Α-carbonic Anhydrase Research Articles

Cyclization of acyl thiosemicarbazides led to new Helicobacter pylori α-carbonic anhydrase inhibitors.

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 concentrationand minimal bactericidal concentration values of the antibiotic, with no synergistic effect.

A cargo sorting receptor mediates chloroplast protein trafficking through the secretory pathway.

Nucleus-encoded chloroplast proteins can be transported via the secretory pathway. The molecular mechanisms underlying the trafficking of chloroplast proteins between the intracellular compartments are largely unclear, and a cargo sorting receptor has not previously been identified in the secretory pathway. Here, we report a cargo sorting receptor that is specifically present in Viridiplantae and mediates the transport of cargo proteins to the chloroplast. Using a forward genetic analysis, we identified a gene encoding a transmembrane protein (MtTP930) in barrel medic (Medicago truncatula). Mutation of MtTP930 resulted in impaired chloroplast function and a dwarf phenotype. MtTP930 is highly expressed in the aerial parts of the plant and is localized to the endoplasmic reticulum (ER) exit sites and Golgi. MtTP930 contains typical cargo sorting receptor motifs, interacts with Sar1, Sec12, and Sec24, and participates in coat protein complex II vesicular transport. Importantly, MtTP930 can recognize the cargo proteins plastidial N-glycosylated nucleotide pyrophosphatase/phosphodiesterase (MtNPP) and α-carbonic anhydrase (MtCAH) in the ER and then transport them to the chloroplast via the secretory pathway. Mutation of a homolog of MtTP930 in Arabidopsis (Arabidopsis thaliana) resulted in a similar dwarf phenotype. Furthermore, MtNPP-GFP failed to localize to chloroplasts when transgenically expressed in Attp930 protoplasts, implying that these cargo sorting receptors are conserved in plants. These findings fill a gap in our understanding of the mechanism by which chloroplast proteins are sorted and transported via the secretory pathway.

Effects of down-regulated carbonic anhydrase 8 on cell survival and glucose metabolism in human colorectal cancer cell lines.

Carbonic anhydrase 8 (CA8) is a member of the α-carbonic anhydrase family but does not catalyze the reversible hydration of carbon dioxide. In the present study, we examined the effects of CA8 on two human colon cancer cell lines, SW480 and SW620, by suppressing CA8 expression through shRNA knockdown. Our results showed that knockdown of CA8 decreased cell growth and cell mobility in SW620 cells, but not in SW480 cells. In addition, downregulated CA8 resulted in a significant decrease of glucose uptake in both SW480 and SW620 cells. Interestingly, stable downregulation of CA8 decreased phosphofructokinase-1 expression but increased glucose transporter 3 (GLUT3) levels in SW620 cells. However, transient downregulation of CA8 fails to up-regulate GLUT3 expression, indicating that the increased GLUT3 observed in SW620-shCA8 cells is a compensatory effect. In addition, the interaction between CA8 and GLUT3 was evidenced by pull-down and IP assays. On the other hand, we showed that metformin, a first-line drug for type II diabetes patients, significantly inhibited cell migration of SW620 cells, depending on the expressions of CA8 and focal adhesion kinase. Taken together, our data demonstrate that when compared to primary colon cancer SW480 cells, metastatic colon cancer SW620 cells respond differently to downregulated CA8, indicating that CA8 in more aggressive cancer cells may play a more important role in controlling cell survival and metformin response. CA8 may affect glucose metabolism- and cell invasion-related molecules in colon cancer, suggesting that CA8 may be a potential target in future cancer therapy.

