.2) inhibitor of the activity of several carbonic anhydrase (CA) isoforms, notably of renal CA II (Ki, 12 nM) and CA IV (Ki, 74 nM). AZM is clinically used for about eighty years in various diseases including epilepsy and glaucoma. Pharmacological AZM increases temporarily the urinary excretion of bicarbonate (HCO3-) and sodium ions (Na+) and sustainably the urinary pH. AZM is excreted almost unchanged over several hours at high rates in the urine. Closely parallel concentrations of circulating and excretory AZM are observed upon administration of therapeutical doses of AZM. In a proof-of-principle study, we investigated the effects of the ingestion of a 250-mg AZM-containing tablet by a healthy volunteer on the urinary excretion of organic and inorganic substances over 5 h (range, 0, 0.5, 1, 1.5, 2, 3, 4, 5 h). Measured analytes included: AZM, amino acids and their metabolites such as guanidinoacetate, i.e. the precursor of creatine, of asymmetrically (ADMA) and symmetrically (SDMA) dimethylated arginine, nitrite (O = N-O-, pKa 3.4) and nitrate (O2N-O-, pKa -1.37), the major metabolites of nitric oxide (NO), the C-H acidic malondialdehyde (MDA; (CHO)2CH2, pKa 4.5), and creatinine for correction of analytes excretion. All analytes were measured by validated isotopologues using gas chromatography-mass spectrometry (GC-MS) methods. AZM excretion in the urine reached its maximum value after 2 h and was fairly stable for the next 3 h. Time series analysis by the ARIMA method was performed. AZM ingestion increased temporarily the urinary excretion of the amino acids Leu + Ile, nitrite and nitrate, decreased temporarily the urinary excretion of other amino acids. AZM decreased sustainably the urinary excretion of MDA, a biomarker of oxidative stress (i.e. lipid peroxidation). Whether this decrease is due to inhibition of the excretion of MDA or attenuation of oxidative stress by AZM is unknown. The acute and chronic effects of AZM on the urinary excretion of electrolytes and physiological substances reported in the literature are discussed in depth in the light of its extraordinary pharmacokinetics and pharmacodynamics. Tolerance development/drug resistance to AZM in chronic use and potential mechanisms are also addressed.

An overview of the latest outlook of sulfamate derivatives as anticancer candidates (2020-2024).

Considering the emergence of new anticancer drugs, in this review we emphasized and highlighted the recent reports and advances related to sulfamate-incorporating compounds with potential anticancer activity during the last 5 years (2020-2024). Additionally, we discussed their structure-activity relationship, clarifying their potent bioactivity as anticancer agents. Sulfamate derivatives hold promise as effective therapeutic candidates against cancer. By targeting biological targets associated with the development of cancer, such as steroid sulfatases (STS), carbonic anhydrases (CAs), microtubules, NEDD8-activating enzyme, small ubiquitin-like modifiers (SUMO)-activating enzyme (SAE), cyclin-dependent kinases (CDKs), breast cancer susceptibility gene 1 (BRCA1), and so on, this can furnish small molecules as anticancer lead candidates serving the drug discovery field. For example, compound 2, an STS inhibitor, demonstrated superior activity compared to its reference, irosustat, by fivefold. In addition, compound 21, an SAE, is under phase I clinical trials. Continued research into sulfamate derivatives holds potential for the development of novel therapeutic agents targeting various diseases.

Sulfonamide-incorporated bis(α-aminophosphonates) as promising carbonic anhydrase inhibitors: Design, synthesis, biological evaluation, and X-ray crystallographic studies.

A novel series of sulfonamide-incorporated bis(α-aminophosphonates) acting as effective carbonic anhydrase (CA, EC 4.2.1.1) inhibitors is reported. The synthesized bivalent ligands were tested against five human (h) isoforms, hCA I, hCA II, hCA VII, hCA IX, and hCA XIII. Such derivatives showed high activity and selectivity against the cancer-related, membrane-bound isoform hCA IX, and among them, compound 5h, tetraisopropyl (1,3-phenylenebis{[(4-sulfamoylphenyl)amino]methylene})bis(phosphonate) showed a KI of 15.1 nM, being highly selective against this isoform over all other investigated ones (hCA I/IX = 42; hCA II/IX = 6, hCA VII/IX = 3, hCA XIII/IX = 5). Therefore, compound 5h could be a potential lead for the development of selective anticancer agents. The newly developed sulfonamides were also found effective inhibitors against the cytosolic hCA XIII isoform. Compound 5i displayed the best inhibition against this isoform with a KI of 17.2 nM, equal to that of the well-known inhibitor acetazolamide (AAZ), but significantly more selective over all other tested isoforms (hCA I/XIII = 239; hCA II/XIII = 23, hCA VII/XIII = 2, hCA IX/XIII = 3) compared to AAZ.

