Carbonic anhydrases: Moiety appended derivatives, medicinal and pharmacological implications.

In the realm of enzymology, Carbonic anhydrase (CA) emerges as a pivotal protagonist orchestrating the rapid conversion of carbon dioxide and water into bicarbonate ions and hydrogen ions, respectively. Carbonic anhydrase inhibitors (CAIs) are the class of drugs that target various isoforms of the enzyme, and these inhibitors play a crucial role in the treatment and management of multiple diseases such as cancer, glaucoma, high altitude sickness, rheumatoid arthritis, obesity, epilepsy, and sleep apnea. Several structural classes of CAIs developed till date possess unique architects of the pharmacophoric requirements around the central core moiety for the selective targeting of various isoforms of the CA. Recent advancements in drug design and development, along with technologies that aid in structure determination, have led to the development of several isoform-selective inhibitors of CA enzymes. However, their clinical development was hampered by the lack of desired therapeutic efficacy, isoform selectivity and safety profile. This review covers the most recent approaches used by different researchers concerned with the development of isoform-selective carbonic anhydrase inhibitors belonging to distinct structural classes like sulphonamides, carbazoles, selenols, coumarin, organotelluride, topiramate, thiophene, triazole, uracil-modified benzylic amines, and thiourea etc. In addition, their structure-activity relationships, biological evaluation, and in silico studies inlcuding the forthcoming avenues of advancements have been discussed. This review serves as a valuable resource for developing potent and efficacious CAIs with remarkable therapeutic implications; offering insights into their potency, specificity, and potential clinical applications.

Integrated approach of machine learning, Mendelian randomization and experimental validation for biomarker discovery in diabetic nephropathy.

AIM: To identify potential biomarkers and explore the mechanisms underlying diabetic nephropathy (DN) by integrating machine learning, Mendelian randomization (MR) and experimental validation. METHODS: Microarray and RNA-sequencing datasets (GSE47184, GSE96804, GSE104948, GSE104954, GSE142025 and GSE175759) were obtained from the Gene Expression Omnibus database. Differential expression analysis identified the differentially expressed genes (DEGs) between patients with DN and controls. Diverse machine learning algorithms, including least absolute shrinkage and selection operator, support vector machine-recursive feature elimination, and random forest, were used to enhance gene selection accuracy and predictive power. We integrated summary-level data from genome-wide association studies on DN with expression quantitative trait loci data to identify genes with potential causal relationships to DN. The predictive performance of the biomarker gene was validated using receiver operating characteristic (ROC) curves. Gene set enrichment and correlation analyses were conducted to investigate potential mechanisms. Finally, the biomarker gene was validated using quantitative real-time polymerase chain reaction in clinical samples from patients with DN and controls. RESULTS: Based on identified 314 DEGs, seven characteristic genes with high predictive performance were identified using three integrated machine learning algorithms. MR analysis revealed 219 genes with significant causal effects on DN, ultimately identifying one co-expressed gene, carbonic anhydrase II (CA2), as a key biomarker for DN. The ROC curves demonstrated the excellent predictive performance of CA2, with area under the curve values consistently above 0.878 across all datasets. Additionally, our analysis indicated a significant association between CA2 and infiltrating immune cells in DN, providing potential mechanistic insights. This biomarker was validated using clinical samples, confirming the reliability of our findings in clinical practice. CONCLUSION: By integrating machine learning, MR and experimental validation, we successfully identified and validated CA2 as a promising biomarker for DN with excellent predictive performance. The biomarker may play a role in the pathogenesis and progression of DN via immune-related pathways. These findings provide important insights into the molecular mechanisms underlying DN and may inform the development of personalized treatment strategies for this disease.

Comparative study of stability and activity of wild-type and mutant human carbonic anhydrase II enzymes using molecular dynamics and docking simulations.

