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Background: Carbonic anhydrase (CA) catalyzes the reversible hydration of CO(2) to bicarbonate and a proton, and alpha-class CA has been reported to facilitate the acid acclimation of Helicobacter pylori (hpalphaCA). The purpose of this study was to characterize the beta-class CA of H. pylori (hpbetaCA) and elucidate the role of this enzyme as a possible drug target for eradication therapy.Methods: We isolated DNA clones of independent H. pylori strains obtained from patients with gastritis (n = 15), gastric ulcer (n = 6), or gastric cancer (n = 16), and then studied genetic polymorphisms. In addition, the susceptibility of H. pylori to sulpiride, an antiulcer drug and efficient inhibitor of both hpalphaCA and hpbetaCA, was studied with an in vitro killing assay.Results: DNA sequences of all 37 hpbetaCA clones encoded a 221 amino acid polypeptide with a variety of polymorphisms (57 types of amino acid substitution at 48 residue positions). There was no polymorphism functionally relevant to the gastric lesion type. One strain included unique residues that were not seen in the other 36 clones from Japanese patients but which were found in a strain obtained from the United Kingdom. Sulpiride had killing effects at concentrations greater than 200 microg/ml for H. pylori, including strains resistant to clarithromycin, metronidazole, or ampicillin.Conclusions: Helicobacter pylori might have evolved independently in the Caucasian and Japanese populations. Dual inhibition of alpha-and beta-class CAs could be applied as alternative therapy for eradication of H. pylori. [ABSTRACT FROM AUTHOR]

A series of 2,4-dichloro-5-{[4-(phenylsulfonyl)piperazin-1-yl]carbonyl}benzenesulfonamides 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 investigated 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 intra ocular pressure (IOP)., (© 2024 The Authors. ChemMedChem published by Wiley-VCH GmbH.)

Lactones, a diverse and abundant class of molecules found in nature, exhibit a wide range of bioactivities, including anti-inflammatory, anticancer, and antibacterial effects. Among them, acyl homoserine lactones (AHSLs) play a crucial role in quorum sensing, influencing bacterial pathogenicity and biofilm formation in Gram-negative bacteria. Paraoxonases (PONs), calcium-containing enzymes known for their lactonase activity, have been shown to hydrolyze AHSLs and reduce the biofilm formation of several pathogenic bacteria. In this study, we explored the potential lactonase activity of a class of zinc(II) enzymes, the carbonic anhydrases (CAs), aiming to uncover new insights into their catalytic versatility. Using LC-MS and MS/MS analyses, we investigated the lactonase activity of CAs and assessed several lactones through a stopped-flow kinetic assay as substrates/inhibitors. Our findings reveal that lactones are novel "prodrug" inhibitors of CAs, with lactones DHC and 6 showing the most promising inhibition constants (K I s) in the low micromolar range against both human and bacterial isozymes., (© 2024 The Author(s). Archiv der Pharmazie published by Wiley‐VCH GmbH on behalf of Deutsche Pharmazeutische Gesellschaft.)

Ambrosia maritima L. (family Asteraceae) is an annual herb widely distributed throughout the Mediterranean region and Africa. The herb is employed in folk medicine for the treatment of many ailments. Herein, we report a comprehensive investigation of the diverse biological potential of two sesquiterpene lactones, damsin and neoambrosin, isolated from Ambrosia maritima. 1D and 2D NMR and HR-ESI-MS/MS were employed to characterize the chemical structures of both compounds. In order to identify biological targets of both compounds we investigated their potential affinity for peroxisome proliferator-activated receptors (PPARs) and transient receptor potential (TRP) channels, which are pleiotropic classes of receptors implicated in essential functions of the body. This was investigated using a luciferase assay and a calcium fluorometric assay. A carbonic anhydrase inhibition assay was also performed using stopped flow CO 2 hydrase spectrophotometric assay. Our analysis revealed that unlike damsin, neoambrosin showed a selective partial agonist effect on PPARγ receptors and TRPA1 channels. Its binding mode was investigated through in silico analysis. Both compounds showed no affinity for the tested carbonic anhydrases. Overall, our study details the chemical properties of neoambrosin and damsin and highlights neoambrosin as novel, cost-effective partial agonist of PPARɣ and TRPA1 receptors despite additional in vivo studies are needed to elucidate its biological and pharmacological properties., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Porphyromonas gingivalis, a key pathogen in periodontal, plays a critical role in systemic pathologiesdiseases by evading host defence mechanisms and invading periodontal tissues. Targeting its virulence mechanisms and overcoming drug resistance are essential steps toward effective therapeutic development. In this study, we focused on the Carbonic Anhydrase (CA, EC: 4.2.1.1) encoded by P. gingivalis as a potential drug target. We determined the crystal structure of PgiCA γ at a resolution of 2.4 Å and conducted kinetic characterization. The structure revealed that active PgiCA γ forms a trimer, with each monomer comprising a left-handed β-helix capped by a C-terminal α-helix and coordinated to a catalytic zinc ion through three histidine residues. Interestingly, one monomer displayed an atypical α-helix conformation, likely due to close interactions with neighbouring trimers within the crystal lattice (a probable crystallographic artefact). These findings provide new insights into the structural and functional properties of PgiCA γ, emphasizing its potential as a target for the development of novel anti-virulence therapies against P. gingivalis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Boronic acids are an interesting but still poorly studied class of carbonic anhydrase inhibitors. Previous investigations proved that derivatives incorporating aromatic, arylalkyl, and arylalkenyl moieties are low micromolar to millimolar inhibitors for several α- and β-CAs involved in pathologic states. Here we report a high-resolution X-ray study on two classes of boronic acids (phenyl and vinyl) in complex with hCA II. Our results unambiguously clarify the binding mode of these molecules to the human carbonic anhydrase active site, which occurs through their tetrahedral anionic form, regardless of the nature of the organic scaffold. Data here presented contribute to the understanding of the inhibition mechanism of boronic acids that can be fruitfully used for the rational design of novel and effective isozyme-specific carbonic anhydrase inhibitors., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