Chemistry of heterocycles as carbonic anhydrase inhibitors: A pathway to novel research in medicinal chemistry review.

Nowadays, the scientific community has focused on dealing with different kinds of diseases by exploring the chemistry of various heterocycles as novel drugs. In this connection, medicinal chemists identified carbonic anhydrases (CA) as one of the biologically active targets for curing various diseases. The widespread distribution of these enzymes and the high degree of homology shared by the different isoforms offer substantial challenges to discovering potential drugs. Medicinal and synthetic organic chemists have been continuously involved in developing CA inhibitors. This review explored the chemistry of different heterocycles as CA inhibitors using the last 11 years of published research work. It provides a pathway for young researchers to further explore the chemistry of a variety of synthetic as well as natural heterocycles as CA inhibitors.

Carbonic anhydrase IX: A tumor acidification switch in heterogeneity and chemokine regulation.

The primary physiological process of respiration produces carbon dioxide (CO2) that reacts with water molecules which subsequently liberates bicarbonate (HCO-3) and protons. Carbonic anhydrases (CAs) are the primary catalyst involved in this conversion. More than 16 isoforms of human CAs show organ or subcellular specific activity. Dysregulation of each CA is associated with multiple pathologies. Out of these members, the overexpression of membrane-bound carbonic anhydrase IX (CAIX) is associated explicitly with hypoxic tumors or various solid cancers. CAIX helps tumors deal with higher CO2 by sequestering it with bicarbonate ions and helping cancer cells to grow in a comparatively hypoxic or acidic environment, thus acting as a pH adaptation switch. CAIX-mediated adaptations in cancer cells include angiogenesis, metabolic alterations, tumor heterogeneity, drug resistance, and regulation of cancer-specific chemokines. This review comprehensively collects and describe the cancer-specific expression mechanism and role of CAIX in cancer growth, progression, heterogeneity, and its structural insight to develop future combinatorial targeted cancer therapies.

Carbonic anhydrases reduce the acidity of the tumor microenvironment, promote immune infiltration, decelerate tumor growth, and improve survival in ErbB2/HER2-enriched breast cancer.

BACKGROUND: Carbonic anhydrases catalyze CO2/HCO3- buffer reactions with implications for effective H+ mobility, pH dynamics, and cellular acid-base sensing. Yet, the integrated consequences of carbonic anhydrases for cancer and stromal cell functions, their interactions, and patient prognosis are not yet clear. METHODS: We combine (a) bioinformatic analyses of human proteomic data and bulk and single-cell transcriptomic data coupled to clinicopathologic and prognostic information; (b) ex vivo experimental studies of gene expression in breast tissue based on quantitative reverse transcription and polymerase chain reactions, intracellular and extracellular pH recordings based on fluorescence confocal microscopy, and immunohistochemical protein identification in human and murine breast cancer biopsies; and (c) in vivo tumor size measurements, pH-sensitive microelectrode recordings, and microdialysis-based metabolite analyses in mice with experimentally induced breast carcinomas. RESULTS: Carbonic anhydrases-particularly the extracellular isoforms CA4, CA6, CA9, CA12, and CA14-undergo potent expression changes during human and murine breast carcinogenesis. In patients with basal-like/triple-negative breast cancer, elevated expression of the extracellular carbonic anhydrases negatively predicts survival, whereas, surprisingly, the extracellular carbonic anhydrases positively predict patient survival in HER2/ErbB2-enriched breast cancer. Carbonic anhydrase inhibition attenuates cellular net acid extrusion and extracellular H+ elimination from diffusion-restricted to peripheral and well-perfused regions of human and murine breast cancer tissue. Supplied in vivo, the carbonic anhydrase inhibitor acetazolamide acidifies the microenvironment of ErbB2-induced murine breast carcinomas, limits tumor immune infiltration (CD3+ T cells, CD19+ B cells, F4/80+ macrophages), lowers inflammatory cytokine (Il1a, Il1b, Il6) and transcription factor (Nfkb1) expression, and accelerates tumor growth. Supporting the immunomodulatory influences of carbonic anhydrases, patient survival benefits associated with high extracellular carbonic anhydrase expression in HER2-enriched breast carcinomas depend on the tumor inflammatory profile. Acetazolamide lowers lactate levels in breast tissue and blood without influencing breast tumor perfusion, suggesting that carbonic anhydrase inhibition lowers fermentative glycolysis. CONCLUSIONS: We conclude that carbonic anhydrases (a) elevate pH in breast carcinomas by accelerating net H+ elimination from cancer cells and across the interstitial space and (b) raise immune infiltration and inflammation in ErbB2/HER2-driven breast carcinomas, restricting tumor growth and improving patient survival.

