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BackgroundMycobacterium tuberculosis (Mtb) carbonic anhydrases (CAs) are critical enzymes that regulate pH by converting CO2 to HCO3−, essential for Mtb’s survival in acidic environments. Inhibiting γ-CAs presents a potential target for novel antituberculosis drugs with unique mechanisms of action.ObjectiveThis study aimed to explore the biological connections underlying Mtb pathogenesis and investigate the mechanistic actions of antituberculosis compounds targeting the Cas9 protein.MethodsWe employed homology modeling and virtual screening to identify compounds with high binding affinities for Cas9 protein. This study used the homology modeling approach employing high-quality AlphaFold DB models for γ-CA. Furthermore, the systems biology approach was used for analyzing the integrated modelling of compounds, integrating data on genes, pathways, phenotypes, and molecular descriptors. Single-cell RNA sequencing was also conducted to profile gene expression.ResultsThree compounds, F10921405, F08060425, and F14437079, potentially binding to Cas9 protein, have been identified. F10921405 and F08060425 showed significant overlap in their effects on pathways related to the immune response, while F14437079 displayed distinct mechanistic pathways. Expression profiling revealed high levels of genes such as PDE4D, ROCK2, ITK, MAPK10, and SYK in response to F1092–1405 and F0806-0425, and MMP2 and CALCRL in response to F1443-7079. These genes, which play a role in immune modulation and lung tissue integrity, are essential to fight against Mtb.ConclusionThe molecular relationship and pathways linked to the mentioned compounds give the study a holistic perspective of targeting Mtb, which is essential in designing specific therapeutic approaches. Subsequent research will involve experimental validation to demonstrate the efficacy of the promising candidates in Mtb infections.

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.

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.

AbstractA 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 CO2 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 KIs 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.

We evaluated the effects of dentine biomodification after pre-treatment with two sulphonamide carbonic anhydrase inhibitors (CAIs) of the N-[4-sulphamoylphenethylcarbamoyl]benzenesulphonamide type, investigating matrix metalloproteases activity, resin–dentine micro tensile bond strength, dentine surface wettability, and antimicrobial activities. Ninety-five sound-extracted human molars were selected for the study. Inhibitory effects were evaluated by gelatinase and collagenase activity tests and collagen degradation FT-IR spectroscopic analysis. Pre-treatment with the two CAIs kept the micro tensile values after 12 months of storage (32.23 ± 5.95) and cariogenic challenge (34.13 ± 2.71) similar to the initial, pre-treatment values (33.56 ± 4.34). A decreased Streptococcus mutans biofilm formation on dentine surfaces and antibacterial activity against planktonic bacteria were observed after CAI treatment. Dentine pre-treatment with sulphonamide CAIs maintained adhesion strength stability, allowed better dentine wettability, maintained matrix collagen, and showed anti-S. mutans activity.

The synthesis of 3H-1,2-benzoxaphosphepine 2-oxides and evaluation of their inhibitory activity against human carbonic anhydrase (hCA) isoforms I, II, IX, and XII are described. The target compounds were obtained via a concise synthesis from commercial salicylaldehydes and displayed low to sub-micromolar inhibition levels against the tumour-associated isoforms hCA IX and XII. All obtained benzoxaphosphepine 2-oxides possess remarkable selectivity for inhibition of hCA IX/XII over the off-target cytosolic hCA isoforms I and II, whose inhibition may lead to side effects.

A series of 1,2,3-benzoxathiazine-2,2-dioxides possessing various substituents in the 5, 7, or 8 position was obtained from corresponding 2-hydroxybenzaldehydes in their reaction with sulfamoyl chloride. 5-, 7-, and 8-aryl substituted 1,2,3-benzoxathiazine-2,2-dioxides were prepared from aryl substituted 2-hydroxybenzaldehydes obtained from 3-, 4-, or 6-bromo-2-hydroxybenzaldehydes via two-step protocol. 1,2,3-Benzoxathiazine-2,2-dioxides were investigated for the inhibition of four human carbonic anhydrase (hCA, EC 4.2.1.1) isoforms, cytosolic hCA I and II and tumour-associated transmembrane hCA IX and XII. Twenty four derivatives of 1,2,3-benzoxathiazine 2,2-dioxide were obtained. Most of them act as nanomolar inhibitors of hCA IX and XII. Almost all compounds except 2d and 5a-e also express nanomolar inhibitory activity for hCA II. hCA I is poorly inhibited or not inhibited by 1,2,3-benzoxathiazine 2,2-dioxides. Some of the new derivatives exhibit promising selectivity towards CA IX/XII over hCA I, although none of the compounds are selective towards CA IX/XII over both hCA I and II.

The present investigation reports the design and synthesis of three series of benzoylthioureido derivatives bearing either benzenesulfonamide 7a–f, benzoic acid 8a–f or ethylbenzoate 9a–f moieties. The synthesised compounds were screened for their carbonic anhydrase inhibitory activity (CAI) against four isoforms hCA I, II, IX, and XII. Compounds 7a, 7b, 7c, and 7f exhibited a potent inhibitory activity towards hCAI (Kis = 58.20, 56.30, 33.00, and 43.00 nM), respectively compared to acetazolamide (AAZ) and SLC-0111 (Kis = 250.00 and 5080.00 nM). Compounds 7a, 7b, 7c, 7e, and 7f elicited selectivity over h CA II (Kis = 2.50, 2.10, 56.60,39.60 and 39.00 nM) respectively, relative to AAZ and SLC-0111(Kis = 12.10 and 960.00 nM). Also, compounds 7c, 7f, and 9e displayed selectivity against the tumour-associated isoform hCA IX (Kis = 31.20, 30.00 and 29.00 nM) respectively, compared to AAZ and SLC-0111 (Kis = 25.70 and 45.00 nM). Additionally, compounds 8a and 8f revealed a moderate to superior selectivity towards hCAXII (Kis = 17.00 and 11.00 nM) relative to AAZ and SLC-0111(Kis = 5.70 and 45.00 nM). Molecular docking and ADME prediction studies were performed on the most active compounds to shed light on their interaction with the hot spots of the active site of CA isoforms, in addition to prediction of their pharmacokinetic and physicochemical properties.

