Carbonic Anhydrase Isoforms Research Articles

Design, Synthesis, Anticancer Screening, and Mechanistic Study of Spiro-N-(4-sulfamoyl-phenyl)-1,3,4-thiadiazole-2-carboxamide Derivatives.

The present study aims to create spiro-N-(4-sulfamoyl-phenyl)-1,3,4-thiadiazole-2-carboxamide derivatives with anticancer activities. The in vitro anticancer evaluation showed that only the novel spiro-acenaphthylene tethered-[1,3,4]-thiadiazole (compound 1) exhibited significant anticancer efficacy as a selective inhibitor of tumor-associated isoforms of carbonic anhydrase. Compound 1 demonstrated considerable efficacy against the renal RXF393, colon HT29, and melanoma LOX IMVI cancer cell lines, with IC50 values of 7.01 ± 0.39, 24.3 ± 1.29, and 9.55 ± 0.51 µM, respectively. In comparison, doxorubicin exhibited IC50 values of 13.54 ± 0.82, 13.50 ± 0.71, and 6.08 ± 0.32 µM for the corresponding cell lines. Importantly, compound 1 exhibited lower toxicity to the normal WI 38 cell line than doxorubicin, with IC50 values of 46.20 ± 2.59 and 18.13 ± 0.93 µM, respectively, indicating greater selectivity of the target compound compared to the standard anticancer agent doxorubicin. Also, mechanistic experiments demonstrated that compound 1 exhibits inhibitory activity against human carbonic anhydrase hCA IX and XII, with IC50 values of 0.477 ± 0.03 and 1.933 ± 0.11 μM, respectively, indicating enhanced selectivity for cancer-associated isoforms over cytosolic isoforms hCA I and II, with IC50 values of 7.353 ± 0.36 and 12.560 ± 0.74 μM, respectively. Cell cycle studies revealed that compound 1 caused G1 phase arrest in RXF393 cells, and apoptosis experiments verified a substantial induction of apoptosis with significant levels of early and late apoptosis, as well as necrosis (11.69%, 19.78%, and 3.66%, respectively), comparable to those induced by the conventional cytotoxic agent doxorubicin, at 9.91%, 23.37%, and 6.16%, respectively. Molecular docking experiments confirmed the strong binding affinity of compound 1 to the active sites of hCA IX and XII, highlighting significant interactions with zinc-binding groups and hydrophobic residues. These findings underscore the target compound's potential as a viable anticancer agent via targeting CA.

Exploring the Potential of Selenium-Containing Amine (Se-AMA) to Enhance Photosynthesis and Leaf Water Content: New Avenues for Carbonic Anhydrase Modulation in Arabidopsis thaliana.

Global changes and growing demands have led to the development of new molecular approaches to improve crop physiological performances. Carbonic anhydrase (CA) enzymes, ubiquitous across various life kingdoms, stand out for their critical roles in plant photosynthesis and water relations. We hypothesize that the modulators of human CAs could affect plant physiology. Our research demonstrated that foliar treatments with a synthetic selenium-containing CA activator (Se-AMA) influenced the physiological performances of Arabidopsis thaliana. Se-AMA increased net photosynthesis (A + 31.7%) and stomatal conductance (gs + 48.2%) at 100 µM, with the most notable effects after 10 days of treatment. Se-AMA at 300 µM proved to be even more effective, boosting A and gs by 19.9% and 55.3%, respectively, already after 3 days of application. Morning treatment with Se-AMA at 300 µM enhanced photosynthetic performances throughout the day, suggesting that the positive effect of Se-AMA lasted for several hours. Additionally, Se-AMA increased water content in plants by 17.1%, suggesting that Se-AMA treatment may have improved plant water absorption and resource management. This effect might be linked to Se-AMA's role in modulating specific CA isoforms working with aquaporins. Although preliminary, these findings suggest that Se-AMA could enhance plant physiological performances under the conditions of non-limiting water availability.

Novel 3-Sulfonamide Dual-Tail Pyrrol-2-one Bridged Molecules as Potent Human Carbonic Anhydrase Isoform Inhibitors: Design, Synthesis, Molecular Modeling Investigation, and Anticancer Activity in MeWo, SK-BR-3, and MG-63 Cell Lines.

