Carbohydrate-modified carbonic anhydrase inhibitors: Advances and challenges.

Heterocyclic compounds represent a prominent class of molecules with diverse pharmacological activities. Among their therapeutic applications, they have gained significant attention as carbonic anhydrase (CA) inhibitors, owing to their potential in the treatment of various diseases such as epilepsy, cancer and glaucoma. CA is a widely distributed zinc metalloenzyme that facilitates the reversible interconversion of carbon dioxide and bicarbonate. This reaction is essential for numerous physiological and pathological processes. In humans, CA exists in sixteen different isoforms, labeled hCA-I to hCA-XV, each distributed across various tissues and organs and involved in crucial physiological functions. Clinically utilized CA inhibitors, such as brinzolamide, dorzolamide and acetazolamide, exhibit poor selectivity, leading to undesirable side effects. A significant challenge in designing effective CA inhibitors is achieving balanced isoform selectivity, prompting the exploration of new chemotypes. This review compiles recent strategies employed by various researchers in developing CAIs across different structural classes, including pyrazoline, quinoline, imidazole, oxadiazole, pyrimidine, coumarin, chalcone, rhodanine, phthalazine, triazole, isatin, and indole. Additionally, the review summarizes structure-activity relationship (SAR) analyses, isoform selectivity evaluations, along with mechanistic and in silico investigations. Insights derived from SAR studies provide crucial directions for the rational design of next-generation heterocyclic CA inhibitors, with improved therapeutic efficacy and reduced side effects. To the best of our knowledge, for the first time, we have comprehensively summarized all known isoforms of CA in relation to various heterocyclic motifs. This review examines the use of different heterocycles as CA inhibitors, drawing on research published over the past 11 years. It offers a valuable resource for early-career researchers, encouraging further exploration of synthetic heterocycles in the development of CA inhibitors.

Carbonic anhydrase: chemistry, physiology, and inhibition.

We present a new approach to carbonic anhydrase II (CA II) inhibitor design that enables close interrogation of the regions of the CA active site where there is the greatest variability in amino acid residues among the different CA isozymes. By appending dual tail groups onto the par excellence CA inhibitor acetazolamide, compounds that may interact with the distinct hydrophobic and hydrophilic halves of the CA II active site were prepared. The dual-tail combinations selected included (i) two hydrophobic moieties, (ii) two hydrophilic moieties, and (iii) one hydrophobic and one hydrophilic moiety. The CA enzyme inhibition profile as well as the protein X-ray crystal structure of compound 3, comprising one hydrophobic and one hydrophilic tail moiety, in complex with CA II is described. This novel dual-tail approach has provided an enhanced opportunity to more fully exploit interactions with the CA active site by enabling these molecules to interact with the distinct halves of the active site. In addition to the dual-tail compounds, a corresponding set of single-tail derivatives was synthesized, enabling a comparative analysis of the single-tail versus dual-tail compound CA inhibition profile.

Synthesis of Novel Carbohydrate Based Enzyme Inhibitor Libraries Utilising Click Chemistry

Carbonic anhydrases (CAs) are metalloenzymes responsible for the reversible hydration of carbon dioxide to bicarbonate, a fundamental reaction involved in various physiological and pathological processes. In the last decades, CAs have been considered as important drug targets for different pathologies such as glaucoma, epilepsy and cancer. The design of potent and selective inhibitors has been an outstanding goal leading to the discovery of new drugs. Among the different strategies developed to date, the design of carbohydrate-based CA inhibitors (CAIs) has emerged as a versatile tool in order to selectively target CAs. The insertion of a glycosyl moiety as a hydrophilic tail in sulfonamide, sulfenamide, sulfamate or coumarin scaffolds allowed the discovery of many different series of sugar-based CAIs, with relevant inhibitory results. This review will focus on carbohydrate-based CAIs developed so far, classifying them in glycosidic and glycoconjugated inhibitors based on the conjugation chemistry adopted.