Plant carbonic anhydrase-like enzymes in neuroactive alkaloid biosynthesis

Plants synthesize numerous alkaloids that mimic animal neurotransmitters1. The diversity of alkaloid structures is achieved through the generation and tailoring of unique carbon scaffolds2,3, yet many neuroactive alkaloids belong to a scaffold class for which no biosynthetic route or enzyme catalyst is known. By studying highly coordinated, tissue-specific gene expression in plants that produce neuroactive Lycopodium alkaloids4, we identified an unexpected enzyme class for alkaloid biosynthesis: neofunctionalized α-carbonic anhydrases (CAHs). We show that three CAH-like (CAL) proteins are required in the biosynthetic route to a key precursor of the Lycopodium alkaloids by catalysing a stereospecific Mannich-like condensation and subsequent bicyclic scaffold generation. Also, we describe a series of scaffold tailoring steps that generate the optimized acetylcholinesterase inhibition activity of huperzine A5. Our findings suggest a broader involvement of CAH-like enzymes in specialized metabolism and demonstrate how successive scaffold tailoring can drive potency against a neurological protein target.

Unique evolution of foraminiferal calcification to survive global changes

Foraminifera, the most ancient known calcium carbonate–producing eukaryotes, are crucial players in global biogeochemical cycles and well-used environmental indicators in biogeosciences. However, little is known about their calcification mechanisms. This impedes understanding the organismal responses to ocean acidification, which alters marine calcium carbonate production, potentially leading to biogeochemical cycle changes. We conducted comparative single-cell transcriptomics and fluorescent microscopy and identified calcium ion (Ca 2+ ) transport/secretion genes and α-carbonic anhydrases that control calcification in a foraminifer. They actively take up Ca 2+ to boost mitochondrial adenosine triphosphate synthesis during calcification but need to pump excess intracellular Ca 2+ to the calcification site to prevent cell death. Unique α-carbonic anhydrase genes induce the generation of bicarbonate and proton from multiple CO 2 sources. These control mechanisms have evolved independently since the Precambrian to enable the development of large cells and calcification despite decreasing Ca 2+ concentrations and pH in seawater. The present findings provide previously unknown insights into the calcification mechanisms and their subsequent function in enduring ocean acidification.

Features of Photosynthesis in Arabidopsis thaliana Plants with Knocked Out Gene of Alpha Carbonic Anhydrase 2.

The knockout of the At2g28210 gene encoding α-carbonic anhydrase 2 (α-CA2) in Arabidopsis thaliana (Columbia) led to alterations in photosynthetic processes. The effective quantum yields of both photosystem II (PSII) and photosystem I (PSI) were higher in α-carbonic anhydrase 2 knockout plants (α-CA2-KO), and the reduction state of plastoquinone pool was lower than in wild type (WT). The electron transport rate in the isolated thylakoids measured with methyl viologen was higher in α-CA2-KO plants. The amounts of reaction centers of PSII and PSI were similar in WT and α-CA2-KO plants. The non-photochemical quenching of chlorophyll a fluorescence in α-CA2-KO leaves was lower at the beginning of illumination, but became slightly higher than in WT leaves when the steady state was achieved. The degree of state transitions in the leaves was lower in α-CA2-KO than in WT plants. Measurements of the electrochromic carotenoid absorbance shift (ECS) revealed that the light-dependent pH gradient (ΔpH) across the thylakoid membrane was lower in the leaves of α-CA2-KO plants than in WT plants. The starch content in α-CA2-KO leaves was lower than in WT plants. The expression levels of the genes encoding chloroplast CAs in α-CA2-KO changed noticeably, whereas the expression levels of genes of cytoplasmic CAs remained almost the same. It is proposed that α-CA2 may be situated in the chloroplasts.

Structural Characterization of Thiadiazolesulfonamide Inhibitors Bound to Neisseria gonorrhoeae α-Carbonic Anhydrase.