The role of carbonic anhydrases isoforms (CAs) IX and XII in the pathogenesis and progression of many types of solid tumors is well known. In this context, selective CA inhibitors (CAIs) towards the mentioned isoforms is a validated strategy for the development of agents to target cancer. For this purpose, novel coumarin derivatives based on the hybridization with arylsulfonamide or biotin scaffolds were synthesized and tested as inhibitors of four different human carbonic anhydrases isoforms: hCA I, II, IX and XII. Coumarin-sulfonamide derived 27, with a thiourea moiety and triazole as linker, showed the highest inhibition activity against hCA XII with an inhibition constant (K I ) of 7.5 nM and afforded a very good selectivity over hCA I. Compound 32 was the most potent inhibitor against hCA IX (K I  = 6.3 nM), 4-fold stronger than the drug acetazolamide AAZ (K I  = 25 nM), used herein as a reference compound, and showed remarkable selectivity over hCA I and II. The coumarin-biotin derivatives 37-39 showed outstanding selectivity towards on-target enzymes (hCA IX and XII) and appear as plausible leads for designing of CAIs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

A novel class of compounds designed to hit two anti-tumour targets, G-quadruplex structures and human carbonic anhydrases (hCAs) IX and XII is proposed. The induction/stabilisation of G-quadruplex structures by small molecules has emerged as an anticancer strategy, disrupting telomere maintenance and reducing oncogene expression. hCAs IX and XII are well-established anti-tumour targets, upregulated in many hypoxic tumours and contributing to metastasis. The ligands reported feature a berberine G-quadruplex stabiliser scaffold connected to a moiety inhibiting hCAs IX and XII. In vitro experiments showed that our compounds selectively stabilise G-quadruplex structures and inhibit hCAs IX and XII. The crystal structure of a telomeric G-quadruplex in complex with one of these ligands was obtained, shedding light on the ligand/target interaction mode. The most promising ligands showed significant cytotoxicity against CA IX-positive HeLa cancer cells in hypoxia, and the ability to stabilise G-quadruplexes within tumour cells.

Toxoplasmosis, induced by the intracellular parasite Toxoplasma gondii , holds considerable implications for global health. While treatment options primarily focusing on folate pathway enzymes have notable limitations, current research endeavours concentrate on pinpointing specific metabolic pathways vital for parasite survival. Carbonic anhydrases (CAs, EC 4.2.1.1) have emerged as potential drug targets due to their role in fundamental reactions critical for various protozoan metabolic processes. Within T. gondii , the Carbonic Anhydrase-Related Protein (TgCA_RP) plays a pivotal role in rhoptry biogenesis. Notably, α-CA (TcCA) from another protozoan, Trypanosoma cruzi , exhibited considerable susceptibility to classical CA inhibitors (CAIs) such as anions, sulphonamides, thiols, and hydroxamates. Here, the recombinant DNA technology was employed to synthesise and clone the identified gene in the T. gondii genome, which encodes an α-CA protein (Tg_CA), with the purpose of heterologously overexpressing its corresponding protein. Tg_CA kinetic constants were determined, and its inhibition patterns explored with inorganic metal-complexing compounds, which are relevant for rational compound design. The significance of this study lies in the potential development of innovative therapeutic strategies that disrupt the vital metabolic pathways crucial for T. gondii survival and virulence. This research may lead to the development of targeted treatments, offering new approaches to manage toxoplasmosis.

A novel series of hydantoins incorporating phthalimides has been synthesised by condensation of activated phthalimides with 1-aminohydantoin and investigated for their inhibitory activity against a panel of human (h) carbonic anhydrase (CA, EC 4.2.1.1): the cytosolic isoforms hCA I, hCA II, and hCA VII, secreted isoform hCA VI, and the transmembrane hCA IX, by a stopped-flow CO 2 hydrase assay. Although all newly developed compounds were totally inactive on hCA I and mainly ineffective towards hCA II, they generally exhibited moderate repressing effects on hCA VI, VII, and IX with K I s values in the submicromolar to micromolar ranges. The salts 3a and 3b , followed by derivative 5 , displayed the best inhibitory activity of all the evaluated compounds and their binding mode was proposed in silico . These compounds can also be considered interesting starting points for the development of novel pharmacophores for this class of enzyme inhibitors.

This study refers to the intricate world of Acinetobacter baumannii , a resilient pathogenic bacterium notorious for its propensity at antibiotic resistance in nosocomial infections. Expanding upon previous findings that emphasised the bifunctional enzyme PaaY, revealing unexpected γ-carbonic anhydrase (CA) activity, our research focuses on a different class of CA identified within the A. baumannii genome, the β-CA, designated as 𝛽-AbauCA (also indicated as CanB), which plays a crucial role in the resistance mechanism mediated by AmpC beta-lactamase. Here, we cloned, expressed, and purified the recombinant 𝛽-AbauCA, unveiling its distinctive kinetic properties and inhibition profile with inorganic anions (classical CA inhibitors). The exploration of 𝛽-AbauCA not only enhances our understanding of the CA repertoire of A. baumannii but also establishes a foundation for targeted therapeutic interventions against this resilient pathogen, promising advancements in combating its adaptability and antibiotic resistance.