A one-pot two-step protocol was developed for the synthesis of a series of novel 4-cyanamidobenzenesulfonamides from easily accessible methyl (4-sulfamoylphenyl)-carbamimidothioate. The new sulphonamides were investigated as inhibitors of the enzyme carbonic anhydrase (CA, EC 4.2.1.1), the human (h) cytosolic isoforms hCA I, II, VII, and XIII, as well as three bacterial enzymes belonging to the β-CA class, MscCA from Mammaliicoccus (Staphylococcus) sciuri and StCA1 and StCA2, from Salmonella enterica (serovar Typhimurium). The human isoforms were generally effectively inhibited by these compounds, with a clear structure-activity relationship privileging long aliphatic chains (C6, C7 and C18) as substituents at the cyanamide functionality. The bacterial CAs were also inhibited by these compounds, but not as effective as the hCAs. The most sensitive enzyme to these inhibitors was StCA1 (KIs of 50.7 − 91.1 nM) whereas SscCA was inhibited in the micromolar range (KIs of 0.86–9.59 µM).

Human Carbonic Anhydrases (hCA) are enzymes that contribute to cancer’s development and progression. Isoforms IX and XII have been identified as potential anticancer targets, and, more specifically, hCA IX is overexpressed in hypoxic tumor cells, where it plays an important role in reprogramming the metabolism. With the aim to find new inhibitors towards IX and XII isoforms, the hybridization of the privileged scaffolds isatin, dihydrothiazole, and benzenesulfonamide was investigated in order to explore how it may affect the activity and selectivity of the hCA isoforms. In this respect, a series of isatin thiazolidinone hybrids have been designed and synthesized and their biological activity and selectivity on hCA I, hCA II, hCA IX, and hCA XII explored. The new compounds exhibited promising inhibitory activity results on isoforms IX and XII in the nanomolar range, which has highlighted the importance of substituents in the isatin ring and in position 3 and 5 of thiazolidinone. In particular, compound 5g was the most active toward hCA IX, while 5f was the most potent inhibitor of hCA XII within the series. When both potency and selectivity were considered, compound 5f appeared as one of the most promising. Additionally, our investigations were supported by molecular docking experiments, which have highlighted the putative binding poses of the most promising compound.

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.

The emergence of tumor-associated human carbonic anhydrases (hCA) as promising therapeutic targets has urged rigorous research into the development of potent and selective hCA IX and XII inhibitors. Rationalization of targeting tumor-specific hCA isoforms is a major challenge that requires a comprehensive understanding of the interactions between inhibitors and the dynamic hCA active site. The benzenesulfonamides and its bioisosteres are currently being used clinically as inhibitors of various hCA isoforms through classical inhibitory mechanism. In addition, several other chemotypes have also been developed with improved potency and selectivity through non-classical inhibitory mechanisms. Coumarin and its derivatives represent highly selective and potent inhibitors of hCA IX and XII. Recently, various other pharmacophores were also proven to have a strong selectivity and potency against hCA IX and XII including pyrazole, 1,2,3-triazole, 4-thiazolidinone, and thiourea. This review navigates through understanding the role of hCA IX and XII in cancer biology, encompassing different inhibition approaches, strategic design methodologies, recent advancements in the rational design of hCA inhibitors, exploration of structure-activity relationships, in-depth mechanistic insights, and PET imaging applications for in vivo visualization of target enzymes.

Research into novel anti-Helicobacter pylori agents represents an important approach for the identification of new treatments for chronic gastritis and peptic ulcers, which are associated with a high risk of developing gastric carcinoma. In this respect, two series of azobenzenesulfonamides were designed, synthesized, and tested against a large panel of human and bacterial CAs to evaluate their inhibitory activity. In addition, computational studies of the novel primary benzenesulfonamides (4a–j) were performed to predict the putative binding mode to both HpCAs. Then, the antimicrobial activity versus H. pylori of the two series was also studied. The best-in-class compounds were found to be 4c and 4e among the primary azobenzenesulfonamides and 5c and 5f belonging to the secondary azobenzenesulfonamides series, showing themselves to exert a promising anti-H. pylori activity, with MIC values of 4–8 μg/mL and MBCs between 4 and 16 μg/mL. Moreover, the evaluation of their toxicity on a G. mellonella larva in vivo model indicated a safe profile for 4c,e and 5c,f. The collected results warrant considering these azobenzenesulfonamides as an interesting starting point for the development of a new class of anti-H. pylori agents.

A new series of piperazine derivatives were synthesized and studied with the aim of obtaining dual inhibitors of P-glycoprotein (P-gp) and carbonic anhydrase XII (hCA XII) to synergistically overcome the P-gp-mediated multidrug resistance (MDR) in cancer cells expressing the two proteins, P-gp and hCA XII. Indeed, these hybrid compounds contain both P-gp and hCA XII binding groups on the two nitrogen atoms of the heterocyclic ring. All compounds showed good inhibitory activity on each protein (P-gp and hCA XII) studied individually, and many of them showed a synergistic effect in the resistant HT29/DOX and A549/DOX cell lines which overexpress both the target proteins. In particular, compound 33 displayed the best activity by enhancing the cytotoxicity and intracellular accumulation of doxorubicin in HT29/DOX and A549/DOX cells, thus resulting as promising P-gp-mediated MDR reverser with a synergistic mechanism. Furthermore, compounds 13, 27 and 32 induced collateral sensitivity (CS) in MDR cells, as they were more cytotoxic in resistant cells than in the sensitive ones; their CS mechanisms were extensively investigated.

