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The α- and β-class carbonic anhydrases (CAs, EC 4.2.1.1) from the pathogenic bacterium Vibrio cholerae, VchCAα, and VchCAβ, were investigated for their activation with natural and non-natural amino acids and amines. The most effective VchCAα activators were L-tyrosine, histamine, serotonin, and 4-aminoethyl-morpholine, which had KAs in the range of 8.21–12.0 µM. The most effective VchCAβ activators were D-tyrosine, dopamine, serotonin, 2-pyridyl-methylamine, 2-aminoethylpyridine, and 2-aminoethylpiperazine, which had KAs in the submicromolar – low micromolar range (0.18–1.37 µM). The two bacterial enzymes had very different activation profiles with these compounds, between each other, and in comparison to the human isoforms hCA I and II. Some amines were selective activators of VchCAβ, including 2-pyridylmethylamine (KA of 180 nm for VchCAβ, and more than 20 µM for VchCAα and hCA I/II). The activation of CAs from bacteria, such as VchCAα/β has not been considered previously for possible biomedical applications. It would be of interest to study in more detail the extent that CA activators are implicated in the virulence and colonisation of the host by such pathogenic bacteria, which for Vibrio cholerae, is highly dependent on the bicarbonate concentration and pH in the surrounding tissue.

The inhibition of α-, β-, γ-, and δ-class carbonic anhydrases (CAs, EC 4.2.1.1) from bacteria (Vibrio cholerae and Porphyromonas gingivalis) and diatoms (Thalassiosira weissflogii) with a panel of N’-aryl-N-hydroxy-ureas is reported. The α-/β-CAs from V. cholerae (VchCAα and VchCAβ) were effectively inhibited by some of these derivatives, with KIs in the range of 97.5 nM – 7.26 µM and 52.5 nM – 1.81 µM, respectively, whereas the γ-class enzyme VchCAγ was less sensitive to inhibition (KIs of 4.75 – 8.87 µM). The β-CA from the pathogenic bacterium Porphyromonas gingivalis (PgiCAβ) was not inhibited by these compounds (KIs > 10 µM) whereas the corresponding γ-class enzyme (PgiCAγ) was effectively inhibited (KIs of 59.8 nM – 6.42 µM). The δ-CA from the diatom Thalassiosira weissflogii (TweCAδ) showed effective inhibition with these derivatives (KIs of 33.3 nM – 8.74 µM). As most of these N-hydroxyureas are also ineffective as inhibitors of the human (h) widespread isoforms hCA I and II (KIs > 10 µM), this class of derivatives may lead to the development of CA inhibitors selective for bacterial/diatom enzymes over their human counterparts and thus to anti-infectives or agents with environmental applications.

A series of symmetric molecules incorporating aryl or pyridyl moieties as central core and 1,4-substituted triazoles as a side bridge was synthesised. The new compounds were investigated as lactate dehydro-genase (LDH, EC 1.1.1.27) inhibitors. The cancer associated LDHA isoform was inhibited with IC50 = 117–174 µM. Seven compounds exhibited better LDHA inhibition (IC50 117–136 µM) compared to known LDH inhibitor – galloflavin (IC50 157 µM).

The activation of a β-class carbonic anhydrase (CAs, EC 4.2.1.1) from Mycobacterium tuberculosis, encoded by the gene Rv3273 (mtCA 3), was investigated using a panel of natural and non-natural amino acids and amines. mtCA 3 was effectively activated by D-DOPA, L-Trp, dopamine and serotonin, with KAs ranging between 8.98 and 12.1 µM. L-His and D-Tyr showed medium potency activating effects, with KAs in the range of 17.6–18.2 µM, whereas other amines and amino acids were relatively ineffective activators, with KAs in the range of 28.9–52.2 µM. As the physiological roles of the three mtCAs present in this pathogen are currently poorly understood and considering that inhibition of these enzymes has strong antibacterial effects, discovering molecules that modulate their enzymatic activity may lead to a better understanding of the factors related to the invasion and colonisation of the host during Mycobacterium tuberculosis infection.