4-Cyanamido-substituted benzenesulfonamides act as dual carbonic anhydrase and cathepsin inhibitors.

A set of novel N-cyano-N-substituted 4-aminobenzenesulfonamide derivatives were synthesized and investigated for their inhibitory activity against four cytosolic carbonic anhydrase (CA, EC 4.2.1.1) isoforms (hCA I, II, VII and XIII) and two cathepsins (S and B). N-alkyl/benzyl-substituted derivatives were revealed to be very potent inhibitors against brain-associated hCA VII, but inactive against both cathepsins. On the other hand, N-acyl-substituted derivatives displayed significant inhibitory activities against cathepsin S, but only moderate to poor inhibitory potency against hCA VII. Both hCA VII and cathepsin S have recently been validated as therapeutic targets in neuropathic pain. This study provided an excellent starting point for further structural optimization of this class of bifunctional compounds to enhance their inhibitory activity and selectivity against hCA VII and cathepsin S and to achieve new compounds with an attractive dual mechanism of action as anti-neuropathic agents.

Substituted furan sulfonamides as carbonic anhydrase inhibitors: Synthesis, biological and in silico studies.

Carbonic Anhydrases (CAs) are a large family of zinc metalloenzymes that catalyze the reversible hydration of carbon dioxide involved in several of biological processes, such as respiration, calcification, acid-base balance, bone resorption, and the formation of aqueous humor, cerebrospinal fluid, saliva, and gastric acid. They show wide diversity in tissue distribution and in their subcellular localization. Fifteen novel furyl sulfonamides were designed, synthesized and evaluated against four human isoforms: hCA I, hCA II, hCA IV and hCA IX. Compounds appeared to be very active mostly against hCAI (8) and hCA IV (11) isoforms being more potent than reference drug acetazolamide (AAZ). It should be mentioned that four compounds were more active than AAZ against hCA IX isoform, with compound 13d to be selective against hCA I (SI 70), hCA II (SI 13.5) and hCA IV (SI 20). Furthermore, docking was performed for some of these compounds on all isoforms I order to understand the possible interactions with the active site. The most active compounds showed good bioavailability and drug likeness scores.

Carbonic anhydrases III and IX are new players in the crosstalk between adrenocortical carcinoma and its altered adipose microenvironment.

PURPOSE: Adrenocortical carcinoma (ACC), a rare malignancy of the adrenocortex, is characterized by a crosstalk between the adipose microenvironment and tumor. Here, we assessed the involvement of carbonic anhydrase (CA) enzymes III and IX (CAIII and CAIX), in the metabolic alterations of the adipose tissue characterizing obesity and in the local crosstalk between the tumor adipose microenvironment and ACC. RESULTS/METHODS: CAIII and CAIX expression is altered in visceral adipose tissue (VAT) in obesity and in ACC. A significant CAIX upregulation was present in ACC at advanced stages (n = 14) (fold increase FI = 7.4 ± 0.1, P < 0.05) associated with lower CAIII levels (FI = 0.25 ± 0.06, P < 0.001), compared with lower stages (n = 9). In vitro coculture between visceral adipose stem cells (ASCs) and ACC cell lines, H295R and MUC-1, mimicking the interaction occurring between VAT and advanced ACC, showed a significant CAIX upregulation in H295R but not in MUC-1 cells, and a decreased expression of CAIII. The effect on adipose cells was different when cocultured with H295R or MUC-1 cells. Coculture did not modulate CAIII expression in ASCs, which, however, was significantly downregulated with H295R (FI = 0.34 ± 0.11, P < 0.05) and upregulated by MUC-1 when cocultured ASCs were induced to differentiate toward adipocytes, with an expression profile similar to what found in VAT of obese subjects. CAIX expression was markedly increased in ASCs cocultured with H295R and to a less extent following adipogenesis induction (FI = 150.9 ± 46.5 and FI = 4.6 ± 1.1, P < 0.01, respectively). CONCLUSION: Our findings highlight a modulation of CAIII and CAIX in the metabolic crosstalk between ACC and its local adipose microenvironment, suggesting that CAs might represent a potential target for novel anticancer therapies.