Abstract An optimization strategy was adopted for designing and synthesizing new series of 2-oxindole conjugates. Selected compounds were evaluated for their antiproliferative effect in vitro against NCI-60 cell lines panel, inhibitory effect on carbonic anhydrase (CA) isoforms (hCAI, II, IX and XII), and protein kinases. Compounds 5 and 7 showed promising inhibitory effects on hCA XII, whereas compound 4d was the most potent inhibitor with low nanomolar CA inhibition against all tested isoforms. These results were rationalized by using molecular docking. Despite its lack of CA inhibitory activity, compound 15c was the most active antiproliferative candidate against most of the 60 cell lines with mean growth inhibition 61.83% and with IC50 values of 4.39, 1.06, and 0.34 nM against MCT-7, DU 145, and HCT-116 cell lines, respectively. To uncover the mechanism of action behind its antiproliferative activity, compound 15c was assessed against a panel of protein kinases (RET, KIT, cMet, VEGFR1,2, FGFR1, PDFGR and BRAF) showing % inhibition of 74%, 31%, 62%, 40%, 73%, 74%, 59%, and 69%, respectively, and IC50 of 1.287, 0.117 and 1.185 μM against FGFR1, VEGFR, and RET kinases, respectively. These results were also explained through molecular docking.

It has been known for some time that Carbonic Anhydrase (CA, EC 4.2.1.1) plays a complex role in vascular function, and in the regulation of vascular tone. Clinically employed CA inhibitors (CAIs) are used primarily to lower intraocular pressure in glaucoma, and also to affect retinal blood flow and oxygen saturation. CAIs have been shown to dilate vessels and increase blood flow in both the cerebral and ocular vasculature. Similar effects of CAIs on vascular function have been observed in the liver, brain and kidney, while vessels in abdominal muscle and the stomach are unaffected. Most of the studies on the vascular effects of CAIs have been focused on the cerebral and ocular vasculatures, and in particular the retinal vasculature, where vasodilation of its vessels, after intravenous infusion of sulfonamide-based CAIs can be easily observed and measured from the fundus of the eye. The mechanism by which CAIs exert their effects on the vasculature is still unclear, but the classic sulfonamide-based inhibitors have been found to directly dilate isolated vessel segments when applied to the extracellular fluid. Modification of the structure of CAI compounds affects their efficacy and potency as vasodilators. CAIs of the coumarin type, which generally are less effective in inhibiting the catalytically dominant isoform hCA II and unable to accept NO, have comparable vasodilatory effects as the primary sulfonamides on pre-contracted retinal arteriolar vessel segments, providing insights into which CA isoforms are involved. Alterations of the lipophilicity of CAI compounds affect their potency as vasodilators, and CAIs that are membrane impermeant do not act as vasodilators of isolated vessel segments. Experiments with CAIs, that shed light on the role of CA in the regulation of vascular tone of vessels, will be discussed in this review. The role of CA in vascular function will be discussed, with specific emphasis on findings with the effects of CA inhibitors (CAI).

Carbonic anhydrases (CAs) from the pathogenic bacteria Nesseria gonorrhoeae and vancomycin-resistant enterococci (VRE) have recently been validated as antibacterial drug targets. Here we explored the inhibition of the α-CA from N. gonorrhoeae (α-NgCA), of α- and γ-class enzymes from Enterococcus faecium (α-EfCA and γ-EfCA) with a panel of aliphatic, heterocyclic and aryl-alkyl primary/secondary monothiocarbamates (MTCs). α-NgCA was inhibited in vitro with KIs ranging from 0.367 to 0.919 µM. The compounds inhibited the α-EfCA and γ-EfCA with KI ranges of 0.195–0.959 µM and of 0.149–1.90 µM, respectively. Some MTCs were also investigated for their inhibitory effects on the growth of clinically-relevant N. gonorrhoeae and VRE strains. No inhibitory effects on the growth of VRE were noted for all MTCs, whereas one compound (13) inhibited the growth N. gonorrhoeae strains at concentrations ranging from 16 to 64 µg/mL. This suggests that compound 13 may be a potential antibacterial agent against N. gonorrhoeae.

In this study, The inhibitory actions of human carbonic anhydrase (CA, EC 4.2.1.1) (hCA) isoforms I, II, IX, and XII are being examined using recently synthesized substituted hydroxyl Schiff derivatives based on the quinazoline scaffold 4–22. Quinazolines 2, 3, 4, 5, 7, 10, 15, and 18 reduce the activity of hCA I isoform effectively to a Ki of 87.6–692.3 nM, which is nearly equivalent to or more potent than that of the standard drug AAZ (Ki, 250.0 nM). Similarly, quinazolines 2, 3, and 5 and quinazoline 14 effectively decrease the inhibitory activity of the hCA II isoform to a KI of 16.9–29.7 nM, comparable to that of AAZ (Ki, 12.0 nM). The hCA IX isoform activity is substantially diminished by quinazolines 2–12 and 14–21 (Ki, 8.9–88.3 nM against AAZ (Ki, 25.0 nM). Further, the activity of the hCA XII isoform is markedly inhibited by the quinazolines 3, 5, 7, 14, and 16 (Ki, 5.4–19.5 nM). Significant selectivity levels are demonstrated for inhibiting tumour-associated isoforms hCA IX over hCAI, for sulfonamide derivatives 6–15 (SI; 10.68–186.29), and 17–22 (SI; 12.52–57.65) compared to AAZ (SI; 10.0). Sulfonamide derivatives 4–22 (SI; 0.50–20.77) demonstrated a unique selectivity in the concurrent inhibition of hCA IX over hCA II compared to AAZ (SI; 0.48). Simultaneously, benzenesulfonamide derivative 14 revealed excellent selectivity for inhibiting hCA XII over hCA I (SI; 60.35), whereas compounds 5–8, 12–14, 16, and 18–22 demonstrated remarkable selectivity for hCA XII inhibitory activity over hCA II (SI; 2.09–7.27) compared to AAZ (SI; 43.86 and 2.10, respectively). Molecular docking studies additionally support 8 to hCA IX and XII binding, thus indicating its potential as a lead compound for inhibitor development.