The human carbonic anhydrase II (HCA II) enzyme is a cytosolic protein located in the membrane of red blood cells that reversible hydration of carbon dioxide (CO2). Considering the critical role of the HCA II and the effects of some mutations on the activity and stability of the enzyme in humans, several computational methods are used to study the structure and dynamics of the wild-type and the mutant enzymes with three ligands, CO2, 4-nitrophenyl acetate and acetazolamide. Our results of MD simulation of a wild-type enzyme with 4-nitrophenyl acetate show that it created essential effects on the fluctuation of this enzyme and made it more unstable and less compact than the same enzyme without ligand. In the MD of the mutant enzyme with 4-nitrophenyl acetate ligand, no significant difference is observed between with and without ligand. The affinity of the wild-type enzyme to the 4-nitrophenyl acetate is notably higher than the mutant enzyme with the same ligand. Furthermore, results showed that wild-type and mutant enzymes with CO2 are more favorable in stability and flexibility than the same enzymes without ligand. The MD results of wild-type with acetazolamide indicate instability compare without ligand, but in MD of mutant enzyme with acetazolamide show that it more stable and compact than the same enzyme without ligand. Finally, Comparing protein trajectories to assess the impact of ligands on the stability and activity of HCA II enzymes can have medical applications and can in the engineering and design of new variants of carbonic anhydrase enzyme.

Carbonic anhydrase-embedded ZIF-8 membrane reactor with improved the recycling and stability for efficient CO2 capture.

Carbonic anhydrase (CA) enzyme-based absorption technology for CO2 capture has been intensively investigated. However, low solubility of CO2 and poor stability of CA severely limits its industrial utilization. Here, hydrolyzed polyacrylonitrile (PAN) membrane (HPAN) was first modified by polyethyleneimine (PEI) with a large number of amino groups, which has a strong affinity for CO2. Then, ZIF-8 was grown in situ on the surface of HPAN/PEI membrane by using the metal chelation of PEI and Zn2+. In this process, CA was embedded inside ZIF-8 by co-precipitation (CA@HPAN/PEI/ZIF-8). The resultant CA@HPAN/PEI/ZIF-8 exhibited high catalytic activity for CO2 capture compared with free CA, which was due to the synergistic enhancement of CO2 capture by PEI and ZIF-8 with high affinity to CO2 and enzymatic catalysis. The yield of CaCO3 by CA@HPAN/PEI/ZIF-8 in the process of one-time conversion of CO2 was 13.6-fold higher than free CA. Furthermore, the CA@HPAN/PEI/ZIF-8 showed better thermal stability, storage and reusability than free CA. Free CA retained only 18.3 % of its original activity after 18 days of storage, whereas CA@HPAN/PEI/ZIF-8 remained 48.7 % of its original activity. The total CaCO3 yield by CA@HPAN/PEI/ZIF-8 was 74.9-fold that of free CA after 8 consecutive rounds of CO2 conversion.

Multi- and poly-pharmacology of carbonic anhydrase inhibitors.