Association between Carbonic Anhydrase VI Gene Copy Number Variations and Dental Caries Experience.

The current study examined the association between the carbonic anhydrase VI (CA VI) copy number variations (CNVs) and dental caries experience in adults. In total, 202 of 35-72 years old subjects participating in the Lithuanian National Oral Health Survey (LNOHS) agreed to provide saliva samples; thus, their data were included in the current study. Information about sociodemographic, environmental, and behavioural determinants was acquired via the self-administered World Health Organization (WHO) questionnaire. Fluoride levels in the drinking water were recorded based on information provided by water suppliers. Dental caries experience was recorded by one calibrated examiner using the WHO criteria for recording caries on smooth (including proximal, buccal, and oral) or occlusal surfaces. Caries experience was measured as the total number of decayed (D3), missing (M), filled (F) surfaces. DNA was extracted from saliva samples to examine CA VI CNVs using the QX200 Droplet Digital PCR system. Negative binomial regression and Poisson regression analyses were employed for data analyses. Based on multivariable regression analyses, higher copy number of CA VI were associated with higher caries experience on smooth surfaces (IRR 1.04, 95% CI: 1.005-1.08) and occlusal surfaces (IRR 1.02, 95% CI: 1.003-1.04). Positive associations between higher copy number of CA VI and higher caries experience on smooth and occlusal surfaces were found, suggesting that the CA VI coding gene may be associated with caries development. Future studies are needed to validate our results and to examine the underlying mechanisms of such associations.

The role of carbonic anhydrases in extinction of contextual fear memory.

Carbonic anhydrases (CAs; EC 4.2.1.1) are metalloenzymes present in mammals with 16 isoforms that differ in terms of catalytic activity as well as cellular and tissue distribution. CAs catalyze the conversion of CO2 to bicarbonate and protons and are involved in various physiological processes, including learning and memory. Here we report that the integrity of CA activity in the brain is necessary for the consolidation of fear extinction memory. We found that systemic administration of acetazolamide, a CA inhibitor, immediately after the extinction session dose-dependently impaired the consolidation of fear extinction memory of rats trained in contextual fear conditioning. d-phenylalanine, a CA activator, displayed an opposite action, whereas C18, a membrane-impermeable CA inhibitor that is unable to reach the brain tissue, had no effect. Simultaneous administration of acetazolamide fully prevented the procognitive effects of d-phenylalanine. Whereas d-phenylalanine potentiated extinction, acetazolamide impaired extinction also when infused locally into the ventromedial prefrontal cortex, basolateral amygdala, or hippocampal CA1 region. No effects were observed when acetazolamide or d-phenylalanine was infused locally into the substantia nigra pars compacta. Moreover, systemic administration of acetazolamide immediately after the extinction training session modulated c-Fos expression on a retention test in the ventromedial prefrontal cortex of rats trained in contextual fear conditioning. These findings reveal that the engagement of CAs in some brain regions is essential for providing the brain with the resilience necessary to ensure the consolidation of extinction of emotionally salient events.

Selective carbonic anhydrase IX and XII inhibitors based around a functionalized coumarin scaffold.