A 3D QSAR selectivity analysis of carbonic anhydrase (CA) inhibitors using a data set of 87 CA inhibitors is reported. After ligand minimization in the binding pockets of CA I, CA II, and CA IV isoforms, selectivity CoMFA and CoMSIA 3D QSAR models have been derived by taking the affinity differences (DeltapKi) with respect to two CA isozymes as independent variables. Evaluation of the developed 3D QSAR selectivity models allows us to determine amino acids in the respective CA isozymes that possibly play a crucial role for selective inhibition of these isozymes. We further combined the ligand-based 3D QSAR models with the docking program AUTODOCK in order to screen for novel CA inhibitors. Correct binding modes are predicted for various CA inhibitors with respect to known crystal structures. Furthermore, in combination with the developed 3D QSAR models we could successfully estimate the affinity of CA inhibitors even in cases where the applied scoring function failed. This novel strategy to combine AUTODOCK poses with CoMFA/CoMSIA 3D QSAR models can be used as a guideline to assess the relevance of generated binding modes and to accurately predict the binding affinity of newly designed CA inhibitors that could play a crucial role in the treatment of pathologies such as tumors, obesity, or glaucoma.

All cells produce carbon dioxide (CO2), which is released as a result of metabolism and must be removed from the body. A large part of this CO2 is converted to bicarbonate by the carbonic anhydrase (CA) enzyme in erythrocytes and is discarded from the body. So, CA has a vital role in red blood cells. In addition to, CA involved in many other pathological and physiological processes and it was determined that the inhibitors of CA were effective in the treatment and diagnosis of many diseases particularly glaucoma. Considering the importance of the CA's inhibitors, in this study it was intended to research the inhibition effects of Eosin Y on CA I and CA II isoenzymes activity purified from human erythrocytes. Eosin Y is a dye molecule commonly used in histological and medical applications. For this purpose, firstly CA I and CA II isoenzymes were purified from human erythrocytes by using Sepharose-4B-L-tyrosine-sulfanilamide affinity chromatography. Then the inhibitory effect of Eosin Y on the activity of these human erythrocyte CA I (hCA I) and CA II (hCA II) isoenzymes was investigated. It was determined that hCA I and hCA II were inhibited by Eosin Y in the millimolar range. IC50 values were found to be 3.78 mM for hCA I and 2.04 mM for hCA II and Ki values were determined as 9.65±0.968 mM and 7.52±2.88 mM for hCA I and hCA II, respectively. In conclusion, it is hoped that the results obtained in this study may be beneficial in the development of new CA inhibitors which may be drug candidates.

Although almost fully automated, the discovery of novel, effective, and safe drugs is still a long-term and highly expensive process. Consequently, the need for fleet, rational, and cost-efficient development of novel drugs is crucial, and nowadays the advanced in silico drug design methodologies seem to effectively meet these issues. The aim of this chapter is to provide a comprehensive overview of some of the current trends and advances in the in silico design of novel drug candidates with a special emphasis on 6-fluoroquinolone (6-FQ) antibacterials as potential novel Mycobacterium tuberculosis DNA gyrase inhibitors. In particular, the chapter covers some of the recent aspects of a wide range of in silico drug discovery approaches including multidimensional machine-learning methods, ligand-based and structure-based methodologies, as well as their proficient combination and integration into an intelligent virtual screening protocol for design and optimization of novel 6-FQ analogs.

Although almost fully automated, the discovery of novel, effective, and safe drugs is still a long-term and highly expensive process. Consequently, the need for fleet, rational, and cost-efficient development of novel drugs is crucial, and nowadays the advanced in silico drug design methodologies seem to effectively meet these issues. The aim of this chapter is to provide a comprehensive overview of some of the current trends and advances in the in silico design of novel drug candidates with a special emphasis on 6-fluoroquinolone (6-FQ) antibacterials as potential novel Mycobacterium tuberculosis DNA gyrase inhibitors. In particular, the chapter covers some of the recent aspects of a wide range of in silico drug discovery approaches including multidimensional machine-learning methods, ligand-based and structure-based methodologies, as well as their proficient combination and integration into an intelligent virtual screening protocol for design and optimization of novel 6-FQ analogs.