Eight genetically distinct families of the enzyme carbonic anhydrase (CA, EC 4.2.1.1) were described in organisms allover the phylogenetic tree. They catalyze the hydration of CO2 to bicarbonate and protons, and are involved in pH regulation, chemosensing and metabolism. The 15 α-CA isoforms present in humans are pharmacological drug targets known for decades, their inhibitors being used as diuretics, antiglaucoma, antiepileptic or antiobesity drugs, as well as for the management of acute mountain sickness, idiopathic intracranial hypertension and recently, as antitumor theragnostic agents. Other potential applications include the use of CA inhibitors (CAIs) in inflammatory conditions, cerebral ischemia, neuropathic pain, or for Alzheimer's/Parkinson's disease management. CAs from pathogenic bacteria, fungi, protozoans and nematodes started to be considered as drug targets in recent years, with notable advances registered ultimately. CAIs have a complex multipharmacology probably unique to this enzyme, which has been exploited intensely but may lead to other relevant applications in the future, due to the emergence of drug design approaches which afforded highly isoform-selective compounds for most α-CAs known to date. They belong to a multitude of chemical classes (sulfonamides and isosteres, (iso)coumarins and related compounds, mono- and dithiocarbamates, selenols, ninhydrines, boronic acids, benzoxaboroles, etc). The polypharmacology of CAIs will also be discussed since drugs originally discovered for the treatment of non-CA related conditions (topiramate, zonisamide, celecoxib, pazopanib, thiazide and high-ceiling diuretics) show efective inhibition against many CAs, which led to their repurposing for diverse pharmacological applications. Significance Statement Carbonic anhydrase inhibitors have multiple pharmacologic applications as diuretics, antiglaucoma, antiepileptic, antiobesity, anti-acute mountain sickness, anti-idiopathic intracranial hypertension and as antitumor drugs. Their use in inflammatory conditions, cerebral ischemia, neuropathic pain, or neurodegenerations started to be investigated recently. Parasite carbonic anhydrases are also drug targets for antiinfectives with novel mechanisms of action which can by pass drug resistance to commonly used such agents. Drugs discovered for the management of other conditions that effectively inhibit these enzymes exert interesting polypharmacologic effects.

Synthesis and in Vitro Antileishmanial Efficacy of Novel Ethylene Glycol Analogues of Benzothiadiazine-1,1-dioxide.

Leishmaniasis is a vector-borne, parasitic disease affecting millions of people and animals worldwide. Current therapeutic options have proven to be ineffective in both treating the disease and preventing its spread. As a result, new drugs must be developed to effectively combat this disease. In this study, a series of 14 ethylene glycol analogues of benzothiadiazine-1,1-dioxide were synthesised to investigate their antileishmanial potential and cytotoxicity. Analogue 9, 2-(2-phenoxyethyl)-2H-benzo[e][1,2,4]thiadiazine-1,1-dioxide, was identified as the most inhibitory compound as it was observed to moderately inhibit the growth of L. major (IC50 103 µM) and L. donovani (IC50 153 µM) promastigotes. However, in general, the series presented with low biological activity, which may be attributed to reduced target affinity and/or undesired cell culture protein binding.

Lasamide Containing Sulfonylpiperazines as Effective Agents for the Management of Glaucoma Associated Symptoms.

A series of 2,4-dichloro-5-{[4-(phenylsulfonyl)piperazin-1-yl]carbonyl}benzenesulfonamide were designed and synthesized through amidation of Lasamide 1 with substituted piperazines. The newly obtained compounds demonstrated remarkable inhibition potency and selectivity for the human (h) expressed Carbonic Anhydrase (CA; EC 4.2.1.1) II isoform. Selected compounds 7 and 9 were considered in an in vivo model of glaucoma and showed relevant performances with the latter being able to last the effect up to 4 hours. The results herein reported are in sustainment of Lasamide derivatives as a new class of compounds potentially exploitable for the management of uncontrolled IOP.

Interaction of Micro- and Nanoplastics with Enzymes: The Case of Carbonic Anhydrase.

Microplastics (MPs) and nanoplastics (NPs) have emerged as significant environmental pollutants with potential detrimental effects on ecosystems and human health. Several studies indicate their interaction with enzymes; this topic represents a multifaceted research field encompassing several areas of interest from the toxicological and ecotoxicological impact of MPs and NPs on humans and wildlife to the biodegradation of plastics by microbial enzymes. This review aims to provide a critical analysis of the state-of-the-art knowledge of the interaction of MPs and NPs on the enzyme carbonic anhydrase (CA), providing recent insights, analyzing the knowledge gaps in the field, and drawing future perspectives of the research and its application. CA is a widespread and crucial enzyme in various organisms; it is critical for various physiological processes in animals, plants, and bacteria. It catalyzes the reversible hydration of CO2, which is essential for respiration, acid-base balance, pH homeostasis, ion transport, calcification, and photosynthesis. Studies demonstrate that MPs and NPs can inhibit CA activity with mechanisms including adsorption to the enzyme surface and subsequent conformational changes. In vitro and in silico studies highlight the role of electrostatic and hydrophobic interactions in these processes. In vivo studies present mixed results, which are influenced by factors like particle type, size, concentration, and organism type. Moreover, the potentiality of the esterase activity of CA for plastic degradation is discussed. The complexity of the interaction between CA and MPs/NPs underscores the need for further research to fully understand the ecological and health impacts of MPs and NPs on CA activity and expression and glimpses of the potentiality and perspectives in this field.