Drug-resistant Neisseria gonorrhoeae is a critical threat to public health, and bacterial carbonic anhydrases expressed by N. gonorrhoeae are potential new therapeutic targets to combat this pathogen. To further expand upon our recent reports of bacterial carbonic anhydrase inhibitors for the treatment of N. gonorrhoeae, our team has solved ligand-bound crystal structures of the FDA-approved carbonic anhydrase inhibitor acetazolamide, along with three analogs, in complex with the essential α-carbonic anhydrase isoform from N. gonorrhoeae. The structural data for the analogs presented bound to N. gonorrhoeae α-carbonic anhydrase supports the observed structure-activity relationship for in vitro inhibition with this scaffold against the enzyme. Moreover, the ligand-bound structures indicate differences in binding poses compared to those traditionally observed with the close human ortholog carbonic anhydrase II. These results present key differences in inhibitor binding between N. gonorrhoeae α-carbonic anhydrase and the human carbonic anhydrase II isoform.

Exploration of 1,2,3-triazole linked benzenesulfonamide derivatives as isoform selective inhibitors of human carbonic anhydrase

A novel series of 1,2,3-triazole benzenesulfonamide substituted 1,3-dioxoisoindolin-5-carboxylate (7a-l) inhibitors of human α-carbonic anhydrase (hCA) was designed using a tail approach. The design method relies on the hybridization of a benzenesulfonamide moiety with a tail of 1,3-dioxoisoindoline-5-carboxylate and a zinc-binding group on a 1,2,3-triazole scaffold. Among the synthesized analogues, 2‑iodophenyl (7f, KI of 105.00 nM and SI of 2.98) and 2‑naphthyl (7h, KI of 32.11 nM and SI of 3.48) analogues (over off-target hCA I) and phenyl (7a, KI of 50.13 nM and SI of 2.74) and 2,6‑dimethylphenyl (7d, KI of 50.60 nM and SI of 3.35) analogues (over off-target hCA II) exhibited a remarkable selectivity for tumor isoforms hCA IX and XII, respectively. Meanwhile, analogue 7a displayed a potent inhibitory effect against the tumor-associated isoform hCA IX (KI of 18.29 nM) compared with the reference drug acetazolamide (AAZ, KI of 437.20 nM), and analogue 7h showed higher potency (KI of 9.22 nM) than AAZ (KI of 338.90 nM) against another tumor-associated isoform hCA XII. However, adding the lipophilic large naphthyl tail to the 1,3-dioxoisoindolin-5-carboxylate analogues increased both the hCA inhibitory and selective activities against the target isoform, hCA XII. Additionally, these analogues (7a-l) showed IC50 values against the human lung (A549) adenocarcinoma cancer cell line ranging from 129.71 to 352.26 μM. The results of the molecular docking study suggested that the sulfonamide moiety fits snugly into the hCAs active sites and interacts with the Zn2+ ion. At the same time, the tail extension engages in various hydrophilic and hydrophobic interactions with the nearby amino acids, which affects the potency and selectivity of the hybrids.

Inhibition studies of the protozoan α-carbonic anhydrase from Trypanosoma cruzi with phenols

The α-class carbonic anhydrase (CA, EC 4.2.1.1) from the protozoan pathogen Trypanosoma cruzi, TcCA, was investigated earlier for its inhibition with anions, sulphonamides, thiols and hydroxamates, well-known classes of CA inhibitors (CAIs). Here we present the first inhibition study of this enzyme with phenols, which possess a diverse CA inhibition mechanism compared to the previously investigated compounds, which are all zinc binders. Indeed, phenols are known to anchor to the zinc coordinated water molecule within the enzyme active site. In a series of 22 diversely substituted phenols, the best inhibitors were simple phenol, pyrocatechol, salicylic acid, 3,5-difluorophenol, 3,4-dihydroxy-benzoic acid, 3,6- dihydroxy-benzoic acid, caffeic acid and its des-hydroxy analog, with KIs of 1.8 − 7.3 µM. The least effective TcCA inhibitor was 3-chloro-4-amino-phenol (KI of 47.9 µM). Although it is not yet clear whether TcCA can be considered as an anti-Chagas disease drug target, as no animal model for investigating the antiprotozoan effects is available so far, finding effective in vitro inhibitors may be a first relevant step towards new antiprotozoal agents.