We present a compact optical programmable ASIC-based tag for the identification of novel drug candidates, requiring no external components. Our tag aims for a time and cost-efficient solution to keep track of compounds during the split and pool synthesis. The ASIC is self-powered by integrated solar cells and optimized for intensive light in the range of 10Mlx. This intensive illumination can cause unwanted leakage currents in p-n junctions. Therefore, extensive care was taken to provide shielding for sensitive parts of the circuit and measurements prove the effectiveness. The ASIC is manufactured in a 0.6µm process including special devices like EEPROM and photodiodes. Our tag contains an oscillator, optical receiver, reference voltage, and a digital controller. A custom optical communication protocol provides an energy-efficient data link to the smart tag with continuous power transfer.

since the 1970s, over 200 studies with acetazolamide have shown it safe and 60–80% effective in acute mountain sickness (AMS). Despite much investigation, our understanding of its action in AMS remains incomplete and more complicated than generally taught. This should come as no surprise

By catalysing a very simple physiological reaction, the hydration of carbon dioxide (CO2) to bicarbonate (HCO3-) and a proton (H+), carbonic anhydrases (CAs), of which five genetically distinct families (α – ϵ) are presently known, are fundamental enzymes in all organisms over the phylogenetic tree. In vertebrates, including humans, the 14 different α-CA isozymes presently known are involved in a host of physiological and pathological processes and modulation of their activity by means of specific inhibitors or activators leads to important pharmacological responses. CA inhibitors (CAIs) – systemic or topically acting – are clinically used ophthalmologic drugs for the management of glaucoma, cystoid macular oedema and retinopathies of diverse nature, being used alone or in combination therapies with many other agents. Topiramate (TPM) and zonisamide are widely used antiepileptics possessing a complex mechanism of action, in which CA inhibition plays a major role. Other neurological or neuromuscular disorders also make use of agents such as acetazolamide, methazolamide, ethoxzolamide or dichlorophenamide, in addition to the classical CAIs. CA activators on the other hand may lead to novel therapies in the treatment of cognitive disorders. Important advances were recently accomplished in the design of CA V-targeted inhibitors, with potential use as antiobesity agents that inhibit a critical step in lipogenesis in which the mitochondrial isozyme V is involved. Isozymes CA IX and CA XII play important roles in pH regulation, cell adhesion, proliferation and differentiation in hypoxic tumours and constitute conceptually novel targets for anticancer therapies. Indeed, many sulfonamide and sulfamate potent CA IX inhibitors were reported, which may open new vistas for such antitumour therapies. Several CA isozymes are complexed with proteins involved in anion transport, such as anion exchangers (AE1) or Na+/HCO3- cotransporter proteins (NBC1 and NBC3), forming metabolons, in which the enzymatic activity of each protein is enhanced by its companion. The understanding of the biochemical mechanisms involved in such interactions may also lead to important novel pharmacological applications.

Carbonic anhydrase (CA) is a metalloenzyme that catalyzes the interconversion between carbon dioxide and water and dissociated ions of carbonic acid. In addition, CA performs various other functions in animals and plants, depending on the part of the living being. CAs have been found in almost all organisms. Besides, CAs are associated with several diseases, such as glaucoma, obesity, epilepsy, cancer, and so on. CAs are also involved in tumor cell growth and angiogenesis. Thus, inhibition of CA may be an attractive way of control of such diseases. Hence, CA inhibitors have been designed and developed to cure CA-associated diseases. Some examples of approved CA inhibitors are dorzolamide, methazolamide, brinzolamide, and dichlorphenamide. Furthermore, various heterocyclic scaffolds were utilized for the design of CA inhibitors. Among those, pyrazole/pyrazoline derivatives have exhibited greater potency toward CA inhibition. Hence, research that took place in the field of drug design and discovery of CA inhibition has been systematically reviewed and collated. Alongside, the structure-activity relationship has been described, followed by a description of the most potent molecules and their structural features.