Novel 3-sulfonamide pyrrol-2-one derivatives containing two sulfonamide groups were synthesized via a one-pot, three-component method using trifluoroacetic acid as a catalyst. Structural confirmation was achieved using spectroscopic techniques. The compounds were tested against four selected human carbonic anhydrase isoforms (hCA I, hCA II, hCA IX, and hCA XII). Most derivatives showed significant selectivity for hCA II, with 4h, 4i, 4n, 4k, and 4j demonstrating enhanced activity due to methoxy and hydroxy group patterns. Compound 4o exhibited strong dual selectivity for hCA II and hCA IX, while 4l was the most effective inhibitor of hCA XII. Additionally, 4e showed a preference for hCA XII inhibition. Biological evaluation on MeWo, SK-BR-3, and MG-63 cancer cells showed that compound 4l was cytotoxic for MeWo cells without significantly affecting normal fibroblasts' viability. Compounds 4e, 4l, and 4o were shown to affect tumor cell viability in combination with doxorubicin in additional testing on MeWo cancer cells.

Exploring aliphatic sulfonamides as multiclass inhibitors of the carbonic anhydrases from the pathogen bacterium Vibrio cholerae.

This study investigates aliphatic sulfonamide derivatives as inhibitors of the α-, β-, and γ-class carbonic anhydrase (CA) isoforms from Vibrio cholerae (VchCAs). A series of 26 compounds bearing a triazole linker and urea- or ether-based tails were described and evaluated for their inhibitory action using a stopped-flow CO2 hydrase technique. These inhibitors demonstrated a preferential efficacy against VchCAβ. Specifically, the ureido derivatives showed the highest inhibitory potency with inhibition constants (KIs) in the submicromolar range (0.67-0.93 µM). Selectivity indices were calculated to assess the selective inhibition of VchCAβ over human CA I and II, as well as other VchCA isozymes. Urea-linked compounds demonstrated a significant 25- to 125-fold selectivity for VchCAβ over hCAs and 14- to 26-fold over other VchCAs. Molecular modeling elucidated the interactions contributing to the efficacy and selectivity of aliphatic sulfonamides as VchCA inhibitors, aligning with and reinforcing the experimental results. The latter suggests that aliphatic sulfonamides could serve as valid targeted therapeutics to treat V. cholerae infections.

Discovery of Sulfanilamide-diazo Derivatives Incorporating Benzoic Acid Moieties as Novel Inhibitors of Human Carbonic Anhydrase II Activity.

Sulfonamides are widely used carbonic anhydrase inhibitors (CAIs) in clinical settings, however, their nonspecific inhibition of multiple carbonic anhydrase isoforms can lead to reduced efficacy and side effects. This study aimed to develop sulfanilamide-diazo derivatives incorporating benzoic acid moieties as novel inhibitors of hCA II activity to reduce side effects and enhance selectivity for different CA isozymes. We investigated the interaction between these derivatives and the hCA II isozyme via various spectroscopic and docking methods. The kinetic data demonstrates that compound 1 (C1) and compound 2 (C2) share a similar inhibitory strength against hCA II, effectively inhibiting its esterase activity through a noncompetitive mechanism with Ki values at low micromolar levels. Fluorescence measurements indicated that the synthesized compounds suppressed the inherent fluorescence of hCA II via a static quenching process, with each compound showing a singular binding site within the enzyme. Thermodynamic evidences highlight the significance of van der Waals interactions and hydrogen bonding in the binding process. The results of molecular docking indicated that both C1 and C2 effectively obstruct the entrance to hCA II's active site, with no significant differences in their binding conformations. While C1 and C2 exhibit CA inhibitory potency lower than that of sulfonamide compounds, this study offers valuable insights that could pave the way for the development of a promising scaffold for designing new carbonic anhydrase inhibitors.

Discovery of the First-in-Class Dual TSPO/Carbonic Anhydrase Modulators with Promising Neurotrophic Activity.

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

Insight on novel sulfamoylphenyl pyrazole derivatives as anticancer carbonic anhydrase inhibitors.

As another part continue for our previous study, variable substituted pyrazoles bearing sulfamoylphenyl moiety were synthesized and screened against two cancer related human carbonic anhydrase (hCA) isoforms and acetazolamide (AAZ) used as a reference standard. Some compounds as 4e and 6c manifested a promising inhibitory activity against both isoforms (KI = 0.072, 0.081 and 0.073, 0.095 µM), respectively. While others as 4a and 5e showed inhibitory activity against hCA IX only (KI = 0.062, 0.04 µM) or against hCA XII only as compound 5b (KI = 0.106 µM) compared to AAZ (KI = 0.065, 0.046 µM), respectively. Also, the anticancer efficacy against 60 cancer cell lines for the target compounds was assessed, and the most promising ones were 4d and 5a-d. Further investigation of the anticancer activity of 5b on MCF-7 cell line explored (IC50 = 5.21 µM) compared to doxorubicin (IC50 = 11.58 µM). Moreover, compound 5b was exposed to cell cycle analysis and apoptotic assay on MCF-7 breast cancer cell line under both normal and hypoxic conditions at its IC50 concentration with elevation of total apoptotic cells % in MCF-7 relative to the control cells; respectively. Finally, molecular modelling simulations rationalized the in vitro testing results.