Unraveling the mechanism of carbonic anhydrase IX inhibition by alkaloids from Ruta chalepensis: A synergistic analysis of in vitro and in silico data.

Due to the pivotal role of carbonic anhydrase IX (CA IX) in pathological conditions, there's a pressing need for novel inhibitors to improve patient outcomes and clinical management. Herein, we investigated the inhibitory efficacy of six alkaloids from Ruta chalepensis against CA IX through in vitro inhibition assay and computational modeling. Skimmianine and maculosidine displayed significant inhibitory activity in vitro, with low IC50 values of 105.2 ± 3.2 and 295.7 ± 14.1 nM, respectively. Enzyme kinetics analyses revealed that skimmianine exhibited a mixed inhibition mode, contrasting with the noncompetitive inhibition mechanism observed for the reference drug (acetazolamide), as indicated by intersecting lines in the Lineweaver-Burk plots. The findings of docking calculations revealed that skimmianine and maculosidine exhibited extensive polar interactions with the enzyme. These alkaloids demonstrate substantial binding interactions and occupy identical binding site as acetazolamide, thereby enhancing their efficacy as inhibitors of CA IX. Utilizing a 100 ns molecular dynamics (MD) simulation, the dynamic interactions between isolated alkaloids and CA IX were intensively assessed. Analysis of diverse MD parameters revealed that skimmianine and maculosidine displayed consistent trajectories and notable energy stabilization during their interaction with CA IX. The findings of MM/PBSA analysis depicted the minimum binding free energy for skimmianine and maculosidine. In addition, the Potential Energy Landscape (PEL) analysis revealed distinct and stable conformational states for the CA IX-ligand complexes, with Skimmianine showing the most stable and lowest energy configuration. These computational findings align with experimental results, emphasizing the potential efficacy of skimmianine and maculosidine as inhibitors of CA IX.

Imaging in Renal Cell Carcinoma Detection.

INTRODUCTION: Imaging in renal cell carcinoma (RCC) is a constantly evolving landscape. The incidence of RCC has been rising over the years with the improvement in image quality and sensitivity in imaging modalities resulting in "incidentalomas" being detected. We aim to explore the latest advances in imaging for RCC. METHODS: A literature search was conducted using Medline and Google Scholar, up to May 2024. For each subsection of the manuscript, a separate search was performed using a combination of the following key terms "renal cell carcinoma", "renal mass", "ultrasound", "computed tomography", "magnetic resonance imaging", "18F-Fluorodeoxyglucose PET/CT", "prostate-specific membrane antigen PET/CT", "technetium-99m sestamibi SPECT/CT", "carbonic anhydrase IX", "girentuximab", and "radiomics". Studies that were not in English were excluded. The reference lists of selected manuscripts were checked manually for eligible articles. RESULTS: The main imaging modalities for RCC currently are ultrasound, computed tomography (CT) and magnetic resonance imaging (MRI). Contrast-enhanced US (CEUS) has emerged as an alternative to CT or MRI for the characterisation of renal masses. Furthermore, there has been significant research in molecular imaging in recent years, including FDG PET, PSMA PET/CT, 99mTc-Sestamibi, and anti-carbonic anhydrase IX monoclonal antibodies/peptides. Radiomics and the use of AI in radiology is a growing area of interest. CONCLUSIONS: There will be significant change in the field of imaging in RCC as molecular imaging becomes increasingly popular, which reflects a shift in management to a more conservative approach, especially for small renal masses (SRMs). There is the hope that the improvement in imaging will result in less unnecessary invasive surgeries or biopsies being performed for benign or indolent renal lesions.