The molecular complexity of cancers and therapy-related side effects often limit efficacy of numerous anti-tumor therapies, and warrant development of new drugs that are specific for certain molecular targets while minimizing the off-target effects. We previously have synthesized a series of benzene-sulfonamides incorporating cyanoacrylamide moieties (tyrphostine analogues), and investigated such new compounds as inhibitors of the metalloenzyme carbonic anhydrase (CA). Specifically, we determined the inhibitory activity of such novel compounds against the cytosolic, house-keeping human (h) isoforms hCA I and II, as well as the transmembrane, tumor-associated ones CA IX and XII. Four compounds, namely CS2, CS6, CS8 and CS13, were very potent CA IX/XII inhibitors whereas they were much less effective as inhibitors of CA I and II. To determine whether these selective tumor-associated CA inhibitors exert antitumor activity, we examined their antiproliferative activity against the human medulloblastoma Daoy cell line, human hepatoma HepG2 cell line and the human epithelial cervical cancer Hela cell line. Compounds CS6 and CS13 showed significant antiproliferative activity against the three cancer cell lines. Daoy was the most sensitive cell line, and compound CS6 was the most potent one. Remarkably, compound CS6 was more potent against Daoy cells than the multi-targeted kinase inhibitor of BCR-ABL and SRC family kinases, dasatinib, since the former exerted smaller IC50 than that exerted by the latter (4.14 versus 7.28 μg/mL). In addition, flow cytometric Annexin-V/propidium iodide assay results showed that cell death induced by compounds CS6 is mediated, at least in part, by apoptosis. Moreover, CS6 significantly inhibited tyrosine kinase activity in Daoy cells, compared to DMSO-treated (control) cells. Taken together, these data indicate that compound CS6 is a novel multiple cancer pathways inhibitor, and warrants further investigation of its antitumor activity in medulloblastoma and other brain tumors. Currently, compounds CS6 and CS13 are being tested for their inhibitory activity against dihydrofolate reductase and thymidylate synthase. Citation Format: Ahmed Mahmoud Alafeefy, Sabry Atia, Sheikh Ahmad, Khairy Zoheir, Abdelkader Ashour, Ashok Kumar. Development of novel selective tumor-associated carbonic anhydrase inhibitors as promising anticancer agents. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-100. doi:10.1158/1538-7445.AM2015-LB-100

Studies were performed to examine the possible effects of carbonic anhydrase (CA) inhibition on the glomerulotubular balance for bicarbonate in anesthetized dogs. Maximal CA inhibition was achieved by acetazolamide infusion and glomerular filtration rate (GFR) was reduced in a stepwise fashion by progressive clamping of the left renal artery. A close relationship (R equals 0.973) was maintained between the amount of filtered and reabsorbed bicarbonate in normal dogs with CA inhibition. A similar relationship was observed between GFR and bicarbonate reabsorption during CA inhibition in normal dogs (R equals 0.957) as well as in sodium bicarbonate loaded dogs (R equals 0.867). In these two groups, GFR in the clamped kidney was reduced to values ranging respectively from 99 to 5% and from 96 to 3%. Distal tubular blockade with ethacrynic acid and chlorothiazide, performed in normal dogs and in sodium bicarbonate loaded dogs, did not abolish glomerulotubular balance for bicarbonate during CA inhibition. This study demonstrates that the glomerulotubular balance for bicarbonate is maintained during CA inhibition whether or not distal tubular blockade is superimposed. A proportionate decrease in both fractions of bicarbonate reabsorption, either CA dependent or not mediated by CA, or an adaptive increase in the fraction of bicarbonate reabsorption not mediated by CA can explain the maintenance of glomerulotubular balance for bicarbonate.