Synthesis and investigation of selective human carbonic anhydrase IX, XII inhibitors using coumarins bearing a sulfonamide or biotin moiety

The role of carbonic anhydrases isoforms (CAs) IX and XII in the pathogenesis and progression of many types of solid tumors is well known. In this context, selective CA inhibitors (CAIs) towards the mentioned isoforms is a validated strategy for the development of agents to target cancer. For this purpose, novel coumarin derivatives based on the hybridization with arylsulfonamide or biotin scaffolds were synthesized and tested as inhibitors of four different human carbonic anhydrases isoforms: hCA I, II, IX and XII. Coumarin-sulfonamide derived 27, with a thiourea moiety and triazole as linker, showed the highest inhibition activity against hCA XII with an inhibition constant (KI) of 7.5 nM and afforded a very good selectivity over hCA I. Compound 32 was the most potent inhibitor against hCA IX (KI = 6.3 nM), 4-fold stronger than the drug acetazolamide AAZ (KI = 25 nM), used herein as a reference compound, and showed remarkable selectivity over hCA I and II. The coumarin-biotin derivatives 37–39 showed outstanding selectivity towards on-target enzymes (hCA IX and XII) and appear as plausible leads for designing of CAIs.

Exploring the Polypharmacological Potential of PCI-27483: A Selective Inhibitor of Carbonic Anhydrases IX and XII.

PCI-27483, originally developed as a potent and selective inhibitor of the serine protease Factor VIIa (FVIIa) in complex with tissue factor (TF), has demonstrated significant promise in cancer therapy. In addition to its primary mechanism of action, the presence of a sulfonamide moiety in the PCI-27483 structure suggests further activities through the inhibition of carbonic anhydrases (CAs), particularly the tumor-associated human (h)CA isoforms hCA IX and XII. This study investigates the inhibitory activity of PCI-27483 against the complete panel of active hCAs, highlighting its polypharmacological potential in cancer treatment. X-ray crystallography and molecular docking studies elucidated the structural features underlying its selective inhibitory activity toward hCA IX and XII, offering insights into its dual-targeting pathway.

Benzimidazolium salts bearing ester group: Synthesis, characterization, crystal structure, molecular docking studies, and inhibitory properties against metabolic enzymes

In this work, the synthesis of several unsymmetrical benzimidazolium salts with an ester (2-ethoxy-2-oxoethyl) group on the nitrogen atom is described. The structures of all compounds were fully characterized by spectroscopic (1H, 13C NMR, FTIR) and analytical (elemental analysis) methods. However, single-crystal X-ray diffraction analysis elucidated the molecular and crystal structures of compounds 1a and 1c. Asymmetric units of both crystal structures contain two crystallographically independent molecules and two bromide anions. All compounds exhibited substantial inhibition against the cytosolic carbonic anhydrase isoforms (hCA I and hCA II) and acetylcholinesterase (AChE) with Ki values 51.00–45.18 nM, 46.94–305.65 nM, and 17.57–65.68 nM, respectively. Notably, compound 1d showed extreme inhibitory effect against hCA I with 51.00±5.31 nM (AZA: 275.44±13.32 nM), hCA II with 46.94±5.44 nM (AZA: 236.55±17.88 nM) and AChE with 17.57±3.14 nM (TAC: 80.44±6.88 nM). In silico approach showed that compound 1d could form stable complexes with target enzymes with a different binding affinity (BE:9.01 kcal/mol for AChE; -7.22 kcal/mol for hCA II and -6.51 kcal/mol for hCA I). The results supported the medical potential of benzimidazolium salts containing ester group. They significantly lighted our knowledge about the chemistry of side groups on phenyl ring especially in the para position as compared to ortho and meta positions.

Benzoxaborinine, New Chemotype for Carbonic Anhydrase Inhibition: Ex Novo Synthesis, Crystallography, In Silico Studies, and Anti-Melanoma Cell Line Activity.