Tumour associated carbonic anhydrases (CAs) IX and XII have been recognised as potential targets for the treatment of hypoxic tumours. Therefore, considering the high pharmacological potential of the chromene scaffold as selective ligand of the IX and XII isoforms, two libraries of compounds, namely 2H-chromene and 7H-furo-chromene derivatives, with diverse substitution patterns were designed and synthesised. The structure of the newly synthesised compounds was characterised and their inhibitory potency and selectivity towards human CA off target isoforms I, II and cancer-associated CA isoforms IX and XII were evaluated. Most of the compounds inhibit CA isoforms IX and XII with no activity against the I and II isozymes. Thus, while the potency was influenced by the substitution pattern along the chromene scaffold, the selectivity was conserved along the series, confirming the high potential of both 2H-chromene and 7H-furo-chromene scaffolds for the design of isozyme selective inhibitors.

A novel library of human carbonic anhydrase (hCA) inhibitors based on the 2-sulfanilamido[1,2,4]triazolo[1,5-a]pyrimidine skeleton modified at its 7-position was prepared by an efficient convergent procedure. These derivatives were evaluated in vitro for their inhibition properties against a representative panel of hCA isoforms (hCA I, II, IV, IX, and XII). The target tumour-associated isoforms hCA IX and XII were potently inhibited with KIs in the low nanomolar range of 5–96 nM and 4–72 nM, respectively. Compounds 1d, 1j, 1v, and 1x were the most potent hCA IX inhibitors with KIs of 5.1, 8.6, 4.7, and 5.1 nM, respectively. Along with derivatives 1d and 1j, compounds 1r and 1ab potently inhibited hCA XII isoform with KIs in a single-digit nanomolar range of 8.8, 5.4, 4.3, and 9.0 nM, respectively. Compounds 1e, 1m, and 1p exhibited the best selectivity against hCA IX and hCA XII isoforms over off-target hCA II, with selectivity indexes ranging from 5 to 14.

A range of 3H-1,2-benzoxaphosphepine 2-oxide aryl derivatives with various substitution patterns at positions 7, 8, or 9 of the scaffold was synthesised in five steps from the commercially available salicylaldehydes. All of the newly obtained compounds were studied for their inhibition potency against carbonic anhydrase (CA) isoforms I, II, IX, and XII. Delightfully, these compounds showed a striking selectivity for the cancer-associated CA IX and XII over the cytosolic CA I and II, whose inhibition may lead to side-effects. Overall, a structure–activity relationship (SAR) revealed that 7- and 8-substituted aryl derivatives were more effective inhibitors of CA IX and XII than 9-substituted derivatives. In addition, the fluorine-containing analogues emerged as the most potent CA IX/XII inhibitors in this series.

A series of phthalimide-capped benzene sulphonamides (1–22) reported by our group for dengue protease inhibitory activity have been evaluated for their carbonic anhydrase (hCA, EC 4.2.1.1) inhibitory activity against hCA I, hCA II. Compounds 1, 3, 10, and 15 showed hCA I inhibition, whereas 1, 4, and 10 showed hCA II inhibition at nanomolar concentrations. Among these compounds, 1 displayed potent inhibitory activity against the hCA I (Ki = 28.5 nM) and hCA II (Ki = 2.2 nM), being 10 and 6 times more potent than acetazolamide, a standard inhibitor (Ki = 250 nM and 12 nM), respectively. Furthermore, this compound displayed 14-fold selectivity towards the hCA II isoform compared to hCA I. Molecular docking and MD simulations were performed to understand the atomic level interactions responsible for the selectivity of compound 1 towards hCA II.

SARS-CoV-2 pandemic in the end of 2019 led to profound consequences on global health and economy. Till producing successful vaccination strategies, the healthcare sectors suffered from the lack of effective therapeutic agents that could control the spread of infection. Thus, academia and the pharmaceutical sector prioritise SARS-CoV-2 antiviral drug discovery. Here, we exploited previous reports highlighting the anti-SARS-CoV-2 activities of isatin-based molecules to develop novel triazolo-isatins for inhibiting main protease (Mpro) of the virus, a crucial enzyme for its replication in the host cells. Particularly, sulphonamide 6b showed promising inhibitory activity with an IC50= 0.249 µM. Additionally, 6b inhibited viral cell proliferation with an IC50 of 4.33 µg/ml, and was non-toxic to VERO-E6 cells (CC50 = 564.74 µg/ml) displaying a selectivity index of 130.4. In silico analysis of 6b disclosed its ability to interact with key residues in the enzyme active site, supporting the obtained in vitro findings.

In this work, new isatin-based sulphonamides (6a-i, 11a-c, 12a-c) were designed and synthesised as potential dual VEGFR-2 and carbonic anhydrase inhibitors with anticancer activities. Firstly, all target isatins were examined for in vitro antitumor action on NCI-USA panel (58 tumour cell lines). Then, the most potent derivatives were examined for the potential CA inhibitory action towards the physiologically relevant hCA isoforms I, II, and tumour-linked hCA IX isoform, in addition, the VEGFR-2 inhibitory activity was evaluated. The target sulphonamides failed to inhibit the CA isoforms that could be attributable to the steric effect of the neighbouring methoxy group, whereas they displayed potent VEGFR-2 inhibitory effect. Following that, isatins 11b and 12b were tested for their influence on the cell cycle disturbance, and towards the apoptotic potential. Finally, detailed molecular modelling analyses, including docking and molecular dynamics, were carried out to assess the binding mode and stability of target isatins.