Hydrogen Sulfide-Releasing Carbonic Anhydrase Inhibitors Effectively Suppress Cancer Cell Growth.

This study proposes a novel therapeutic strategy for cancer management by combining the antitumor effects of hydrogen sulfide (H2S) and inhibition of carbonic anhydrases (CAs; EC 4.2.1.1), specifically isoforms IV, IX, and XII. H2S has demonstrated cytotoxicity against various cancers at high concentrations. The inhibition of tumor-associated CAs leads to lethal intracellular alkalinization and acidification of the extracellular tumor microenvironment and restores tumor responsiveness to the immune system, chemotherapy, and radiotherapy. The study proposes H2S donor-CA inhibitor (CAI) hybrids for tumor management. These compounds effectively inhibit the target CAs, release H2S consistently, and exhibit potent antitumor effects against MDA-MB-231, HCT-116, and A549 cancer cell lines. Notably, some compounds display high cytotoxicity across all investigated cell lines. Derivative 30 shows a 2-fold increase in cytotoxicity (0.93 ± 0.02 µM) under chemically induced hypoxia in HCT-116 cells. These compounds also disturb the cell cycle, leading to a reduction in cell populations in G0/G1 and S phases, with a notable increase in G2/M and Sub-G1. This disruption is correlated with induced apoptosis, with fold increases of 37.2, 24.5, and 32.9 against HCT-116 cells and 14.2, 13.1, and 19.9 against A549 cells compared to untreated cells. These findings suggest the potential of H2S releaser-CAI hybrids as effective and versatile tools in cancer treatment.

Entrapment of Cyanase from Thermomyces lanuginosus Using Biomimetic Silica and Its Application for Cyanate Bioremediation.

Cyanate, a toxic product from the chemical oxidation treatment of highly toxic cyanide, can be converted to harmless ammonia and carbon dioxide by cyanase (EC 4.2.1.104). Cyanase from Thermomyces lanuginosus was entrapped in biomimetic silica to improve stability and reusability. After entrapment, the enzyme's activity increased by two-fold, and the residual activity after 30-min of incubation at 60 °C also increased by two-fold, compared to the free enzyme. After being stored at room temperature for 28 days, the entrapped cyanase retained 79% of the initial activity, while the free form retained 61%. The immobilized cyanase was successfully applied to cyanate detoxification; the co-entrapment of carbonic anhydrase from Sulfurihydrogenibium azorense decreased the amount of bicarbonate necessary for cyanate detoxification by 50%. The cyanate degradation retained 53% of the initial value after the co-entrapped cyanate and carbonic anhydrase were reused five times.

Exploration of 1,3,5-trisubstituted pyrazoline derivatives as human carbonic anhydrase inhibitors: Synthesis, biological evaluation and in silico studies.

The human carbonic anhydrase (hCA) IX and XII isoforms are overexpressed in hypoxic conditions, contributing to cancer. Lack of isoform selectivity has been one of the main challenges associated with the existing drugs targeting hCAs. Hence, the development of alternative approaches, such as tail approach to develop more selective hCA IX and XII inhibitors is need of the hour. In the present work, we designed and synthesized 24 new 1,3.5-trisubstituted-pyrazoline derivatives with diverse substitutions. The synthesized analogs were evaluated for their hCA inhibitory activities against hCA I, II, IX, and XII isoforms. Among the tested compounds, derivative 8 displayed good inhibitory activity against hCA IX (Ki = 331 nM) and XII (Ki = 96.7 nM). In addition, 9a-g also exhibited some inhibitory activities against hCA IX and XII, with Kis ranging from 574-799 nM and 137-369 nM, respectively. Molecular modelling studies of compound 8 displayed metal coordination with zinc ion and hydrophobic, hydrophilic interactions with adjacent amino acid residues, and maintained stable interactions throughout 100 ns. In addition, ADMET studies demonstrated that compound 8 obeyed the Lipinski's rule of five and was found to be druggable and non-toxic. Hence, compound 8 was identified as potential lead for further development.