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

Novel Schiff Bases: Synthesis, Characterization, Bioactivity, Cytotoxicity, and Computational Evaluations

Hypopharyngeal cancer is rare subtype of head and neck cancers with relatively poor prognosis. Current therapeutic modalities lack the potential to provide patients with better clinical outcome and quality of life. This study was conducted on the synthesis of 2-methoxy-4-((3-alkyl(aryl)-5-oxo-1H-1,2,4-triazol-4(5H)-ylimino)-methyl)-phenyl-4-(2-methoxy-4-((3-alkyl(aryl)-5-oxo-1H-1,2,4-triazol-4(5H)-ylimino)-methyl)-phenyl)-4-oxobutanoates (3) using biologically important 1,2,4-triazole. The condensation of 3-alkyl(aryl)-4-amino-4,5-dihydro-1H-1,2,4-triazol-5-ones (1) with 4-formyl-2-methoxyphenyl-4-(4-formyl-2-methoxyphenyl)-4-oxobutanoate yielded the biologically active 2-methoxy-4-((3-alkyl(aryl)-5-oxo-1H-1,2,4-triazol-4(5H)-ylimino)-methyl)-phenyl-4-(2-methoxy-4-((3-alkyl(aryl)-5-oxo-1H-1,2,4-triazol-4(5H)-ylimino)-methyl)-phenyl)-4-oxobutanoates (3). The compounds obtained were analyzed via FT-IR,1H-/13C-NMR spectrometers, elemental analysis, and HRMS spectroscopic techniques. Furthermore, we aimed at investigating the potential of compounds 3a-g against FaDu hypopharyngeal cancer cells. We demonstrated that compounds 3c, 3e, and 3 g had relatively lower IC50 values compared to the remaining tested compounds and more importantly their IC50 values were comparable to 5-FU, which suggests them as important therapeutic agent candidates. These newly synthesized compounds were assessed for their inhibitory activities toward two human carbonic anhydrase isoforms I and II (hCA I and II). Then, molecular docking calculations were made to compare the biological activities of studied molecules against cancer proteins. Compound 3c has a docking score of −7.15 against squamous cell carcinoma protein with ID: 2DO4 and −5.49 docking score against squamous cell carcinoma protein with ID:5PJZ. ADME/T analysis was performed to examine the drug properties of studied molecules.

Novel thiazolotriazole and triazolothiadiazine scaffolds as selective tumor associated carbonic anhydrase inhibitors endowed with cathepsin B inhibition.

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

The physiological significance of plasma-accessible carbonic anhydrase in the respiratory systems of fishes.

Carbonic anhydrase (CA) activity is ubiquitously found in all vertebrate species, tissues and cellular compartments. Most species have plasma-accessible CA (paCA) isoforms at the respiratory surfaces, where the enzyme catalyzes the conversion of plasma bicarbonate to carbon dioxide (CO2) that can be excreted by diffusion. A notable exception are the teleost fishes that appear to lack paCA at their gills. The present review: (i) recapitulates the significance of CA activity and distribution in vertebrates; (ii) summarizes the current evidence for the presence or absence of paCA at the gills of fishes, from the basal cyclostomes to the derived teleosts and extremophiles such as the Antarctic icefishes; (iii) explores the contribution of paCA to organismal CO2 excretion in fishes; and (iv) the functional significance of its absence at the gills, for the specialized system of O2 transport in most teleosts; (v) outlines the multiplicity and isoform distribution of membrane-associated CAs in fishes and methodologies to determine their plasma-accessible orientation; and (vi) sketches a tentative time line for the evolutionary dynamics of branchial paCA distribution in the major groups of fishes. Finally, this review highlights current gaps in the knowledge on branchial paCA function and provides recommendations for future work.

Structure based exploration of mitochondrial alpha carbonic anhydrase inhibitors as potential leads for anti-obesity drug development.