The production and biochemical characterization of α-carbonic anhydrase from Lactobacillus rhamnosus GG

We report the production and biochemical characterization of an α-carbonic anhydrase (LrhCA) from gram-positive probiotic bacteria Lactobacillus rhamnosus GG. CAs form a family of metalloenzymes that catalyze hydration of CO2/interconversion between CO2 and water to bicarbonate ions and protons. They are divided into eight independent gene families (α, β, γ, δ, ζ, η, θ, and ι). Interestingly, many pathogens have been identified with only β- and/or γ-CAs, which can be targeted with CA-specific inhibitors (CAIs) acting as anti-pathogen drugs. Since it is important to study the potential off-target effects of CAIs for both the human body and its commensal bacteria, we took L. rhamnosus GG as our study subject. To date, only a single α-CA has been identified in L. rhamnosus GG, which was successfully produced and biochemically characterized. LrhCA showed moderate catalytic activity with the following kinetic parameters: kcat of 9.86 × 105 s−1 and kcat/KM of 1.41 × 107 s−1 M−1. Moderate inhibition was established with 11 of the 39 studied sulfonamides. The best inhibitors were 5-((4-aminophenyl)sulfonamido)-1,3,4-thiadiazole-2-sulfonamide, 4-(2-hydroxymethyl-4-nitrophenyl-sulfonamidoethyl)-benzenesulfonamide, and benzolamide with Ki values of 319 nM, 378 nM, and 387 nM, respectively. The other compounds showed weaker inhibitory effects. The Ki of acetazolamide, a classical CAI, was 733 nM. In vitro experiments with acetazolamide showed that it had no significant effect on cell growth in L. rhamnosus GG culture. Several sulfonamides, including acetazolamide, are in use as clinical drugs, making their inhibition data highly relevant to avoid any adverse off-target effects towards the human body and its probiotic organisms.Key points• The α-carbonic anhydrase from Lactobacillus rhamnosus GG (LrhCA) is 24.3 kDa.• LrhCA has significant catalytic activity with a kcat of 9.9 × 105 s-1.• Acetazolamide resulted in a marginal inhibitory effect on cell growth.

Coumarins inhibit η-class carbonic anhydrase from Plasmodium falciparum

Coumarins were discovered to act as inhibitors of α-carbonic anhydrases (CAs, EC 4.2.1.1) after undergoing hydrolysis mediated by the esterase activity of the enzyme to the corresponding 2-hydroxycinnamic acids. Other classes of CAs among the eight currently known do not possess esterase activity or this activity was poorly investigated. Hence, we decided to look at the potential of coumarins as inhibitors of the η-CA from the malaria-producing protozoan Plasmodium falciparum, PfaCA. A panel of simple coumarins incorporating hydroxyl, amino, ketone or carboxylic acid ester moieties in various positions of the ring system acted as low to medium micromolar PfaCA inhibitors, whereas their affinities for the cytosolic off-target human isoforms hCA I and II were in a much higher range. Thus, we confirm that η-CAs possess esterase activity and that coumarins effectively inhibit this enzyme. Elaboration of the simple coumarin scaffolds investigated here may probably lead to more effective PfaCA inhibitors.

Coumarins effectively inhibit bacterial α-carbonic anhydrases

Coumarins are known to act as prodrug inhibitors of mammalian α-carbonic anhydrases (CAs, EC 4.2.1.1) but they were not yet investigated for the inhibition of bacterial α-CAs. Here we demonstrate that such enzymes from the bacterial pathogens Neisseria gonorrhoeae (NgCAα) and Vibrio cholerae (VchCAα) are inhibited by a panel of simple coumarins incorporating hydroxyl, amino, ketone or carboxylic acid ester moieties in various positions of the ring system. The nature and the position of the substituents in the coumarin ring were the factors which strongly influenced inhibitory efficacy. NgCAα was inhibited with KIs in the range of 28.6–469.5 µM, whereas VchCAα with KIs in the range of 39.8–438.7 µM. The two human (h)CA isoforms included for comparison reason in the study, hCA I and II, were less prone to inhibition by these compounds, with KIs of 137–948.9 µM for hCA I and of 296.5–961.2 µM for hCA II, respectively. These findings are relevant for discovering coumarin bacterial CA inhibitors with selectivity for the bacterial over human isoform, with potential applications as novel antibacterial agents.