Considering the unrecognised physio-pathological role of human carbonic anhydrase III (hCA III), a structure-based drug design was set up to identify the first-in-class potent and selective inhibitors of this neglected isoform. hCA III targeting was planned considering a unique feature of its active site among the other hCA isoforms, i.e. the Leu198/Phe198 substitution which interferes with the binding of aromatic/heterocyclic sulfonamides and other inhibitors. Thus, new aliphatic primary sulfonamides possessing long and flexible (CH2)nSO2NH2 moieties were designed to coordinate the zinc(II) ion, bypassing the bulky Phe198 residue. They incorporate 1,2,3-triazole linkers which connect the tail moieties to the sulfonamide head, enhancing thus the contacts at the active site entrance. Some of these compounds act as nanomolar and selective inhibitors of hCA III over other isoforms. Docking/molecular dynamics simulations were used to investigate ligand/target interactions for these sulfonamides which might improve our understanding of the physio-pathological roles of hCA III.

Multiple inhibitions of CA, COX-2 and 5-LOX enzymes has been recognised as a useful strategy for the development of anti-inflammatory drugs that can avoid the disadvantages of using NSAIDs alone. Here, we report new pyridazine-based sulphonamides (5a-c and 7a-f) as potential multi-target anti-inflammatory candidates. First, the furanone heterocycle in the dual CA/COX-2 inhibitor Polmacoxib was replaced with the pyridazinone one. Then, a hydrophobic tail was appended through benzylation of the 3-hydroxyl group of the pyridazinone scaffold to afford benzyloxy pyridazines 5a-c. Furthermore, the structures were adorned with the polar sulphonate functionality, in pyridazine sulphonates 7a-f, that are expected to be engaged in interactions with the hydrophilic half of the CA binding sites. All of the disclosed pyridazinones were tested for inhibitory activities against 4 hCA isoforms (I, II, IX, and XII), as well as against COX-1/2, and 5-LOX. Furthermore, in vivo anti-inflammatory and analgesic effects of pyridazinones 7a and 7b were examined.

Design and synthesis of three novel series of aryl enaminones (3a–f and 5a–c) and pyrazole (4a-c) linked compounds with sulphonamides, sulfaguanidine, or carboxylic acid functionalities were reported as carbonic anhydrase inhibitors (CAIs) using the “tail approach” strategy in their design to achieve the most variable amino acids in the middle/outer rims of the hCAs active site. The synthesised compounds were assessed in vitro for their inhibitory activity against the following human (h) isoforms, hCA I, II, IX, and XII using stopped-flow CO2 hydrase assay. Enaminone sulphonamide derivatives (3a–c) potently inhibited the target tumour-associated isoforms hCA IX and hCA XII (KIs 26.2–63.7 nM) and hence compounds 3a and 3c were further screened for their in vitro cytotoxic activity against MCF-7 and MDA-MB-231 cancer cell lines under normoxic and hypoxic conditions. Derivative 3c showed comparable potency against both MCF-7 and MDA-MB-231 cancer cell lines under both normoxic ((IC50 = 4.918 and 12.27 µM, respectively) and hypoxic (IC50 = 1.689 and 5.898 µM, respectively) conditions compared to the reference drug doxorubicin under normoxic (IC50 = 3.386 and 4.269 µM, respectively) and hypoxic conditions (IC50 = 1.368 and 2.62 µM, respectively). Cell cycle analysis and Annexin V-FITC and propidium iodide double staining methods were performed to reinforce the assumption that 3c may act as a cytotoxic agent through the induction of apoptosis in MCF-7 cancer cells.

Vibrio cholerae causes life-threatening infections in low-income countries due to the rise of antibacterial resistance. Innovative pharmacological targets have been investigated and carbonic anhydrases (CAs, EC: 4.2.1.1) encoded by V. cholerae (VchCAs) emerged as a valuable option. Recently, we developed a large library of para- and meta-benzenesulfonamides characterised by moieties with a different flexibility degree as CAs inhibitors. Stopped flow-based enzymatic assays showed strong inhibition of VchαCA for this library, while lower affinity was detected against the other isoforms. In particular, cyclic urea 9c emerged for a nanomolar inhibition of VchαCA (KI = 4.7 nM) and high selectivity with respect to human isoenzymes (SI≥ 90). Computational studies revealed the influence of moiety flexibility on inhibitory activity and isoform selectivity and allowed accurate SARs. However, although VchCAs are involved in the bacterium virulence and not in its survival, we evaluated the antibacterial activity of such compounds, resulting in no direct activity.

A series of 20 newly designed (E)-1-(4-sulphamoylphenylethyl)-3-arylidene-5-aryl-1H-pyrrol-2(3H)-ones was synthesised and assessed as carbonic anhydrase (CA, EC 4.2.1.1) inhibitors towards four human isoforms of pharmaceutical interest, that is, hCA I, II, IX and XII. The compounds displayed low to high nanomolar potency against all the isoforms. Introducing strong electron withdrawing groups at the para position of the arylidene ring increased the binding affinity to the enzyme. All compounds showed acceptable pharmacokinetic range and physicochemical characteristics as determined by computational ADMET analysis. Density Functional Theory (DFT) calculations of 3n were carried to gain understanding on the stability of the E and Z isomers. The energy values clearly indicate the stability of E isomer over Z isomer by −8.2 kJ mol−1. Our findings indicate that these molecules are useful as leads for discovering new CA inhibitors.

