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A series of coumarins and the corresponding 2-thioxocoumarines were prepared and tested for their inhibition profiles against four physiologically relevant human carbonic anhydrases (hCAs, EC 4.2.1.1), isoforms hCA I, II, IX, and XII. The X-ray crystal structure of 6-hydroxy-2-thioxocoumarin bound to hCA II revealed an unprecedented and unexpected inhibition mechanism for this new class of inhibitors when compared to isostructural coumarins. Unlike coumarins which are hydrolyzed by the esterase CA activity to the corresponding 2-hydroxy-cinnamic acid derivatives, the 2-thioxocoumarin was observed intact when bound to hCA II, with its exo-sulfur atom anchored to the zinc-coordinated water molecule, whereas the scaffold establishing favorable contacts with amino acid residues from the active site. This inhibition mechanism is very different from the one observed for hydrolyzed coumarins, which occlude the entrance of the active site cavity. This versatility in the binding mode of coumarins/thioxocoumarins has important consequences for the design of isoform-selective CA inhibitors, some of which are in clinical use or clinical development for various pathologies, among which glaucoma, edema, epilepsy, neuropathic pain, and hypoxic tumors. [ABSTRACT FROM AUTHOR]

Sulfonamides and coumarins incorporatingarylsulfonylureido tails were prepared and assayed as inhibitors ofthe metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1). Some derivativesincorporating 3-pyridinesulfonamide and arylsulfonylureoido fragmentswere low nanomolar inhibitors of isoforms CA II and XII (upregulatedor overexpressed in glaucoma) and showed effective in vivo intraocularpressure lowering effects in an animal model of the disease, whichwere several times better compared to those of the antiglaucoma drugdorzolamide. By means of X-ray crystallography of adducts of severalsulfonamides with CA II, the effective inhibitory properties wererationalized at the molecular level. The coumarins were ineffectiveas hCA I and II inhibitors but showed low nanomolar activity for theinhibition of the tumor-associated isoforms hCA IX and XII. The presenceof arylsulfonylureido tails in these CA inhibitors possessing quitedifferent mechanisms of action led to highly effective and isoform-selectivecompounds targeting enzymes involved in severe pathologies such asglaucoma or cancer. [ABSTRACT FROM AUTHOR]

The crystal structures of sulfanilamide (4-aminobenzene-sulfonamide) complexed to the enzyme variant human carbonic anhydrase I Michigan 1 (CA I M1) and to its (Zn)2 adduct refined at 2.2 and 2.6 A˚ resolution respectively are described. Comparisons among these structures and the corresponding sulfonamide adduct of human CA I show significant differences in the orientation of the inhibitor molecule and in its interactions with active site residues such as His200, Thr199, Leu198, Gln92, and Arg/His67 which are known to play important roles in substrate or inhibitor recognition and binding. In CA I M1 molecule B a lengthening of the Zn&z.sbnd;N1 sulfanilamide bond distance and a corresponding shortening of the distance between the sulfonamido group and Thr199 are observed compared with the values for native CA I. When the second Zn(II) ion is present in the active site, the p-amino group and the aromatic ring of the inhibitor molecule appear to tilt towards Gln92 and Arg67, moving away from residues His200 and Leu198. The structural differences in inhibitor binding between the CA I isozyme and the CA I M1 variant are discussed in terms of the different inhibition constants measured for a variety of aromatic and heterocyclic sulfonamides. [Copyright &y& Elsevier]

Streptococcus pneumoniae hyaluronate lyase (spnHL) is a pathogenic bacterial spreading factor and cleaves hyaluronan, an important constituent of the extra-cellular matrix of connective tissues, through an enzymatic Β-elimination process, different from the hyaluronan degradation by hydrolases in animals. The mechanism of hyaluronan binding and degradation was proposed based on the 1.56 Å resolution crystal structure, substrate modeling and mutugenesis studies on spnHL. Five mutants, R243V, N349A, H399A, Y408F and N580G, were constructed and their activities confirmed our mechanism hypothesis. The important roles of Tyr408, Asn349 and His399 in enzyme catalysis were proposed, explained and confirmed by mutant studies. The remaining weak enzymatic activity of the H399A mutant, the role of the free carboxylate group on the glucuronate residue, the enzymatic behavior on chondroitin and chondroitin sulfate, and the small activity increase in the N580G mutant were explained based on this mechanism. A possible function of the C-terminal Β-sheet domain is to modulate enzyme activity through binding to calcium ions. [ABSTRACT FROM AUTHOR]