Obesity has emerged as a major health challenge globally in the last two decades. Dysregulated fatty acid metabolism and de novo lipogenesis are prime causes for obesity development which ultimately trigger other co-morbid pathological conditions thereby risking life longevity. Fatty acid metabolism and de novo lipogenesis involve several biochemical steps both in cytosol and mitochondria. Reportedly, the high catalytically active mitochondrial carbonic anhydrases (CAVA/CAVB) regulate the intercellular depot of bicarbonate ions and catalyze the rapid carboxylation of pyruvate and acetyl-co-A to acetyl-co-A and malonate respectively, which are the precursors of fatty acid synthesis and lipogenesis. Several in vitro and in vivo investigations indicate inhibition of mitochondrial carbonic anhydrase isoforms interfere in the functioning of pyruvate, fatty acid and succinate pathways. Targeting of mitochondrial carbonic anhydrase isoforms (CAVA/CAVB) could thereby modulate gluconeogenetic as well as lipogenetic pathways and pave way for designing of novel leads in the development pipeline of anti-obesity medications. The present review unveils a diverse chemical space including synthetic sulphonamides, sulphamates, sulfamides and many natural bioactive molecules which selectively inhibit the mitochondrial isoform CAVA/CAVB with an emphasis on major state-of-art drug design strategies. More than 60% similarity in the structural framework of the carbonic anhydrase isoforms has converged the drug design methods towards the development of isoform selective chemotypes. While the benzene sulphonamide derivatives selectively inhibit CAVA/CAVB in low nanomolar ranges depending on the substitutions on the phenyl ring, the sulpamates and sulpamides potently inhibit CAVB. The virtual screening and drug repurposing methods have also explored many non-sulphonamide chemical scaffolds which can potently inhibit CAVA. The review could pave way for the development of novel and effective anti-obesity drugs which can modulate the energy metabolism.

Characterization of two carbonic anhydrase isoforms in the pulmonate snail (Lymnaea Stagnalis) and their involvement in Molluskan calcification

Calcifying organisms are suffering from negative impacts induced by climate change, such as CO2-induced acidification, which may impair external calcified structures. Freshwater mollusks have the potential to suffer more from CO2-induced acidification than marine calcifiers due to the lower buffering capacity of many freshwater systems. One of the most important enzymes contributing to the biomineralization reaction is carbonic anhydrase (CA), which catalyzes the reversible conversion of CO2 to bicarbonate, the major carbon source of the calcareous structure in calcifiers. In this study we characterized two α-CA isoforms (LsCA1 and LsCA4) from the freshwater snail Lymnaea stagnalis using a combination of gene sequencing, gene expression, phylogenetic analysis and biochemical assays. Both CA isoforms demonstrated high expression levels in the mantle tissue, the major site for biomineralization. Furthermore, expression of LsCA4 during development parallels shell formation. The primary protein structure analysis, active site configuration and the catalytic activity of LsCA4 together suggest that the LsCA4 is embedded in the apical and basolateral membranes of mantle cells; while LsCA1 is proposed to be cytosolic and might play an important role in acid-base regulation. These findings of LsCA isoforms form a strong basis for a more detailed physiological understanding of the effects of elevated CO2 on calcification in freshwater mollusks.

Glucoimines incorporating classical and non-classical carbonic anhydrase pharmacophores: Design, synthesis, conformational analysis, biological evaluation, and docking simulations

In this report, a series of glucoimines has been prepared by reaction of d-glucosamine and aromatic aldehydes incorporating classical and non-classical carbonic anhydrase pharmacophores. The conformational behavior of veratrole glucoimine was studied in solution and in crystalline form, by NMR and X-ray diffraction analysis, which revealed that pyranose ring adopted a 4C1 conformation. All glucoimines were tested against four isozymes of carbonic anhydrase: hCAs I and II (cytosolic, ubiquitous isozymes) and hCAs IX and XII (tumor-associated isozymes). In this study, per-O-acetylated and deprotected sulfonamide were identified as potent inhibitors of tumor-associated carbonic anhydrase isoforms. Molecular docking simulation was performed inside the active site of hCA II to evaluate the binding modes of veratrole and sulfonamide glucoimines. The last ones demonstrated the most favorable docking scores, indicating strong binding affinity towards hCAII in correlation with experimental inhibition activities. Interestingly, interaction analyses revealed distinct binding modes for ligand-hCAII complexes primarily due to the sulfonamide group.

Overexpression of native carbonic anhydrases increases carbon conversion efficiency in the methanotrophic biocatalyst Methylococcus capsulatus Bath.