Hypoxia in tumors contributes to chemotherapy resistance, worsened by acidosis driven by carbonic anhydrases ( h CA IX and XII). Targeting these enzymes can mitigate acidosis, thus enhancing tumor sensitivity to cytotoxic drugs. Herein, novel 4-(pyrazolyl)benzenesulfonamide ureas ( SH7a-t ) were developed and evaluated for their inhibitory activity against h CA IX and XII. They showed promising results ( h CA IX: K I = 15.9-67.6 nM, h CA XII: K I = 16.7-65.7 nM). Particularly, SH7s demonstrated outstanding activity ( K I s = 15.9 nM for h CA IX and 55.2 nM for h CA XII) and minimal off-target kinase inhibition over a panel of 258 kinases. In NCI anticancer screening, SH7s exhibited broad-spectrum activity with an effective growth inhibition full panel GI 50 (MG-MID) value of 3.5 μM and a subpanel GI 50 (MG-MID) range of 2.4-6.3 μM. Furthermore, SH7s enhanced the efficacy of Taxol and 5-fluorouracil in cotreatment regimens under hypoxic conditions in HCT-116 colorectal cancer cells, indicating its potential as a promising anticancer agent.

Acinetobacter baumannii is a Gram-negative opportunistic pathogen responsible for severe hospital-associated infections. Owing to its ability to develop resistance to a wide range of antibiotics, novel therapeutic strategies are urgently needed. One promising approach is to target bacterial carbonic anhydrases (CAs; EC 4.2.1.1), which are enzymes critical for various metabolic processes. The genome of A. baumannii encodes a β-CA (βAbauCA), which is essential for producing bicarbonate ions required in the early stages of uridine triphosphate (UTP) synthesis, a precursor for the synthesis of peptidoglycans, which are vital components of the bacterial cell wall. This study aimed to inhibit βAbauCA in vitro, with the potential to impair the vitality of the pathogen in vivo. We conducted sequence and structural analyses of βAbauCA to explore its differences from those of human CAs. Additionally, kinetic and inhibition studies were performed to investigate the catalytic efficiency of βAbauCAβ and its interactions with sulfonamides and their bioisosteres, classical CA inhibitors. Our results showed that βAbauCA has a turnover rate higher than that of hCA I but lower than that of hCA II and displays distinct inhibition profiles compared to human α-CAs. Based on the obtained data, there are notable differences between the inhibition profiles of the human isoforms CA I and CA II and bacterial βAbauCA. This could open the door to designing inhibitors that selectively target bacterial β-CAs without affecting human α-CAs, as well as offer a novel strategy to weaken A. baumannii and other multidrug-resistant pathogens.

In searching for putative new therapeutic strategies to treat neurodegenerative diseases, the mitochondrial 18 kDa translocator protein (TSPO) and cerebral isoforms of carbonic anhydrase (CA) were exploited as potential targets. Based on the structures of a class of highly affine and selective TSPO ligands and a class of CA activators, both developed by us in recent years, a small library of 2-phenylindole-based dual TSPO/CA modulators was developed, able to bind TSPO and activate CA VII in the low micromolar/submicromolar range. The interaction with the two targets was corroborated by computational studies. Biological investigation on human microglia C20 cells identified derivative 3 as a promising lead compound worthy of future optimization due to its (i) lack of cytotoxicity, (ii) ability to stimulate TSPO steroidogenic function and activate CA VII, and (iii) ability to effectively upregulate gene expression of the brain-derived neurotrophic factor.

A series of novel 4-alkylthio-2-chloro-5-[(2-arylmethylidene)hydrazinecarbonyl]benzenesulfonamide derivatives 3-22 were synthesized and evaluated for their inhibitory activity against human carbonic anhydrase isozymes hCA I, hCA II, hCA IX, and hCA XII. These compounds showed varying degrees of activity against the studied isoenzymes. However, the importance of substituent choice in designing potent carbonic anhydrase inhibitors is highlighted by the strong inhibition profiles of compounds 3 and 10 against hCA IX and the low average K I values for compounds 9 and 10 (134 nM and 77 nM, respectively). All the synthesized compounds were evaluated for their antiproliferative activity toward HeLa, HCT-116, and MCF-7 cell lines. Compounds 9 and 19 exhibited significant activity, particularly against the MCF-7 cell line (IC 50 values of 4 μM and 6 μM, respectively). Notably, compound 9 demonstrated a high selectivity index (SI = 8.2) for MCF-7 cells. The antiproliferative effects of compounds 9 and 19 were linked to the induction of cell cycle arrest and apoptosis via the mitochondrial pathway and involved the activation of the MAPK/ERK signaling pathway. Inhibition of MAPK/ERK activity reduced the compounds' ability to induce cell cycle arrest and apoptosis, indicating the critical role of this pathway. These findings suggest that compounds 9 and 19 are promising candidates for further development as specific and potent anticancer agents targeting the MAPK/ERK pathway., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Medical University of Gdansk. Published by Elsevier Ltd.. All rights reserved.)