Inhibition of specific carbonic anhydrase (CA) enzymes is a validated strategy for the development of agents to target cancer. The CA isoforms IX and XII are overexpressed in various human solid tumors wherein they play a critical role in regulating extracellular tumor acidification, proliferation, and progression. A series of novel sulfonamides based on the coumarin scaffold were designed, synthesized and characterized as potent and selective CA inhibitors. Selected compounds show significant activity and selectivity over CA I and CA II to target the tumor-associated CA IX and CA XII with high inhibition activity at the single digit nanomolar level. Twelve compounds were identified to be more potent compared with acetazolamide (AAZ) control to inhibit CA IX while one was also more potent than AAZ to inhibit CA XII. Compound 18f (Ki's = 955 nM, 515 nM, 21 nM and 5 nM for CA's I, II, IX, and XII, respectively) is highlighted as a novel CA IX and XII inhibitor for further development.

Differential CMS-Related Expression of Cell Surface Carbonic Anhydrases IX and XII in Colorectal Cancer Models-Implications for Therapy.

Tumor-associated carbonic anhydrases IX (CAIX) and XII (CAXII) have long been in the spotlight as potential new targets for anti-cancer therapy. Recently, CAIX/CAXII specific inhibitor SLC-0111 has passed clinical phase I study and showed differential response among patients with colorectal cancer (CRC). CRC can be classified into four different consensus molecular subgroups (CMS) showing unique expression patterns and molecular traits. We questioned whether there is a CMS-related CAIX/CAXII expression pattern in CRC predicting response. As such, we analyzed transcriptomic data of tumor samples for CA9/CA12 expression using Cancertool. Protein expression pattern was examined in preclinical models comprising cell lines, spheroids and xenograft tumors representing the CMS groups. Impact of CAIX/CAXII knockdown and SLC-0111 treatment was investigated in 2D and 3D cell culture. The transcriptomic data revealed a characteristic CMS-related CA9/CA12 expression pattern with pronounced co-expression of both CAs as a typical feature of CMS3 tumors. Protein expression in spheroid- and xenograft tumor tissue clearly differed, ranging from close to none (CMS1) to strong CAIX/CAXII co-expression in CMS3 models (HT29, LS174T). Accordingly, response to SLC-0111 analyzed in the spheroid model ranged from no (CMS1) to clear (CMS3), with moderate in CMS2 and mixed in CMS4. Furthermore, SLC-0111 positively affected impact of single and combined chemotherapeutic treatment of CMS3 spheroids. In addition, combined CAIX/CAXII knockdown and more effective treatment with SLC-0111 reduced clonogenic survival of CMS3 modelling single cells. In conclusion, the preclinical data support the clinical approach of targeted CAIX/CAXII inhibition by showing linkage of expression with response and suggest that patients with CMS3-classified tumors would most benefit from such treatment.

Conformationally Restricted Glycoconjugates Derived from Arylsulfonamides and Coumarins: New Families of Tumour-Associated Carbonic Anhydrase Inhibitors.

The involvement of carbonic anhydrases (CAs) in a myriad of biological events makes the development of new inhibitors of these metalloenzymes a hot topic in current Medicinal Chemistry. In particular, CA IX and XII are membrane-bound enzymes, responsible for tumour survival and chemoresistance. Herein, a bicyclic carbohydrate-based hydrophilic tail (imidazolidine-2-thione) has been appended to a CA-targeting pharmacophore (arylsulfonamide, coumarin) with the aim of studying the influence of the conformational restriction of the tail on the CA inhibition. For this purpose, the coupling of sulfonamido- or coumarin-based isothiocyanates with reducing 2-aminosugars, followed by the sequential acid-promoted intramolecular cyclization of the corresponding thiourea and dehydration reactions, afforded the corresponding bicyclic imidazoline-2-thiones in good overall yield. The effects of the carbohydrate configuration, the position of the sulfonamido motif on the aryl fragment, and the tether length and substitution pattern on the coumarin were analysed in the in vitro inhibition of human CAs. Regarding sulfonamido-based inhibitors, the best template turned out to be a d-galacto-configured carbohydrate residue, meta-substitution on the aryl moiety (9b), with Ki against CA XII within the low nM range (5.1 nM), and remarkable selectivity indexes (1531 for CA I and 181.9 for CA II); this provided an enhanced profile in terms of potency and selectivity compared to more flexible linear thioureas 1-4 and the drug acetazolamide (AAZ), used herein as a reference compound. For coumarins, the strongest activities were found for substituents devoid of steric hindrance (Me, Cl), and short linkages; derivatives 24h and 24a were found to be the most potent inhibitors against CA IX and XII, respectively (Ki = 6.8, 10.1 nM), and also endowed with outstanding selectivity (Ki > 100 µM against CA I, II, as off-target enzymes). Docking simulations were conducted on 9b and 24h to gain more insight into the key inhibitor-enzyme interactions.