As part of our ongoing endeavour to identify novel inhibitors of cancer-associated CA isoforms IX and XII as possible anticancer candidates, here we describe the design and synthesis of small library of 2-aryl-quinazolin-4-yl aminobenzoic acid derivatives (6a–c, 7a–c, and 8a–c) as new non-classical CA inhibitors. On account of its significance in the anticancer drug discovery and in the development of effective CAIs, the 4-anilinoquinazoline privileged scaffold was exploited in this study. Thereafter, the free carboxylic acid functionality was appended in the ortho (6a–c), meta (7a–c), or para-positon (8a–c) of the anilino motif to furnish the target inhibitors. All compounds were assessed for their inhibitory activities against the hCA I, II (cytosolic), IX, and XII (trans-membrane, tumour-associated) isoforms. Moreover, six quinazolines (6a–c, 7b, and 8a–b) were chosen by the NCI-USA for in vitro anti-proliferative activity evaluation against 59 human cancer cell lines representing nine tumour subpanels.

In the relentless search for new cancer treatments, organoselenium compounds, and carbonic anhydrase (CA) inhibitors have emerged as promising drug candidates. CA isoforms IX and XII are overexpressed in many types of cancer, and their inhibition is associated with potent antitumor/antimetastatic effects. Selenium-containing compounds, particularly selenols, have been shown to inhibit tumour-associated CA isoforms in the nanomolar range since the properties of the selenium atom favour binding to the active site of the enzyme. In this work, two series of selenoesters (1a–19a and 1b–19b), which gathered NSAIDs, carbo/heterocycles, and fragments from natural products, were evaluated against hCA I, II, IX, and XII. Indomethacin (17b) and flufenamic acid (19b) analogs exhibited selectivity for tumour-associated isoform IX in the low micromolar range. In summary, selenoesters that combine NSAIDs with fragments derived from natural sources have been developed as promising nonclassical inhibitors of the tumour-associated CA isoforms.

Carbonic anhydrases (CAs) are important regulators of pH homeostasis and participate in many physiological and pathological processes. CA activators (CAAs) are becoming increasingly important in the biomedical field since enhancing CA activity may have beneficial effects at neurological level. Here, we investigate selected antihistamines, phenothiazine-based antipsychotics, and tricyclic antidepressants (TCAs) as potential activators of human CAs I, II, IV, and VII. Our findings indicate that these compounds are more effective at activating hCA II and VII compared to hCA I and IV. Overall, hCA VII was the most efficiently activated isoform, particularly by phenothiazines and TCAs. This is especially relevant since hCA VII is the most abundant isoform in the central nervous system (CNS) and is implicated in neuronal signalling and bicarbonate balance regulation. This study offers additional insights into the pharmacological profiles of clinically employed drugs and sets the ground for the development of novel optimised CAAs.

The Carbonic anhydrase isoforms IX and XII play a significant role in regulating the intracellular and extracellular pH in hypoxic tumours abetting the metastasis of solid tumours. Selective and potent inhibitors targeting carbonic anhydrase IX and XII reduce the activity of these isoforms in hypoxic tumours, representing an antitumor and antimetastatic mechanism. Coumarin-based derivatives are selective inhibitors of CA isoforms IX and XII. In this study, we report the design and synthesis of new 3-substituted coumarin derivatives with different functional moieties and their inhibitory activity against various carbonic anhydrase isoforms. We found that the tertiary sulphonamide derivative 6c showed selective inhibition against CA IX with IC50 of 4.1 µM. Similarly, the carbothioamides 7c, 7b and oxime ether derivative 20a exhibited good inhibition against CA IX and CA XII. Additionally, the binding mode was predicted and validated using molecular docking and dynamic simulations.

A novel series of twelve aromatic bis-ureido-substituted benzenesulfonamides was synthesised by conjugation of aromatic aminobenzenesulfonamides with aromatic bis-isocyanates. The obtained bis-ureido-substituted derivatives were tested against four selected human carbonic anhydrase isoforms (hCA I, hCA II, hCA IX and hCA XII). Most of the new compounds showed an effective inhibitory profile against isoforms hCA IX and hCA XII, also having some selectivity with respect to hCA I and hCA II. The inhibition constants of these compounds against isoforms hCA IX and XII were in the range of 6.73–835 and 5.02–429 nM, respectively. Since hCA IX and hCA XII are important drug targets for anti-cancer/anti-metastatic drugs, these effective inhibitors reported here may be considered of interest for cancer related studies in which these enzymes are involved.

Schistosoma mansoni is an intestinal parasite with one β-class carbonic anhydrase, SmaBCA. We report the sequence enhancing, production, catalytic activity, and inhibition results of the recombinant SmaBCA. It showed significant catalytic activity on CO2 hydration in vitro with kcat 1.38 × 105 s−1 and kcat/Km 2.33 × 107 M−1 s−1. Several sulphonamide inhibitors, from which many are clinically used, showed submicromolar or nanomolar inhibitory effects on SmaBCA. The most efficient inhibitor with a KI of 43.8 nM was 4-(2-amino-pyrimidine-4-yl)-benzenesulfonamide. Other effective inhibitors with KIs in the range of 79.4–95.9 nM were benzolamide, brinzolamide, topiramate, dorzolamide, saccharin, epacadostat, celecoxib, and famotidine. The other tested compounds showed at least micromolar range inhibition against SmaBCA. Our results introduce SmaBCA as a novel target for drug development against schistosomiasis, a highly prevalent parasitic disease.

Carbonic anhydrases (CAs) are a zinc metalloenzymes that catalyse the reversible hydration of carbon dioxide to bicarbonate and proton, pivotal for a wide range of biological processes. CAs are involved in numerous pathologies and thus represent valuable drug targets in the treatments of several diseases such as glaucoma, obesity, tumour, neuropathic pain, cerebral ischaemia, or as antiinfectives. In the last two decades, several efforts have been made to achieve selective CA inhibitors (CAIs) employing different drug design approaches. However, N-substitutions on primary sulphonamide groups still remain poorly investigated. Here, we reported for the first time the co-crystallisation of a N-nitro sulphonamide derivative with human (h) CA II pointing out the binding site and mode of inhibition of this class of CAIs. The thorough comprehension of the ligand/target interaction might be valuable for a further CAI optimisation for achieving new potent and selective derivatives.