The activation of the δ-class carbonic anhydrase (CAs, EC 4.2.1.1) from the diatom Thalassiosira weissflogii (TweCAδ) was investigated using a panel of natural and non-natural amino acids and amines. The most effective activator of TweCAδ was d-Tyr (KA of 51 nM), whereas several other amino acids and amines, such as L-His, L-Trp, d-Trp, dopamine and serotonin were submicromolar activators (KAs from 0.51 to 0.93 µM). The most ineffective activator of TweCAδ was 4-amino-l-Phe (18.9 µM), whereas d-His, l-/d-Phe, l-/d-DOPA, l-Tyr, histamine, some pyridyl-alkylamines, l-adrenaline and aminoethyl-piperazine/morpholine were moderately potent activators (KAs from 1.34 to 8.16 µM). For any δ-CA, there are no data on the crystal structure, homology modelling and the amino acid residues that are responsible for proton transfer to the active site are currently unknown making it challenging to provide a detailed rational for these findings. However, these data provide further evidence that this class of underexplored CA deserves more attention.

The β-class carbonic anhydrase (CA, EC 4.2.1.1) from the pathogenic bacterium Burkholderia pseudomallei, BpsCAβ, that is responsible for the tropical disease melioidosis was investigated for its activation with natural and non-natural amino acids and amines. Previously, the γ-CA from this bacterium has been investigated with the same library of 19 amines/amino acids, which show very potent activating effects on both enzymes. The most effective BpsCAβ activators were L- and D-DOPA, L- and D-Trp, L-Tyr, 4-amino-L-Phe, histamine, dopamine, serotonin, 2-pyridyl-methylamine, 1-(2-aminoethyl)-piperazine and L-adrenaline with KAs of 0.9–27 nM. Less effective activators were D-His, L- and D-Phe, D-Tyr, 2-(2-aminoethyl)pyridine and 4-(2-aminoethyl)-morpholine with KAs of 73 nM–3.42 µM. The activation of CAs from bacteria, such as BpsCAγ/β, has not been considered previously for possible biomedical applications. It would be of interest to perform studies in which bacteria are cultivated in the presence of CA activators, which may contribute to understanding processes connected with the virulence and colonization of the host by pathogenic bacteria.

Elevated blood glucose and increased activities of secreted phospholipase A2 (sPLA2) are strongly linked to coronary heart disease. In this report, our goal was to develop small heterocyclic compound that inhibit sPLA2. The title compounds were also tested against α-glucosidase and α-amylase. This array of enzymes was selected due to their implication in blood glucose regulation and diabetic cardiovascular complications. Therefore, two distinct series of quinoxalinone derivatives were synthesised; 3-[N′-(substituted-benzylidene)-hydrazino]-1H-quinoxalin-2-ones 3a–f and 1-(substituted-phenyl)-5H-[1,2,4]triazolo[4,3-a]quinoxalin-4-ones 4a–f. Four compounds showed promising enzyme inhibitory effect, compounds 3f and 4b–d potently inhibited the catalytic activities of all of the studied proinflammatory sPLA2. Compound 3e inhibited α-glucosidase (IC50 = 9.99 ± 0.18 µM); which is comparable to quercetin (IC50 = 9.93 ± 0.66 µM), a known inhibitor of this enzyme. Unfortunately, all compounds showed weak activity against α-amylase (IC50 > 200 µM). Structure-based molecular modelling tools were utilised to rationalise the SAR compared to co-crystal structures with sPLA2-GX as well as α-glucosidase. This report introduces novel compounds with dual activities on biochemically unrelated enzymes mutually involved in diabetes and its complications.

The carbonic anhydrase superfamily (CA, EC 4.2.1.1) of metalloenzymes is present in all three domains of life (Eubacteria, Archaea, and Eukarya), being an interesting example of convergent/divergent evolution, with its seven families (α-, β-, γ-, δ-, ζ-, η-, and θ-CAs) described so far. CAs catalyse the simple, but physiologically crucial reaction of carbon dioxide hydration to bicarbonate and protons. Recently, our groups characterised the α-CA from the thermophilic bacterium, Sulfurihydrogenibium yellowstonense finding a very high catalytic activity for the CO2 hydration reaction (kcat = 9.35 × 105 s−1 and kcat/Km = 1.1 × 108 M−1 s−1) which was maintained after heating the enzyme at 80 °C for 3 h. This highly thermostable SspCA was covalently immobilised within polyurethane foam and onto the surface of magnetic Fe3O4 nanoparticles. Here, we describe a one-step procedure for immobilising the thermostable SspCA directly on the surface membrane of Escherichia coli, using the INPN domain of Pseudomonas syringae. This strategy has clear advantages with respect to other methods, which require as the first step the production and the purification of the biocatalyst, and as the second step the immobilisation of the enzyme onto a specific support. Our results demonstrate that thermostable SspCA fused to the INPN domain of P. syringae ice nucleation protein (INP) was correctly expressed on the outer membrane of engineered E. coli cells, affording for an easy approach to design biotechnological applications for this highly effective thermostable catalyst.