A small series of arylsulfonamide derivatives was designed and synthesized to study linear and cyclic inhibitors targeting human Carbonic Anhydrases (hCAs EC 4.2.1.1) as essential enzymes regulating (patho)-physiological processes. Particularly, the synthesis of these ten compounds was inspired to the well-known arylsulfonamides having flexible or constrained linkers able to maintain the two crucial moieties, anchoring zinc group and hydrophobic tail, in the optimized orientation within CA cavities of tumor-expressed isoforms hCA IX and hCA XII. The synthesized imine derivatives and related cyclic 1,3-thiazin-4-ones were screened in a stopped-flow carbon dioxide hydrase assay and proved to be effective inhibitors against hCA IX and hCA XII isoforms with K i values ranging of 3.7-215.7 nM and 5.7-415.0 nM, respectively. Molecular docking studies of both series of arylsulfonamides were conducted to propose their binding mode within hCA IX and hCA XII active sites thus highlighting their distinct ability to occupy the two catalytic cavities. Moreover, the 4-[(3-cyanophenyl)methylidene]aminobenzene-1-sulfonamide 7 proved to reduce the cell viability of breast carcinoma (MCF-7) and colon rectal carcinoma (HCT-116) human cell lines under the fixed doses of 10 μM. These results encouraged us to continue our efforts in developing potent and efficient arylsulfonamides targeting hCA IX and hCA XII isoforms., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Ciprofloxacin (CPX) is one of the most employed antibiotics in clinics to date. However, the rise of drug-resistant bacteria is dramatically impairing its efficacy, especially against life-threatening pathogens, such as Pseudomonas aeruginosa . This Gram-negative bacterium is an opportunistic pathogen, often infecting immuno-compromised patients with severe or fatal outcomes. The evidence of the possibility of exploiting Carbonic Anhydrase (CA, EC: 4.2.1.1) enzymes as pharmacological targets along with their role in P. aeruginosa virulence inspired the derivatization of CPX with peculiar CA-inhibiting chemotypes. Thus, a large library of CPX derivatives was synthesized and tested on a panel of bacterial CAs and human isoenzymes I and II. Selected derivatives were evaluated for antibacterial activity, revealing bactericidal and antibiofilm properties for some compounds. Importantly, promising preliminary absorption, distribution, metabolism, and excretion (ADME) properties in vitro were found and no cytotoxicity was detected for some representative compounds when tested in Galleria mellonella larvae.

Carbonic anhydrase (CA) IV is a membrane-bound enzyme involved in important physio-pathological processes, such as excitation-contraction coupling in heart muscle, central nervous system (CNS) extracellular buffering, and mediation of inflammatory response after stroke. Known since the mid-1980s, this isoform is still largely unexplored when compared to other isoforms, mostly for the current lack of inhibitors targeting selectively this isoform. The discovery of selective CA IV inhibitors is thus largely awaited. In this work, we report β-(di) fluoropropyl diamines as effective CA IV inhibitors, opening real perspectives for a new mode of selective inhibition of this isoform. Inhibition data reveal that the essential structure core to ensure a potent and selective inhibition of CA IV is the N -propyldiamine. Molecular modeling studies were employed to understand the binding mode of the synthesized amines. Conformational searches within the active site space carried out in an implicit solvent (water) model were also conducted.

Diseases caused by protozoan parasites represent a huge challenge to global health care, due to the lack of selective and efficient treatments for the management and spreading of such complex pathologies. The protozoans Trypanosoma cruzi (T. cruzi) and Leishmania spp. are the etiological agents of the so-called neglected tropical diseases (NTDs), that is, Chagas disease (CD) and leishmaniasis, respectively. In such a context, the metalloenzymes carbonic anhydrases (CAs; EC 4.2.1.1) emerged as potential protozoan druggable enzymes, being involved in the parasites' life cycle. Several studies suggested the relevance of the protozoan-expressed CAs as future candidates for the management of NTDs., (© 2024 The Author(s). Archiv der Pharmazie published by Wiley‐VCH GmbH on behalf of Deutsche Pharmazeutische Gesellschaft.)

Background: Sulfonamide derivatives are well-reported hCA IX inhibitors; however, they inhibit all types of hCA without any selectivity, leading to severe adverse effects. Hence, developing a novel nonsulfonamide class of tumor-associated hCA IX inhibitors through non-classical inhibition may provide greater selectivity and better pharmacokinetics., Objective: The objective of this study was to develop non-sulfonamide derivatives as potential human carbonic anhydrase (hCA) inhibitors and develop a new series of chromene-linked bis-indole derivatives., Methods: We synthesized and characterized the chromene-linked bis-indole derivatives and further evaluated them against four hCA isoforms, i.e., hCA I, hCA II, hCA IX, and hCA XII, and determined the ADMET parameters by the In-silico method., Results: Most of the compounds showed significantly greater affinity and selectivity towards the tumorassociated hCA IX over other hCA isoforms within the lower micromolar to submicromolar range. In particular, the bromo-substituted bis-indole derivative 6t showed an excellent inhibition of hCA IX isoform with an affinity (Ki) of 2.61 μM. In contrast, the cyano group substituted bis-indole derivative 6s and also displayed a strong inhibition of hCA IX isoform with an affinity (Ki) of 2.73 μM. Many other potential candidates, including 6g, 6i, 6k, 6m, 6o, 6p, and 6r, showed higher affinity at tumor-associated hCA IX with lower than 10 μM compared to other hCA isoforms., Conclusion: Therefore, the chromene-linked bis-indole derivatives can serve as a novel non-sulfonamide class of tumor-associated hCA IX inhibitors., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Human carbonic anhydrases (hCAs) have essential roles in respiration, acid-base balance, and fluid secretion, with implications in diseases such as glaucoma, epilepsy, obesity, and cancer. Of the fifteen known hCAs, human CA I (hCA I) is particularly abundant in erythrocytes, playing a critical role in CO 2 transport. Despite extensive research on hCA I, the impact of post-translational modifications (PTMs), particularly phosphorylation, on its catalytic activity and inhibitor binding remains poorly understood. Although multiple phosphorylation sites have been identified in hCA I in vivo through high-throughput proteomics studies including at the highly conserved Ser51 residue, the functional consequences of these modifications are not well characterized. We investigated the effects of a phosphomimetic mutation at Ser51 on hCA I, examining its catalytic efficiency and susceptibility to inhibition by sulfonamides and anions. Using a recombinant expression system and a stopped-flow kinetic assay, we characterized the CO 2 hydration activity and inhibition profiles of S51E hCA I compared to the wild type enzyme. Our results demonstrate that the S51E mutation increases the catalytic turnover rate (k cat ) from 2.0 × 10 5 s -1 to 2.6 × 10 5 s -1 but significantly decreases substrate affinity, raising the Michaelis constant (K M ) from 4.0 mM to 13.9 mM, reducing overall catalytic efficiency by over 50 %. Inhibition studies with a panel of 41 sulfonamides revealed that the S51E mutation dramatically alters inhibitor sensitivity, particularly for the most effective inhibitors. For example, 15 of the 16 most effective sulfonamide inhibitors for hCA I (with K I s <350 nM) were an average of over 35-fold less effective in inhibiting S51E hCA I than the wild type. The K I of the anticonvulsant zonisamide increased from 31 nM for the wild type hCA I to 4.0 μM. The inhibition profile with a panel of 37 small anions further indicated that the S51E mutant exhibited significantly reduced susceptibility to inhibition by 24 out of 37 tested anions, with some K I values increasing by up to 11,000-fold for inhibitors like hydrogen sulfide. This study underscores the significant impact that phosphorylation may have on hCA I function and inhibition. By characterizing the effects of phosphorylation on the CO 2 hydration activity and inhibitor sensitivity of hCA I, these findings represent early steps in developing more selective proteoform-specific inhibitors, which could lead to more effective treatments for diseases involving carbonic anhydrases., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