Prediction of in silico ADMET Properties and Molecular Docking Study of Substituted Thiadiazole for Screening of Antibacterial and Antifungal Activities against Protein Targets Helicobacter pylori α-Carbonic Anhydrase and Trypanosoma brucei Pteridine Reductase

The aim of this work was to evaluate the physico-chemical, pharmacokinetic parameters (absorption, distribution, metabolism, excretion and toxicity) and pharmacodynamic parameters (bioactivity and adverse reactions) of substituted thiadiazole by means of in silico computational prediction. Online softwares such as Pre-ADMET, Molinspiration and rule of five were used for the analysis. Substituted thiadiazole fits the characteristics of drug-likeness, pharmacokinetic properties appropriate to the predicted patterns and activities within the scope for the treatment of infection in the stomach or duodenum (first part of the small intestine), gastritis and trypanosomiasis. Therefore, in silico results allow us to conclude that substituted thiadiazole is predicted to be a potential future drug candidate, due to its relevant Drug-likeness profile, bioavailability, excellent liposolubility and adequate pharmacokinetics, including at the level of CNS, penetrating the blood-brain barrier. Molecular docking studies have also been performed to screen the antibacterial and antifungal activities of the 50 designed compounds against protein targets Helicobacter pylori α-carbonic anhydrase (PDB: 5TUO) and Trypanosoma brucei Pteridine Reductase (PTR1) (PDB: 4WCD) respectively. Among all the compounds C11 exhibited the most significant affinity score against Helicobacter pylori α-carbonic anhydrase and C37 exhibited the most significant affinity score against Trypanosoma brucei pteridine reductase (PTR1) best significant hydrogen bonds interaction at the active site of protein.

Dithiocarbamates effectively inhibit the α-carbonic anhydrase from Neisseria gonorrhoeae

Recently, inorganic anions and sulphonamides, two of the main classes of zinc-binding carbonic anhydrase inhibitors (CAIs), were investigated for inhibition of the α-class carbonic anhydrase (CA, EC 4.2.1.1) from Neisseria gonorrhoeae, NgCA. As an extension to our previous studies, we report that dithiocarbamates (DTCs) derived from primary or secondary amines constitute a class of efficient inhibitors of NgCA. KIs ranging between 83.7 and 827 nM were measured for a series of 31 DTCs that incorporated various aliphatic, aromatic, and heterocyclic scaffolds. A subset of DTCs were selected for antimicrobial testing against N. gonorrhoeae, and three molecules displayed minimum inhibitory concentration (MIC) values less than or equal to 8 µg/mL. As NgCA was recently validated as an antibacterial drug target, the DTCs may lead to development of novel antigonococcal agents.

Surface display of carbonic anhydrase on Escherichia coli for CO2 capture and mineralization