A small library of substituted cyclic guanidine incorporated benzothiazole-6-sulphonamides was synthesized. All obtained compounds were investigated for their inhibitory activity against the key brain-associated human carbonic anhydrase isoform hCA VII (a promising target for the treatment of neuropathic pain) and three isoforms expressed in brain and other tissues, hCA I, II, and IV. Sulphaguanidine derivatives 9a–d were inactive on the all investigated isoforms while the primary sulphonamide containing guanidines 6a–c and 7a–c were inactive towards hCA IV but displayed inhibiting properties on hCA I, II, and VII with KIs values in the low nanomolar to micromolar ranges. The results indicated that isoforms hCA II and VII were potently and selectively inhibited by these compounds, whereas the cytosolic hCA I was less sensitive to inhibition. The derivatives reported in this study might be useful for design of more potent and selective inhibitors of hCA II and VII.

A series of 6-ureido/amidocoumarins (5a–p and 7a–c) has been designed and synthesised to develop potent and isoform- selective carbonic anhydrase hCA XI and XII inhibitors. All coumarin derivatives were investigated for their CA inhibitory effect against hCA I, II, IX, and XII. Interestingly, target coumarins potently inhibited both tumour-related isoforms hCA IX (KIs: 14.7–82.4 nM) and hCA XII (KIs: 5.9–95.1 nM), whereas the cytosolic off-target hCA I and II isoforms have not inhibited by all tested coumarins up to 100 μM. These findings granted the target coumarins an excellent selectivity profile towards both hCA IX and hCA XII isoforms, supporting their development as promising anticancer candidates. Moreover, all target molecules were evaluated for their anticancer activities against HCT-116 and MCF-7 cancer cells. The 3,5-bis-trifluoromethylphenyl ureidocoumarin 5i, exerted the best anticancer activity. Overall, ureidocoumarins, particularly compound 5i, could serve as a promising prototype for the development of potent anticancer CAIs.

A series of 4-methyl-1,2,3-benzoxathiazine-2,2-dioxides with various substituents in 5, 6 or 7 positions was obtained from corresponding 2’-hydroxyacetophenones in their reaction with sulphamoyl chloride. 6- and 7-aryl substituted 4-methyl-1,2,3-benzoxathiazine-2,2-dioxides were obtained from aryl substituted 2’-hydroxyacetophenonesprepared from 4- or 5-bromo-2’-hydroxyacetophenones via two-step protocol. 4-Methyl-1,2,3-benzoxathiazine-2,2-dioxides were investigated as inhibitors of four human (h) carbonic anhydrase (hCA, EC 4.2.1.1) isoforms, off-target cytosolic hCA I and II, and target transmembrane, tumour-associated hCA IX and XII. Twenty derivatives of 4-methyl-1,2,3-benzoxathiazine 2,2-dioxide were obtained. With one exception (compound2a), they mostly act as nanomolar inhibitors of target hCA IX and XII. Basically, all screened compounds express none or low inhibitory properties towards off-target hCA I. hCA II is inhibited in micromolar range. Overwhelming majority of 4-methyl-1,2,3-benzoxathiazine 2,2-dioxides express excellent selectivity towards CA IX/XII over hCA I as well as very good selectivity towards CA IX/XII over hCA II.

The γ-carbonic anhydrase (CA, EC 4.2.1.1) from the pathogenic bacterium, Mammaliicoccus (Staphylococcus) sciuri (MscCAγ) was recently cloned and purified by our groups. Here we investigated inhibition of this enzyme with (in)organic simple and complex anions, in the search of inhibitors with potential applications. The most effective inhibitors (KIs in the micromolar range) were peroxydisulfate and trithiocarbonate, whereas submillimolar inhibition was observed with N,N-diethyldithiocarbamate and phenylboronic acid (KIs of 0.5–0.9 mM). Thiocyanate, hydrogensulfide, bisulphite, stannate, divanadate, tetraborate, perrhenate, perruthenate, hexafluorophosphate, triflate and iminodisulfonate showed KIs of 1.0–13.7 mM. Cyanate, cyanide, azide, carbonate, nitrate, tellurate, selenocyanide, tetrafluoroborate, sulfamide, sulphamic acid and phenylarsonic acid were weaker inhibitors, with KIs in the range of 25.2–95.5 mM, whereas halides, bicarbonate, nitrite, sulphate, perchlorate and fluorosulfonate did not show inhibitory action up until 100 mM concentrations in the assay system. Finding more effective MscCAγ inhibitors may be helpful to fight drug resistance to antibiotics.

A β-class carbonic anhydrase (CA, EC 4.2.1.1) present in the genome of the Monogenean platyhelminth Gyrodactylus salaris, a fish parasite, GsaCAβ, has been investigated for its inhibitory effects with a panel of sulphonamides and sulfamates, some of which in clinical use. Several effective GsaCAβ inhibitors were identified, belonging to simple heterocyclic sulphonamides, the deacetylated precursors of acetazolamide and methazolamide (KIsof 81.9–139.7 nM). Many other simple benezene sulphonamides and clinically used agents, such as acetazolamide, methazolamide, ethoxzolamide, dorzolamide, benzolamide, sulthiame and hydrochlorothiazide showed inhibition constants

Click chemistry reactions constitute an important and relatively new approach in the medicinal chemistry toolbox and offer substantial advantages to medicinal chemists in terms of overcoming the limitations of facile chemical synthesis, increased throughput, yields and improved quality of compound libraries. Over the last two decades, click chemistry has been widely used in different aspects of carbonic anhydrase (CA, EC 4.2.1.1) modulators drug design. This review provides an overview of the general principles and applications of click chemistry in CA-related research, including the design of selective CA inhibitors (CAIs), their applications against several diseases such as innovative anticancer and anti-infective agents, fluorescent and radiopharmaceutical probes as new theranostic agents, as well as their application in protein labelling.