In previous studies, we demonstrated the potent activity of a library of 25 N,N'-disubstituted diamines (NNDDA) toward Trypanosomatid and Apicomplexa parasites. Considering the structure similarity between this collection and SQ109, an antituberculosis compound, and its compelling antiparasitic properties, we aimed to repurpose this library for tuberculosis treatment. We assayed this collection against Mycobacterium tuberculosis H37Rv and M. avium, obtaining several compounds with MIC values below 10 µM. The most active analogs were also evaluated against M. smegmatis, a non-pathogenic species, and the non-tuberculosis mycobacteria M. abscessus, M. kansasii, and M. fortuitum. 3c stands out as the lead mycobacterial compound of the collection, with potent activity against M. tuberculosis (minimal inhibitory concentration [MIC] = 3.4 µM) and moderate activity against M. smegmatis, M. kansasii, and M. fortuitum (all with MIC values of 26.8 µM). To unravel the mechanism of action, we employed the web-based platform Polypharmacology Browser 2 (PPB2), obtaining carbonic anhydrases as potential drug targets. Nevertheless, none of the compounds displayed experimental inhibition. In summary, our study confirms the validity of the repurposing approach and underscores the antimycobacterial potential of NNDDA compounds, especially the analog 3c, setting a stepping stone for further studies., (© 2024 Deutsche Pharmazeutische Gesellschaft.)

Sulfonamides are promising classical carbonic anhydrase (CA; EC 4.2.1.1) inhibitors, being used for several medical purposes such as diuretics, anticonvulsants, topically acting antiglaucoma agents, for antiobesity and anticancer therapies. Herein, a series of chalcone-based benzenesulfonamides (3a‒m) was synthesized and assessed for its inhibitory activity against a panel of four human carbonic anhydrases (hCA isoforms I, II, IX, and XII). Most compounds displayed single- to double-digit nanomolar inhibition constants (K i s), with some derivatives being more potent and/or selective than the standard drug acetazolamide (AAZ). Among the synthesized compounds, 3g compound demonstrated the highest inhibitory activity against the hCA II isoform (K i  = 2.5 nM) with 30-, 9-, and 11-fold selectivity for hCA II over the I, IX, and XII isoforms, respectively. Structure-activity relationships for different substitution patterns were analyzed. Additionally, a molecular docking study showed that compound 3g bound to hCA II by coordinating with the zinc ion through the deprotonated benzenesulfonamide moiety, in addition to a hydrogen bond formed between an oxygen of the sulfonamide moiety and Thr199. Moreover, the chalcone core participated in van der Waals interactions with some active site residues, such as Ile91, Val121, and Leu198. Consequently, this report introduces a successful approach toward identifying compound 3g as a highly potent and selective chalcone-based benzenesulfonamide inhibitor of hCA II worthy of further investigation., (© 2024 The Author(s). Archiv der Pharmazie published by Wiley‐VCH GmbH on behalf of Deutsche Pharmazeutische Gesellschaft.)

The eradication of Helicobacter pylori, the etiologic agent of gastric ulcer and adenocarcinoma, is a big concern in clinics due to the increasing drug resistance phenomena and the limited number of efficacious treatment options. The exploitation of the H. pylori carbonic anhydrases (HpCAs) as promising pharmacological targets has been validated by the antibacterial activity of previously reported CA inhibitors due to the role of these enzymes in the bacterium survival in the gastric mucosa. The development of new HpCA inhibitors seems to be on the way to filling the existing antibiotics gap. Due to the recent evidence on the ability of the coumarin scaffold to inhibit microbial α-CAs, a large library of derivatives has been developed by means of a pH-regulated cyclization reaction of coumarin-bearing acyl thiosemicarbazide intermediates. The obtained 1,3,4-thiadiazoles (10-18a,b) and 1,2,4-triazole-3-thiones (19-26a,b) were found to strongly and selectively inhibit HpαCA and computational studies were fundamental to gaining an understanding of the interaction networks governing the enzyme-inhibitor complex. Antibacterial evaluations on H. pylori ATCC 43504 highlighted some compounds that maintained potency on a resistant clinical isolate. Also, their combinations with metronidazole decreased both the minimal inhibitory concentration and minimal bactericidal concentration values of the antibiotic, with no synergistic effect., (© 2024 The Author(s). Archiv der Pharmazie published by Wiley‐VCH GmbH on behalf of Deutsche Pharmazeutische Gesellschaft.)