Overview on bacterial carbonic anhydrase genetic families.

Bacterial carbonic anhydrases (BCAs, EC 4.2.1.1) are indispensable enzymes in microbial physiology because they facilitate the hydration of carbon dioxide (CO2) to bicarbonate ions (HCO3-) and protons (H+), which are crucial for various metabolic processes and cellular homeostasis. Their involvement spans from metabolic pathways, such as photosynthesis, respiration, to organic compounds production, which are pivotal for bacterial growth and survival. This chapter elucidates the diversity of BCA genetic families, categorized into four distinct classes (α, ß, γ, and ι), which may reflect bacterial adaptation to environmental niches and their metabolic demands. The diversity of BCAs is essential not only for understanding their physiological roles but also for exploring their potential in biotechnology. Knowledge of their diversity enables researchers to develop innovative biocatalysts for industrial applications, including carbon capture technologies to convert CO2 emissions into valuable products. Additionally, BCAs are relevant to biomedical research and drug development because of their involvement in bacterial pathogenesis and microbial survival within the host. Understanding the diversity and function of BCAs can aid in designing targeted therapeutics that interfere with bacterial metabolism and potentially reduce the risk of infections.

Sulfonamide inhibitors of bacterial carbonic anhydrases.

The increasing prevalence of antibiotic-resistant bacteria necessitates the exploration of novel therapeutic targets. Bacterial carbonic anhydrases (CAs) have been known for decades, but only in the past ten years they have garnered significant interest as drug targets to develop antibiotics having a diverse mechanism of action compared to the clinically used drugs. Significant progress has been made in the field in the past three years, with the validation in vivo of CAs from Neisseria gonorrhoeae, and vancomycin-resistant enterococci as antibiotic targets. This chapter compiles the state-of-the-art research on sulfonamide derivatives described as inhibitors of all known bacterial CAs. A section delves into the mechanisms of action of sulfonamide compounds with the CA classes identified in pathogenic bacteria, specifically α, ß, and γ classes. Therefore, the inhibitory profiling of the bacterial CAs with classical and clinically used sulfonamide compounds is reported and analyzed. Another section covers various other series of sulfonamide CA inhibitors studied for the development of new antibiotics. By synthesizing current research findings, this chapter highlights the potential of sulfonamide inhibitors as a novel class of antibacterial agents and paves the way for future drug design strategies.

Enterococci carbonic anhydrase inhibition.

Carbonic anhydrase metalloenzymes are encoded in genomes throughout all kingdoms of life with a conserved function catalyzing the reversible conversion of CO2 to bicarbonate. Carbonic anhydrases have been well-investigated in humans, but are still relatively understudied in bacterial organisms, including Enterococci. Studies over the past decade have presented bacterial carbonic anhydrases as potential drug targets, with some chemical scaffolds potently inhibiting the Enterococcus carbonic anhydrases in vitro and displaying antimicrobial efficacy against Enterococcus organisms. While carbonic anhydrases in Enterococci still have much to be explored, hypotheses may be drawn from similar Gram-positive organisms for which known information exists about carbonic anhydrase function and relevance. Within this chapter is reported information and rational hypotheses regarding the subcellar locations, potential physiological roles, essentiality, structures, and kinetics of carbonic anhydrases in Enterococci.

Helicobacter pylori CAs inhibition.