A library of novel alkyl/benzyl (4-sulphamoylphenyl)carbamimidothioates was synthesised by selective S-alkylation of the easily accessible 4-thioureidobenzenesulphonamide. The compounds were assayed as inhibitors of four human (h) carbonic anhydrase isoforms hCA I, II, VII, and XIII, as well as three bacterial enzymes belonging to the β-CA class, MscCA from Mammaliicoccus (Staphylococcus) sciuri and StCA1 and StCA2, from Salmonella enterica (serovar Typhimurium). Most compounds investigated here exhibited moderate to low nanomolar inhibition constants against hCA I, II, and VII. The cytosolic hCA XIII was also inhibited by these compounds, but not as effective as hCA I, II, and VII. Several compounds were very effective against MscCA and StCA1. StCA2 was less inhibited compared to MscCA and StCA1. Some compounds showed considerable selectivity for inhibiting some CA isoforms. They may thus be considered as interesting starting points for the discovery and development of novel therapeutic agents belonging to this class of enzyme inhibitors.

A small library of novel thiazolone-benzenesulphonamides has been prepared and evaluated for their ability to inhibit three human cytosolic carbonic anhydrases (hCA I, hCA II, and hCA VII) and three bacterial carbonic anhydrases (MscCAβ, StCA1, and StCA2). All investigated hCAs were inhibited by the prepared compounds 4a–4j in the low nanomolar range. These compounds were effective hCA I inhibitors (KIs of 31.5–637.3 nM) and excellent hCA II (KIs in the range of 1.3–13.7 nM) and hCA VII inhibitors (KIs in the range of 0.9–14.6 nM). The most active analog in the series, 4-((4-oxo-5-propyl-4,5-dihydrothiazol-2-yl)amino)benzenesulphonamide 4d, strongly inhibited bacterial MscCAβ, with KI of 73.6 nM, considerably better than AAZ (KI of 625 nM). The tested compounds displayed medium inhibitory potency against StCA1 (KIs of 69.2–163.3 nM) when compared to the standard drug (KI of 59 nM). However, StCA2 was poorly inhibited by the sulphonamides reported here, with KIs in the micromolar range between 275.2 and 4875.0 nM.

Natural products and analogues are a source of antibacterial drug discovery. Considering drug resistance levels emerging for antibiotics, identification of bacterial metalloenzymes and the synthesis of selective inhibitors are interesting for antibacterial agent development. Peptide nucleic acids are attractive antisense and antigene agents representing a novel strategy to target pathogens due to their unique mechanism of action. Antisense inhibition and development of antisense peptide nucleic acids is a new approach to antibacterial agents. Due to the increased resistance of biofilms to antibiotics, alternative therapeutic options are necessary. To develop antimicrobial strategies, optimised in vitro and in vivo models are needed. In vivo models to study biofilm-related respiratory infections, device-related infections: ventilator-associated pneumonia, tissue-related infections: chronic infection models based on alginate or agar beads, methods to battle biofilm-related infections are discussed. Drug delivery in case of antibacterials often is a serious issue therefore this review includes overview of drug delivery nanosystems.

Carbonic anhydrases (CAs) are widespread metalloenzymes which catalyse the reversible hydration of carbon dioxide (CO2) to bicarbonate (HCO3−) and a proton, relevant in many physiological processes. In the last few years, the involvement of CA activation in different metabolic pathways in the human brain addressed the research to the discovery of novel CA activators. Here, a new series of isoxazoline-based amino alcohols as CA activators was investigated. The synthesis and the CA activating effects towards four human CA isoforms expressed in the human brain, that are hCAs I, II, IV and VII, were reported. The best results were obtained for the (methyl)-isoxazoline-amino alcohols 3 and 5 with KA values in the submicromolar range (0.52–0.86 µM) towards hCA VII, and a good selectivity over hCA I. Being hCA VII involved in brain function and metabolism, the newly identified CA activators might be promising hit compounds with potential therapeutic applications in ageing, epilepsy or neurodegeneration.

While the carbonic anhydrase (CA, EC 4.2.1.1) superfamily of enzymes has been described primarily as involved only in pH regulation for decades, it also has many other important functions. CO2, bicarbonate, and protons, the physiological substrates of CA, are indeed the main buffering system in organisms belonging to all life kingdoms; however, in the last period, relevant progress has been made in the direction of elucidating the involvement of the eight genetically distinct CA families in chemical sensing, metabolism, and several other crucial physiological processes. Interference with CA activity, both by inhibiting and activating these enzymes, has thus led to novel applications for CA inhibitors and activators in the field of innovative biomedicine and environment and health. In this perspective article, I will discuss the recent advances which have allowed for a deeper understanding of the biochemistry of these versatile enzymes and various applications of their modulators of activity.

Vancomycin-resistant enterococci (VRE), consisting of pathogenic Enterococcus faecalis and E. faecium, is a leading cause of hospital-acquired infections (HAIs). We recently repurposed the FDA-approved human carbonic anhydrase (CA) inhibitor acetazolamide (AZM) against VRE agent with the likely mechanism of action for the molecules being inhibition of one, or both, of the bacterial CA isoforms expressed in VRE. To elucidate how inhibitor binding to the enzymes relates to MIC, we further characterised the inhibition constants (Ki) against the E. faecium α-CA (Efα-CA) and γ-CA (Efγ-CA), as well as against human CA I (hCAI) and human CA II (hCAII) to assess selectivity. We have also utilised homology modelling and molecular dynamics (MD) simulations to gain a better understanding of the potential interactions the molecules are making with the targets. In this paper, we elaborate on the SAR for the AZM analogs as it pertains to MIC and Ki for each CA.