In the present study, the design and synthesis of novel coumarin derivatives 8a-h, 11a-d and 16a-c as potential selective inhibitors for the tumor associated human carbonic anhydrase isoforms (hCA IX and XII) was reported. All the newly synthesized derivatives showed potent to mild activity against the targeted CA IX (K I  = 0.08-9.57 µM), with selectivity indices over CA I (SI = 2.0-21.9) and over CA II (SI = 1.1-15.7). They showed similar activities against CA XII (K I  = 0.06-9.48 µM) with selectivity indices over CA I (SI = 1.4-21.2) and CA II (SI = 0.9-15.5). Compound 16b featuring sulfonamide function possessed promising inhibitory activities against the targeted isoforms CA IX and XII with K I values of 0.08 and 0.06 µM, respectively. Interestingly, it was found that using compound 16b at a nontoxic concentration as an adjuvant with Doxorubicin against MCF-7 cells enhanced the cytotoxicity under hypoxia by almost 3.5 folds; IC 50 decreased from 25.74 to 7.43 µM. Therefore, compound 16b restored the cytotoxicity of Doxorubicin against MCF-7 cells under hypoxia, almost as normoxia. Furthermore, flow cytometry analysis of a combination treatment of compound 16b and Doxorubicin to the MCF7 cell line revealed an increase in cell cycle arrest at the G2/M phase and a more efficient apoptotic effect than Doxorubicin alone. Furthermore, compound 16b showed no cytotoxicity against normal breast MCF-10A cell line (IC 50  = 296.25 µM)., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

In today's medical field, there is a growing trend of exploiting a single small molecule to target two different molecular targets concurrently. This approach is proving to be highly effective in fighting against cancer. The 4-anilinoquinazoline scaffold, known for its potential in cancer therapy and its effectiveness as a leading class of tyrosine kinase inhibitors, was employed to develop a novel series of anilinoquinazoline-sulfonamides (AQSs) (8a-d, 9a-f, and 10a-d) as dual inhibitors of the tumor-associated carbonic anhydrases (CA) IX/XII and EGFR. 2-(3-Methoxyphenyl)quinazoline bearing p-sulfanilamide 10b elicited superior hCA IX and XII inhibition in the low nanomolar range (K I s = 38.4 and 8.9 nM, respectively). Also, 10b shined as a potent and selective EGFR inhibitor, boasting an impressive IC 50 value of 51.2 ± 0.97 nM, surpassing the reference EGFR inhibitor Erlotinib (IC 50  = 80 ± 2.0 nM). Compound 10b exhibited broadest-spectrum antiproliferative activity against the NCI-tumor panel with a mean GI% value of 68 %. Of special interest, 10b demonstrated potent growth inhibition (GI% ≥ 80-97 %) toward cell lines reported to express high levels of EGFR belonging to renal, colon, breast, and lung cancers. Compound 10b's molecular docking in the CA IX/XII and EGFR active sites revealed binding modes that justify its potent enzyme inhibitory effects. Additionally, molecular dynamic simulations demonstrated strong and stable interactions of 10b with the binding sites of these targets., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

We synthesized herein 16 compounds (SUT1-SUT16) as potential carbonic anhydrase (CA) inhibitors utilizing the tail-approach design. Based on this strategy, we connected benzenesulfonamide, the zinc-binding scaffold, to different urea moieties with the 1,3,4-thiadiazole ring as a linker. We obtained the target compounds by the reaction of 4-(5-amino-1,3,4-thiadiazol-2-yl)benzenesulfonamide with aryl isocyanates. Upon confirmation of their structures, the compounds were screened for their ability to inhibit the tumor-related human (h) isoforms human carbonic anhydrase (hCA) IX and XII, as well as the physiologically dominant hCA I and II. Most of the molecules demonstrated K i values ≤ 10 nM with different selectivity profiles. The binding modes of SUT9, SUT10, and SUT5, the most effective inhibitors of hCA II, IX, and XII, respectively, were predicted by molecular docking. SUT16 (4-{5-[3-(naphthalen-1-yl)ureido]-1,3,4-thiadiazol-2-yl}benzenesulfonamide) was found to be the most selective inhibitor of the cancer-associated isoforms hCA IX and XII over the off-target isoforms, hCAI and II. The interaction dynamics and stability of SUT16 within hCA IX and XII were investigated by molecular dynamics simulations as well as dynophore analysis. Based on computational data, increased hydrophobic contacts and hydrogen bonds in the tail part of these molecules within hCA IX and XII were found as favorable interactions leading to effective inhibitors of cancer-related isoforms., (© 2024 The Author(s). Archiv der Pharmazie published by Wiley‐VCH GmbH on behalf of Deutsche Pharmazeutische Gesellschaft.)