Antimicrobial and Antibiofilm Activities of Carvacrol, Amoxicillin and Salicylhydroxamic Acid Alone and in Combination vs. Helicobacter pylori: Towards a New Multi-Targeted Therapy.

The World Health Organization has indicated Helicobacter pylori as a high-priority pathogen whose infections urgently require an update of the antibacterial treatments pipeline. Recently, bacterial ureases and carbonic anhydrases (CAs) were found to represent valuable pharmacological targets to inhibit bacterial growth. Hence, we explored the underexploited possibility of developing a multiple-targeted anti-H. pylori therapy by assessing the antimicrobial and antibiofilm activities of a CA inhibitor, carvacrol (CAR), amoxicillin (AMX) and a urease inhibitor (SHA), alone and in combination. Minimal Inhibitory (MIC) and Minimal Bactericidal (MBC) Concentrations of their different combinations were evaluated by checkerboard assay and three different methods were employed to assess their capability to eradicate H. pylori biofilm. Through Transmission Electron Microscopy (TEM) analysis, the mechanism of action of the three compounds alone and together was determined. Interestingly, most combinations were found to strongly inhibit H. pylori growth, resulting in an additive FIC index for both CAR-AMX and CAR-SHA associations, while an indifferent value was recorded for the AMX-SHA association. Greater antimicrobial and antibiofilm efficacy of the combinations CAR-AMX, SHA-AMX and CAR-SHA against H. pylori were found with respect to the same compounds used alone, thereby representing an innovative and promising strategy to counteract H. pylori infections.

Pharmacological Properties of Sulfonamide Derivatives, New Inhibitors of Carbonic Anhydrase.

Selective blocking of individual isoforms of carbonic anhydrase (CA) is now one of the main directions in the development of its inhibitors. The new 1,2,4-oxadiazole-containing sulfonamides B12 and B13 predominantly block CA II and CA IX. The study of acute toxicity of B12 and B13 showed their safety. Substance B13 caused a relatively short-term, but rapid (within 30 min) decrease in the intraocular pressure in rabbits, which indicates the promise of its use for the emergency decrease in the intraocular pressure in medical practice. Analysis of the effects of sulfonamides on the functions of CNS showed that compound B12 probably exhibit tranquilizing activity; B13 is promising for the creation of drugs that have an antidepressant effect and at the same time increase the mental and physical performance.

Loss of luminal carbonic anhydrase XIV results in decreased biliary bicarbonate output, liver fibrosis, and cholangiocyte proliferation in mice.

Carbonic anhydrase XIV (Car14) is highly expressed in the hepatocyte, with predominance in the canalicular membrane and its active site in the extracellular milieu. The aim of this study is to determine the physiological relevance of Car14 for biliary fluid and acid/base output, as well as its role in the maintenance of hepatocellular and cholangiocyte integrity. The common bile duct of anesthetized car14-/- and car14+/+ mice was cannulated and hepatic HCO3- output was measured by microtitration and bile flow gravimetrically before and during stimulation with intravenously applied tauroursodeoxycholic acid (TUDCA). Morphological alterations and hepatic damage were assessed histologically and immunohistochemically in liver tissue from 3- to 52-week-old car14-/- and car14+/+ mice, and gene and/or protein expression was measured for pro-inflammatory cytokines, fibrosis, and cholangiocyte markers. Biliary basal and more so TUDCA-stimulated HCO3- output were significantly reduced in car14-/- mice of all age groups, whereas bile flow and hepatic and ductular morphology were normal at young age. Car14-/- mice developed fibrotic and proliferative changes in the small bile ducts at advanced age, which was accompanied by a reduction in bile flow, and an upregulation of hepatic cytokeratin 19 mRNA and protein expression. Membrane-bound Car14 is essential for biliary HCO3- output, and its loss results in gradual development of small bile duct disease and hepatic fibrosis. Bile flow is not compromised in young adulthood, suggesting that Car14-deficient mice may be a model to study the protective role of biliary canalicular HCO3- against luminal noxi to the cholangiocyte.