The blue laccase from the white-rot basidiomycete fungus Panus tigrinus, an enzyme involved in lignin biodegradation, has been crystallized. P. tigrinus laccase crystals grew within one week at 296 K using the sitting-drop vapour- diffusion method in 22%(w/v) PEG 4000, 0.2 M CaCl2, 100 mM Tris-HCl pH 7.5. The crystals belong to the monoclinic space group P21, with unit-cell parameters a = 54.2, b = 111.6, c = 97.1, β = 97.7°, and contain 46% solvent. A complete native data set was collected to 1.4 Å resolution at the copper edge. Molecular replacement using the Coprinus cinereus laccase structure (PDB code 1 hfu) as a starting model was performed and initial electron-density maps revealed the presence of a full complement of copper ions. Model refinement is in progress. The P. tigrinus laccase structural model exhibits the highest resolution available to date and will assist in further elucidation of the catalytic mechanism and electron-transfer processes for this class of enzymes. [ABSTRACT FROM AUTHOR]

3-Chlorocatechol 1,2-dioxygenase (3-ClC1,2DO), a key enzyme of a new modified ortho-pathway, was isolated from a variant of the Gram-positive bacterium Rhodococcus opacus 1CP utilizing 2-chlorophenol as the sole energy and carbon source via a 3-chlorocatechol branch of a modified ortho-pathway. 3-ClC1,2DO catalyzes the intradiol cleavage of 3-chlorocatechol. The enzyme contains Fe[sup III] ions essential to the catalytic activity; it is a homodimer with a molecular weight of about 58 kDa composed of two identical subunits in an (αFe)[sub 2]-type quaternary structure. Its physicochemical properties are intermediate between those of the pyrocatechase from the ordinary pathway and those of the chloro-pyrocatechase from the modified pathway described previously for this strain. 3-ClC1,2DO was crystallized using the sitting-drop vapour-diffusion method. After 2 d, prismatic crystals grew in 15% PEG 8000, 0.3 M magnesium acetate, 100 mM HEPES pH 7.5, 5% glycerol. X-ray diffraction data were collected from a frozen crystal to a maximum resolution of 2.0 Å using 25% PEG 400 as cryoprotectant at the Elettra synchrotron source, Trieste, Italy, at a wavelength of 1.01 Å using a MAR CCD detector. The crystals belong to space group P1, with unit-cell parameters a = 83.18, b = 86.61, c = 93.44 Å. Assuming a reasonable range for V[sub M], the asymmetric unit could contain from three to five (αFe[sup III])[sub 2] dimers. A peak present in the κ = 180° and κ = 90° sections is consistent with a fourfold axis and four dimers in the asymmetric unit. Comparison of the crystal structure of this enzyme with that of the 4chlorocatechol 1,2-dioxygenase recently crystallized from the same bacterium (Ferraroni et al., 2002) may reveal important details of the influence of the active-site conformation and the amino-acid substitutions involved in substrate selectivity. [ABSTRACT FROM AUTHOR]