In the search of new cymantrenyl- and ferrocenyl-sulfonamides as potencial inhibitors of human carbonic anhydrases (hCAs), four compounds based on N-ethyl or N-methyl benzenesulfonamide units have been obtained. These cymantrenyl (1a-b) and ferrocenyl (2a-b) derivatives were prepared by the reaction between aminobenzene sulfonamides ([NH 2 -(CH 2 ) n -(C 6 H 4 )-SO 2 -NH 2 )], where n = 1, 2) with cymantrenyl sulfonyl chloride (P1) or ferrocenyl sulfonyl chloride (P2), respectively. All compounds were characterized by conventional spectroscopic techniques and cyclic voltammetry. In the solid state, the molecular structures of compounds 1a, 1b, and 2b were determined by single-crystal X-ray diffraction. Biological evaluation as carbonic anhydrases inhibitors were carried out and showed derivatives 1b y 2b present a higher inhibition than the drug control for the Human Carbonic Anhydrase (hCA) II and IX isoforms (K I  = 7.3 nM and 5.8 nM, respectively) and behave as selective inhibition for hCA II isoform. Finally, the docking studies confirmed they share the same binding site and interactions as the known inhibitors acetazolamide (AAZ) and agree with biological studies., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Rodrigo Arancibia reports financial support was provided by National Agency for Research and Development. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

In the present study, we focus on dinuclear cobalt-based CO-RMs with the aim of elucidating their CO release mechanism, as well as to understand how structural changes targeted to modify the electronic properties of these compounds can modulate CO delivery. To this end, we specifically synthesized a set of phenyl-propargyl-based CO-RMs bearing -NO 2 , -H, and -OCH 3 as para-substituents (R) with varying mesomeric influence (M) and different heteroatoms (X = NH, O, or S) linking the propargyl tail and the aromatic ring. The effects of R and X in modulating CO release were assessed by using several experimental and computational techniques to obtain a coherent picture and to shed light on the stability and release properties of Co-based CO-RMs.

The benzoxaborinine scaffold, a homologue of benzoxaborole with an additional carbon atom in the boracycle, shows significant potential in developing new therapeutic agents. This study reports the synthesis, inhibition assays against four human carbonic anhydrase (hCA, EC 4.2.1.1) isoforms, and anti-melanoma evaluation of 7-aryl(thio)ureido-substituted benzoxaborinines. Some derivatives, particularly compound 11 , exhibited potent inhibitory activity (below 65 nM) against hCA IX and XII and stronger antiproliferative effects than SLC-0111 on human melanoma cells under hypoxia. Crystallographic studies of benzoxaborinine 3 adducts with hCA I and II demonstrated the binding mode of this chemotype, revealing that although both benzoxaborinine 3 and benzoxaborole 10 share a similar zinc-binding mode, the expanded ring in benzoxaborinine led to a different orientation within the active site. These findings suggest that benzoxaborinines hold promise for designing novel carbonic anhydrase inhibitors.

PCI-27483, originally developed as a potent and selective inhibitor of the serine protease Factor VIIa (FVIIa) in complex with tissue factor (TF), has demonstrated significant promise in cancer therapy. In addition to its primary mechanism of action, the presence of a sulfonamide moiety in the PCI-27483 structure suggests further activities through the inhibition of carbonic anhydrases (CAs), particularly the tumor-associated human (h)CA isoforms hCA IX and XII. This study investigates the inhibitory activity of PCI-27483 against the complete panel of active hCAs, highlighting its polypharmacological potential in cancer treatment. X-ray crystallography and molecular docking studies elucidated the structural features underlying its selective inhibitory activity toward hCA IX and XII, offering insights into its dual-targeting pathway., Competing Interests: The authors declare no competing financial interest., (© 2024 American Chemical Society.)

Novel chalcogen-containing amides and esters bearing the benzenesulfonamide moiety have been synthesised upon nucleophilic conjugate addition of thiols and selenols to suitable electron-deficient alkenes. The activity of the synthesised compounds as Carbonic Anhydrases inhibitors has been investigated in vitro and the inhibition mechanism has been elucidated by X-rays studies., (© 2024 Wiley-VCH GmbH.)

Tuberculosis (TB) is an infectious disease that primarily affects the lungs of humans and accounts for Mycobacterium tuberculosis (Mtb) bacteria as the etiologic agent. In this study, we introduce a computational framework designed to identify the important chemical features crucial for the effective inhibition of Mtb β-CAs. Through applying a mechanistic model, we elucidated the essential features pivotal for robust inhibition. Using this model, we engineered molecules that exhibit potent inhibitory activity and introduce relevant novel chemistry. The designed molecules were prioritized for synthesis based on their predicted pKi values via the QSAR (Quantitative Structure-Activity Relationship) model. All the rationally designed and synthesized compounds were evaluated in vitro against different carbonic anhydrase isoforms expressed from the pathogen Mtb; moreover, the off-target and widely human-expressed CA I and II were also evaluated. Among the reported derivatives, 2, 4, and 5 demonstrated the most valuable in vitro activity, resulting in promising candidates for the treatment of TB infection. All the synthesized molecules exhibited favorable pharmacokinetic and toxicological profiles based on in silico predictions. Docking analysis confirmed that the zinc-binding groups bind effectively into the catalytic triad of the Mtb β-Cas, supporting the in vitro outcomes with these binding interactions. Furthermore, molecules with good prediction accuracies according to previously established mechanistic and QSAR models were utilized to delve deeper into the realm of systems biology to understand their mechanism in combating tuberculotic pathogenesis. The results pointed to the key involvement of the compounds in modulating immune responses via NF-κβ1, SRC kinase, and TNF-α to modulate granuloma formation and clearance via T cells. This dual action, in which the pathogen's enzyme is inhibited while modulating the human immune machinery, represents a paradigm shift toward more effective and comprehensive treatment approaches for combating tuberculosis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