We cloned, expressed, purified, and determined the kinetic constants of the recombinant α-carbonic anhydrase (rec-MgaCA) identified in the mantle tissue of the bivalve Mediterranean mussel, Mytilus galloprovincialis. In metazoans, the α-CA family is largely represented and plays a pivotal role in the deposition of calcium carbonate biominerals. Our results demonstrated that rec-MgaCA was a monomer with an apparent molecular weight of about 32 kDa. Moreover, the determined kinetic parameters for the CO2 hydration reaction were kcat = 4.2 × 105 s−1 and kcat/Km of 3.5 × 107 M−1 ×s−1. Curiously, the rec-MgaCA showed a very similar kinetic and acetazolamide inhibition features when compared to those of the native enzyme (MgaCA), which has a molecular weight of 50 kDa. Analysing the SDS-PAGE, the protonography, and the kinetic analysis performed on the native and recombinant enzyme, we hypothesised that probably the native MgaCA is a multidomain protein with a single CA domain at the N-terminus of the protein. This hypothesis is corroborated by the existence in mollusks of multidomain proteins with a hydratase activity. Among these proteins, nacrein is an example of α-CA multidomain proteins characterised by a single CA domain at the N-terminus part of the entire protein.

Quinoxalines are benzopyrazine derivatives with significant therapeutic impact in the pharmaceutical industry. They proved to be useful against inflammation, bacterial, fungal, viral infection, diabetes and other applications. Very recently, in January 2024, the FDA approved new quinoxaline containing drug, erdafitinib for treatment of certain carcinomas. Despite the diverse biological activities exhibited by quinoxaline derivatives and the role of secretory phospholipase A2 (sPLA2) in diabetes-related complications, the potential of sPLA2-targeting quinoxaline-based inhibitors to effectively address these complications remains unexplored. Therefore, we designed novel sPLA2- and α-glucosidase-targeting quinoxaline-based heterocyclic inhibitors to regulate elevated post-prandial blood glucose linked to patients with diabetes-related cardiovascular complications. Compounds 5a–d and 6a–d were synthesised by condensing quinoxaline hydrazides with various aryl sulphonyl chlorides. Biological screening revealed compound 6a as a potent sPLA2 inhibitor (IC50 = 0.0475 µM), whereas compound 6c most effectively inhibited α-glucosidase (IC50 = 0.0953 µM), outperforming the positive control acarbose. Moreover, compound 6a was the best inhibitor for both enzymes. Molecular docking revealed pharmacophoric features, highlighting the importance of a sulfonohydrazide moiety in the structural design of these compounds, leading to the development of potent sPLA2 and α-glucosidase inhibitors. Collectively, our findings helped identify promising candidates for developing novel therapeutic agents for treating diabetes mellitus.

Chagas disease and leishmaniasis are neglected tropical disorders caused by the protozoans Trypanosoma cruzi and Leishmania spp. Carbonic anhydrases (CAs, EC 4.2.1.1) from these protozoans (α-TcCA and β-LdcCA) have been validated as promising targets for chemotherapic interventions. Many anti-protozoan agents, such as nitroimidazoles, nifurtimox, and benznidazole possess a nitro aromatic group in their structure which is crucial for their activity. As a continuation of our previous work on N-nitrosulfonamides as anti-protozoan agents, we investigated benzenesulfonamides bearing a nitro aromatic moiety against TcCA and LdcCA, observing selective inhibitions over human off-target CAs. Selected derivatives were assessed in vitro in different developmental stages of T. cruzi and Leishmania spp. A lack of significant growth inhibition has been found, which has been connected to the low permeability of this class of derivatives through cell membranes. Further strategies necessarily need to be designed for targeting Chagas disease and leishmaniasis with nitro-containing CA inhibitors.