Mineralization catalyzed by carbonic anhydrase (CA) is one of the most promising technologies for capturing CO2. In this work, Escherichia coli BL21(DE3) was used as the host, and the N-terminus of ice nucleation protein (INPN) was used as the carrier protein. Different fusion patterns and vectors were used to construct CA surface display systems for α-carbonic anhydrase (HPCA) from Helicobacter pylori 26695 and α-carbonic anhydrase (SazCA) from Sulfurihydrogenibium azorense. The surface display system in which HPCA was fused with INPN via a flexible linker and intermediate repeat sequences showed higher whole-cell enzyme activity, while the enzyme activity of the SazCA expression system was significantly higher than that of the HPCA expression system. The pET22b vector with the signal peptide PelB was more suitable for the cell surface display of SazCA. Cell fractionation and western-blot analysis indicated that SazCA and INPN were successfully anchored on the cell's outer membrane as a fusion protein. The enzyme activity of the surface display strain E-22b-IRLS (11.43 U·mL−1OD600−1) was significantly higher than that of the intracellular expression strain E-22b-S (8.355 U·mL−1OD600−1) under optimized induction conditions. Compared with free SazCA, E-22b-IRLS had higher thermal and pH stability. The long-term stability of SazCA was also significantly improved by surface display. When the engineered strain and free enzyme were used for CO2 mineralization, the amount of CaCO3 deposition catalyzed by the strain E-22b-IRLS on the surface (241 mg) was similar to that of the free SazCA and was significantly higher than the intracellular expression strain E-22b-S (173 mg). These results demonstrate that the SazCA surface display strain can serve as a whole-cell biocatalyst for CO2 capture and mineralization.

Carbonic Anhydrases: An Ancient Tool in Calcareous Sponge Biomineralization.

Enzymes of the α-carbonic anhydrase gene family (CAs) are essential for the deposition of calcium carbonate biominerals. In calcareous sponges (phylum Porifera, class Calcarea), specific CAs are involved in the formation of calcite spicules, a unique trait and synapomorphy of this class. However, detailed studies on the CA repertoire of calcareous sponges exist for only two species of one of the two Calcarea subclasses, the Calcaronea. The CA repertoire of the second subclass, the Calcinea, has not been investigated so far, leaving a considerable gap in our knowledge about this gene family in Calcarea. Here, using transcriptomic analysis, phylogenetics, and in situ hybridization, we study the CA repertoire of four additional species of calcareous sponges, including three from the previously unsampled subclass Calcinea. Our data indicate that the last common ancestor of Calcarea had four ancestral CAs with defined subcellular localizations and functions (mitochondrial/cytosolic, membrane-bound, and secreted non-catalytic). The evolution of membrane-bound and secreted CAs involved gene duplications and losses, whereas mitochondrial/cytosolic and non-catalytic CAs are evidently orthologous genes. Mitochondrial/cytosolic CAs are biomineralization-specific genes recruited for biomineralization in the last common ancestor of calcareous sponges. The spatial–temporal expression of these CAs differs between species, which may reflect differences between subclasses or be related to the secondary thickening of spicules during biomineralization that does not occur in all species. With this study, we extend the understanding of the role and the evolution of a key biomineralization gene in calcareous sponges.

Evaluation of actin as a reference for quantitative gene expression studies in Emiliania huxleyi (Prymnesiophyceae) under ocean acidification conditions

ABSTRACT Gene expression studies of marine phytoplankton under ocean acidification conditions are frequently based on relative measurements, with actin commonly used as a reference gene. Evidence from other organisms suggests that actin gene expression may be regulated by environmental conditions, compromising the role of actin as a reference gene. In this work the reliability of actin as a reference gene for ocean acidification experimental conditions (high CO2 vs low CO2) in two different metabolic states (acclimated metabolism vs perturbed metabolism) for the coccolithophore Emiliania huxleyi was tested. The transcriptional response of the actin (act) is compared with the expression of specific target genes associated with inorganic carbon uptake (α-carbonic anhydrase: αca1) and assimilation (RuBisCO: rbcL), which was regulated under the experimental conditions. Our results showed act expression instability in experimental conditions, evidencing that act is not a reliable reference gene for studies assessing the effect of ocean acidification on Emiliania huxleyi. Furthermore, when the act-based normalization was quantitatively tested, rbcL and αca1 expression were compromised, leading us to conclude that absolute gene expression quantification should be considered as a potentially reliable alternative for studying gene expression under ocean acidification conditions.