A series of thirteen cyclic sulfonyl guanidines were prepared and evaluated against tumor-associated human (h) carbonic anhydrase (CA, EC 4.2.1.1) isoforms hCA IX and hCA XII, as well as against off-target cytosolic isoforms hCA I and hCA II. The compounds reported here were generally inactive against both off-target isoforms (K I >100 μM), while all of them moderately inhibited both target isoforms hCA IX and XII in the submicromolar to micromolar ranges in which K I values spanned from 0.57 to 8.4 μM against hCA IX and from 0.34 to 9.7 against hCA XII. Due to the notable selectivity of the title compounds toward isoforms hCA IX and XII, they can be considered as useful scaffolds for further chemical optimization to develop new highly selective antitumor agents., (© 2024 Wiley-VCH GmbH.)

The present research focused on the tail-approach synthesis of novel extended thiazolotriazoles (8a-8j) and triazolothiadiazines (11a-11j) including aminotriazole intermediate 10. After successful synthesis, all the compounds were evaluated for their inhibition potential against cytosolic isoforms of human carbonic anhydrase (hCA I, II), tumor-linked transmembrane isoforms (hCA IX, XII), and cathepsin B. As per the inhibition data, the newly synthesized compounds showed poor inhibition against hCA I. Many of the compounds showed effective inhibition toward hCA IX and/or XII in low nanomolar concentration. Despite the strong to moderate inhibition of hCA II by these compounds, more than half of them demonstrated better inhibition against hCA IX and/or XII, comparatively. Further, insights of CA inhibition data of these extended analogs and their comparison with earlier reported thiazolotriazole and triazolothiadiazine derivatives might help in the rational design of novel potent and selective hCA IX and XII inhibitors. The novel compounds were also found to possess anti-cathepsin B potential at a low concentration of 10 -7  M. Broadly, compounds of series 11a-11j presented more effective inhibition against cathepsin B than their counterparts in series 8a-8j. Moreover, these in vitro results with respect to cathepsin B inhibition were also supported by the in silico insights obtained via molecular modeling studies., (© 2024 Deutsche Pharmazeutische Gesellschaft.)

The design and synthesis of a library of 21 novel benzenesulfonamide-bearing 3-functionalized pyrazole-linked 1,2,3-triazole derivatives as dual inhibitors of cathepsin B and carbonic anhydrase enzymes are reported. The target 1,2,3-triazole-linked pyrazolic esters (16) were synthesized by the condensation of 1,2,3-triazolic diketo esters with 4-hydrazinobenzenesulfonamide hydrochloride, and these were further converted into the corresponding carboxylic acid (17) and carboxamide (18) analogs. The synthesized compounds were assayed in vitro for their inhibition potential against human carbonic anhydrase (hCA) isoforms I, II, IX, and XII. They were found to be potent inhibitors at the low nanomolar level against the cancer-related hCA IX and XII and to be selective towards the cytosolic isoform hCA I. The physiologically important isoform hCA II was potently inhibited by all the newly synthesized compounds showing K I values ranging between 0.8 and 561.5 nM. The ester derivative 16c having 4-fluorophenyl (K I  = 5.2 nM) was the most potent inhibitor of hCA IX, and carboxamide derivative 18b (K I  = 2.2 nM) having 4-methyl substituted phenyl was the most potent inhibitor of hCA XII. The newly synthesized compounds exhibited potent cathepsin B inhibition at 10 -7  M concentration. In general, the carboxamide derivatives (18) showed higher % inhibition as compared with the corresponding ester derivatives (16) and carboxylic acid derivatives (17) for cathepsin B. The interactions of the target compounds with the active sites of cathepsin B and CA were studied through molecular docking studies. Further, the in silico absorption, distribution, metabolism, excretion, and toxicity (ADMET) and drug-likeness properties of the target compounds were also studied., (© 2024 Deutsche Pharmazeutische Gesellschaft.)

Lasamide is a synthetic precursor and a contaminant of the diuretic Furosemide manufacturing process and represents a highly valuable building block for fragment-based drug discovery approaches. We assessed the ability of Lasamide to inhibit in vitro the human-expressed Carbonic Anhydrases by means of the stopped-flow technique, and we assessed its binding modes within hCAs II and XII-mimic catalytic clefts by X-ray crystallography. Interestingly, an unprecedented crystal form for the hCA IX mimic H-tag is reported and discussed herein., Competing Interests: The authors declare no competing financial interest., (© 2024 American Chemical Society.)

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 CO 2 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., (Copyright © 2024 American Society for Pharmacology and Experimental Therapeutics.)

This study introduces a novel multitargeting strategy that combines carbonic anhydrase (CA) activators and cholinesterase (ChE) inhibitors to enhance cognitive functions. A series of tacrine-based derivatives with amine/amino acid moieties were synthesized and evaluated for their dual activity on brain CA isoforms and ChEs (AChE and BChE). Several derivatives, notably compounds 26 , 30 , 34 , and 40 , demonstrated potent CA activation, particularly of hCA II and VII, and strong ChE inhibition with subnanomolar to low nanomolar IC 50 values. In vivo studies using a mouse model of social recognition memory showed that these derivatives significantly improved memory consolidation at doses 10-100 times lower than the reference compounds (either alone or in combination). Molecular modeling and ADMET predictions elucidated the compound binding modes and confirmed favorable pharmacokinetic and safety profiles. The findings suggest that dual modulation of CA and ChE activities is a promising strategy for treating cognitive deficits associated with neurodegenerative and psychiatric disorders.