Methanotrophic bacteria play a vital role in the biogeochemical carbon cycle due to their unique ability to use CH4 as a carbon and energy source. Evidence suggests that some methanotrophs, including Methylococcus capsulatus, can also use CO2 as a carbon source, making these bacteria promising candidates for developing biotechnologies targeting greenhouse gas capture and mitigation. However, a deeper understanding of the dual CH4 and CO2 metabolism is needed to guide methanotroph strain improvements and realize their industrial utility. In this study, we show that M. capsulatus expresses five carbonic anhydrase (CA) isoforms, one α-CA, one γ-CA, and three β-CAs, that play a role in its inorganic carbon metabolism and CO2-dependent growth. The CA isoforms are differentially expressed, and transcription of all isoform genes is induced in response to CO2 limitation. CA null mutant strains exhibited markedly impaired growth compared to an isogenic wild-type control, suggesting that the CA isoforms have independent, non-redundant roles in M. capsulatus metabolism and physiology. Overexpression of some, but not all, CA isoforms improved bacterial growth kinetics and decreased CO2 evolution from CH4-consuming cultures. Notably, we developed an engineered methanotrophic biocatalyst overexpressing the native α-CA and β-CA with a 2.5-fold improvement in the conversion of CH4 to biomass. Given that product yield is a significant cost driver of methanotroph-based bioprocesses, the engineered strain developed here could improve the economics of CH4 biocatalysis, including the production of single-cell protein from natural gas or anaerobic digestion-derived biogas.IMPORTANCEMethanotrophs transform CH4 into CO2 and multi-carbon compounds, so they play a critical role in the global carbon cycle and are of interest for biotechnology applications. Some methanotrophs, including Methylococcus capsulatus, can also use CO2 as a carbon source, but this dual one-carbon metabolism is incompletely understood. In this study, we show that M. capsulatus carbonic anhydrases are critical for this bacterium to optimally utilize CO2. We developed an engineered strain with improved CO2 utilization capacity that increased the overall carbon conversion to cell biomass. The improvements to methanotroph-based product yields observed here are expected to reduce costs associated with CH4 conversion bioprocesses.

Physiological modeling of the metaverse of the Mycobacterium tuberculosis β-CA inhibition mechanism

Tuberculosis (TB) is an infectious disease that primarily affects the lungs of humans and accounts for Mycobacterium tuberculosis (Mtb) bacteria as the etiologic agent. In this study, we introduce a computational framework designed to identify the important chemical features crucial for the effective inhibition of Mtb β-CAs. Through applying a mechanistic model, we elucidated the essential features pivotal for robust inhibition. Using this model, we engineered molecules that exhibit potent inhibitory activity and introduce relevant novel chemistry. The designed molecules were prioritized for synthesis based on their predicted pKi values via the QSAR (Quantitative Structure-Activity Relationship) model. All the rationally designed and synthesized compounds were evaluated in vitro against different carbonic anhydrase isoforms expressed from the pathogen Mtb; moreover, the off-target and widely human-expressed CA I and II were also evaluated. Among the reported derivatives, 2, 4, and 5 demonstrated the most valuable in vitro activity, resulting in promising candidates for the treatment of TB infection. All the synthesized molecules exhibited favorable pharmacokinetic and toxicological profiles based on in silico predictions. Docking analysis confirmed that the zinc-binding groups bind effectively into the catalytic triad of the Mtb β-Cas, supporting the in vitro outcomes with these binding interactions. Furthermore, molecules with good prediction accuracies according to previously established mechanistic and QSAR models were utilized to delve deeper into the realm of systems biology to understand their mechanism in combating tuberculotic pathogenesis. The results pointed to the key involvement of the compounds in modulating immune responses via NF-κβ1, SRC kinase, and TNF-α to modulate granuloma formation and clearance via T cells. This dual action, in which the pathogen's enzyme is inhibited while modulating the human immune machinery, represents a paradigm shift toward more effective and comprehensive treatment approaches for combating tuberculosis.

Study of Chalcogen Aspirin Derivatives with Carbonic Anhydrase Inhibitory Properties for Treating Inflammatory Pain.

Carbonic anhydrase (CA) inhibitors represent intriguing tools for treating pain. This study aims at studying the pharmacological profile of chalcogen bioisosteres of aspirin, as inhibitors of CA isoforms (hCA I, II, IV, VII, IX, and XII). Our results show that selenoaspirin (5) displayed markedly superior inhibitory potency across all tested isoforms compared to thioaspirin (7) and aspirin, with a strong selectivity against the isoform CA IX. X-ray crystallography confirmed that both compounds bind effectively within the active site of hCA II, revealing unique structural characteristics compared to those of aspirin. In a preclinical model of inflammatory pain, compound 7 exhibited a longer lasting antihyperalgesic effect than aspirin, though with a lower potency. Conversely, compound 5 exhibited both lower potency and efficacy than aspirin in reducing pain, which entailed both adverse effects. Nevertheless, the therapeutic potential of chalcogen-based aspirin derivatives as novel CA inhibitors deserves to be further explored for clinical applications.