Infections from Helicobacter pylori (Hp) are endangering Public Health safety worldwide, due to the associated high risk of developing severe diseases, such as peptic ulcer, gastric cancer, diabetes, and cardiovascular diseases. Current therapies are becoming less effective due to the rise of (multi)drug-resistant phenotypes and an urgent need for new antibacterial agents with innovative mechanisms of action is pressing. Among the most promising pharmacological targets, Carbonic Anhydrases (EC: 4.2.1.1) from Hp, namely HpαCA and HpßCA, emerged for their high druggability and crucial role in the survival of the pathogen in the host. Thereby, in the last decades, the two isoenzymes were isolated and characterized offering the opportunity to profile their kinetics and test different series of inhibitors.

Neisseria gonorrhoeae carbonic anhydrase inhibition.

Carbonic anhydrases (CAs) are ubiquitous enzymes that are found in all kingdoms of life. Though different classes of CAs vary in their roles and structures, their primary function is to catalyze the reaction between carbon dioxide and water to produce bicarbonate and a proton. Neisseria gonorrhoeae encodes for three distinct CAs (NgCAs) from three different families: an α-, a ß-, and a γ-isoform. This chapter details the differences between the three NgCAs, summarizing their subcellular locations, roles, essentiality, structures, and enzyme kinetics. These bacterial enzymes have the potential to be drug targets; thus, previous studies have investigated the inhibition of NgCAs-primarily the α-isoform. Therefore, the classes of inhibitors that have been shown to bind to the NgCAs will be discussed as well. These classes include traditional CA inhibitors, such as sulfonamides, phenols, and coumarins, as well as non-traditional inhibitors including anions and thiocarbamates.

Challenges for developing bacterial CA inhibitors as novel antibiotics.

Acetazolamide, methazolamide, ethoxzolamide and dorzolamide, classical sulfonamide carbonic anhydrase (CA) inhibitors (CAIs) designed for targeting human enzymes, were also shown to effectively inhibit bacterial CAs and were proposed for repurposing as antibacterial agents against several infective agents. CAs belonging to the α-, ß- and/or γ-classes from pathogens such as Helicobacter pylori, Neisseria gonorrhoeae, vacomycin resistant enterococci (VRE), Vibrio cholerae, Mycobacterium tuberculosis, Pseudomonas aeruginosa and other bacteria were considered as drug targets for which several classes of potent inhibitors have been developed. Treatment of some of these pathogens with various classes of such CAIs led to an impairment of the bacterial growth, reduced virulence and for drug resistant bacteria, a resensitization to clinically used antibiotics. Here I will discuss the strategies and challenges for obtaining CAIs with enhanced selectivity for inhibiting bacterial versus human enzymes, which may constitute an important weapon for addressing the drug resistance to ß-lactams and other clinically used antibiotics.

Bacterial ß-carbonic anhydrases.

ß-Carbonic anhydrases (ß-CA; EC 4.2.1.1) are widespread zinc metalloenzymes which catalyze the interconversion of carbon dioxide and bicarbonate. They have been isolated in many pathogenic and non-pathogenic bacteria where they are involved in multiple roles, often related to their growth and survival. ß-CAs are structurally distant from the CAs of other classes. In the active site, located at the interface of a fundamental dimer, the zinc ion is coordinated to two cysteines and one histidine. ß-CAs have been divided in two subgroups depending on the nature of the fourth ligand on the zinc ion: class I have a zinc open configuration with a hydroxide ion completing the metal coordination, which is the catalytically active species in the mechanism proposed for the ß-CAs similar to the well-known of α-CAs, while in class II an Asp residue substitute the hydroxide. This latter active site configuration has been showed to be typical of an inactive form at pH below 8. An Asp-Arg dyad is thought to play a key role in the pH-induced catalytic switch regulating the opening and closing of the active site in class II ß-CAs, by displacing the zinc-bound solvent molecule. An allosteric site well-suited for bicarbonate stabilizes the inactive form. This bicarbonate binding site is composed by a triad of well conserved residues, strictly connected to the coordination state of the zinc ion. Moreover, the escort site is a promiscuous site for a variety of ligands, including bicarbonate, at the dimer interface, which may be the route for bicarbonate to the allosteric site.