Anion inhibition studies of the α-carbonic anhydrases from Neisseria gonorrhoeae

The bacterial pathogen Neisseria gonorrhoeae encodes for an α-class carbonic anhydrase (CA, EC 4.2.1.1), NgCA, which was investigated for its inhibition with a series of inorganic and organic anions. Perchlorate and hexafluorophosphate did not significantly inhibit NgCA CO2 hydrase activity, whereas the halides, azide, bicarbonate, carbonate, stannate, perosmate, diphosphate, divanadate, perruthenate, and trifluoromethanesulfonate showed inhibition constants in the range of 1.3–9.6 mM. Anions/small molecules such as cyanate, thiocyanate, nitrite, nitrate, bisulphite, sulphate, hydrogensulfide, phenylboronic acid, phenylarsonic acid, selenate, tellurate, tetraborate, perrhenate, peroxydisulfate, selenocyanate, iminodisulfonate, and fluorosulfonate showed KIs in the range of 0.15–1.0 mM. The most effective inhibitors detected in this study were sulfamide, sulfamate, trithiocarbonate and N,N-diethyldithiocarbamate, which had KIs in the range of 5.1–88 µM. These last compounds incorporating the CS2 - zinc-binding group may be used as leads for developing even more effective NgCA inhibitors in addition to the aromatic/heterocyclic sulphonamides, as this enzyme was recently validated as an antibacterial drug target for obtaining novel antigonococcal agents

External α-carbonic anhydrase and solute carrier 4 are required for bicarbonate uptake in a freshwater angiosperm.

The freshwater monocot Ottelia alismoides is the only known species to operate three CO2-concentrating mechanisms (CCMs): constitutive bicarbonate (HCO3-) use, C4 photosynthesis, and facultative Crassulacean acid metabolism, but the mechanism of HCO3- use is unknown. We found that the inhibitor of an anion exchange protein, 4,4'-diisothio-cyanatostilbene-2,2'-disulfonate (DIDS), prevented HCO3- use but also had a small effect on CO2 uptake. An inhibitor of external carbonic anhydrase (CA), acetazolamide (AZ), reduced the affinity for CO2 uptake but also prevented HCO3- use via an effect on the anion exchange protein. Analysis of mRNA transcripts identified a homologue of solute carrier 4 (SLC4) responsible for HCO3- transport, likely to be the target of DIDS, and a periplasmic α-carbonic anhydrase 1 (α-CA1). A model to quantify the contribution of the three different pathways involved in inorganic carbon uptake showed that passive CO2 diffusion dominates inorganic carbon uptake at high CO2 concentrations. However, as CO2 concentrations fall, two other pathways become predominant: conversion of HCO3- to CO2 at the plasmalemma by α-CA1 and transport of HCO3- across the plasmalemma by SLC4. These mechanisms allow access to a much larger proportion of the inorganic carbon pool and continued photosynthesis during periods of strong carbon depletion in productive ecosystems.

Optimization of Acetazolamide-Based Scaffold as Potent Inhibitors of Vancomycin-Resistant Enterococcus.

Vancomycin-resistant enterococci (VRE) are the second leading cause of hospital-acquired infections (HAIs) attributed to a drug-resistant bacterium in the United States, and resistance to the frontline treatments is well documented. To combat VRE, we have repurposed the FDA-approved carbonic anhydrase drug acetazolamide to design potent antienterococcal agents. Through structure-activity relationship optimization we have arrived at two leads possessing improved potency against clinical VRE strains from MIC = 2 μg/mL (acetazolamide) to MIC = 0.007 μg/mL (22) and 1 μg/mL (26). Physicochemical properties were modified to design leads that have either high oral bioavailability to treat systemic infections or low intestinal permeability to treat VRE infections in the gastrointestinal tract. Our data suggest the intracellular targets for the molecules are putative α-carbonic and γ-carbonic anhydrases, and homology modeling and molecular dynamics simulations were performed. Together, this study presents potential anti-VRE therapeutic options to provide alternatives for problematic VRE infections.