4-Chlorocatechol 1,2-dioxygenase (4-ClC1,2DO) from the Gram- positive bacterium Rhodococcus opacus (erythropolis) 1CP, an enzyme involved in the aerobic biodegradation of chloroaromatic compounds, has been crystallized. 4-ClC1,2DO, which specifically catalyzes the intradiol cleavage of 4-substituted catechols, which are intermediates in the degradation of a variety of aromatic pollutants, to the corresponding maleylacetates, has recently been purified to homogeneity. The enzyme is an homodimer composed of two identical subunits in an α2-type quaternary structure; it has a molecular weight of about 29 kDa per monomer and contains one FeIII and one MnIII ion per homodimer. Hexagonal crystals grown in 1.6 M ammonium sulfate, 0.1 M sodium chloride, 100 mM Tris-HCl pH 9.0, 5-15% glycerol were successfully frozen under liquid nitrogen, adding 30% glycerol to the mother-liquor solution as a cryoprotectant. A complete data set was collected at 2.8 Å resolution using the EMBL beamline BW7A at the DORIS storage ring, DESY, Hamburg, Germany with a MAR CCD detector and a wavelength of 1.01 Å. The crystals belong to the primitive hexagonal space group P6322, with unit-cell parameters a = 90.4, c = 307.5 Å. This is the first intradiol dioxygenase which specifically catalyzes the cleavage of chlorocatechols in Gram-positive bacteria to give diffraction-quality crystals. [ABSTRACT FROM AUTHOR]

Carbonic anhydrase inhibitors (CAIs) of the sulfonamide, sulfamate and coumarin classes bearing the phenylureido tail found in the clinically used drug Sorafenib, a multikinase inhibitor actually used for the management of hepatocellular carcinomas, are reported. All compounds were assayed on human (h) CA isoforms I, II, VII and IX, involved in various pathologies. Among the sulfonamides, several compounds were selective for inhibiting hCA IX, with K I values in the low nanomolar ranges (i.e. 0.7–30.2 nM). We explored the binding modes of such compounds by means of X-ray crystallographic studies on isoform hCA I in adduct with one sulfonamide and a sulfamate inhibitor. Antiproliferative properties of some sulfamates on breast tumor cell lines were also investigated. Image 1 • CAIs by merging the multikinase inhibitor sorafenib with the benzene sulfonamide hCA IX inhibitor SLC-0111 are reported. • Sulfamates 23 and 24 decrease proliferation breast cancer cell lines at concentrations above 10 μM. • The binding modes of compounds 17 and 23 on the hCA I isoform by means of X-ray crystallographic studies are reported. [ABSTRACT FROM AUTHOR]

The intravascular parasitic worm Schistosoma mansoni is a causative agent of schistosomiasis, a disease of great global public health significance. Here we identify an α-carbonic anhydrase (SmCA) that is expressed at the schistosome surface as determined by activity assays and immunofluorescence/immunogold localization. Suppressing SmCA expression by RNAi significantly impairs the ability of larval parasites to infect mice, validating SmCA as a rational drug target. Purified, recombinant SmCA possesses extremely rapid CO2 hydration kinetics (kcat: 1.2 × 106 s-1; kcat/Km: 1.3 × 108 M-1s-1). The enzyme's crystal structure was determined at 1.75 Å resolution and a collection of sulfonamides and anions were tested for their ability to impede rSmCA action. Several compounds (phenylarsonic acid, phenylbaronic acid, sulfamide) exhibited favorable Kis for SmCA versus two human isoforms. Such selective rSmCA inhibitors could form the basis of urgently needed new drugs that block essential schistosome metabolism, blunt parasite virulence and debilitate these important global pathogens. Akram Da'dara et al. report the biochemical characterization of an α-carbonic anhydrase (SmCA) expressed at the surface of the parasitic worm Schistosoma mansoni. Along with the crystal structure of SmCA, they show the function of selective inhibitors in blocking essential schistosome metabolism. [ABSTRACT FROM AUTHOR]

Herein, we describe the design and synthesis of novel aryl pyrimidine benzenesulfonamides APBSs 5a-n , 6a-c , 7a-b , and 8 as pazopanib analogues to explore new potent and selective inhibitors for the CA IX. All APBSs were examined in vitro for their promising inhibition activity against a small panel of hCAs (isoforms I, II, IX, and XII). The X-ray crystal structure of CA I in adduct with a representative APBS analogue was solved. APBS - 5m , endowed with the best hCA IX inhibitory efficacy and selectivity, was evaluated for antiproliferative activity against a small panel of different cancer cell lines, SK-MEL-173, MDA-MB-231, A549, HCT-116, and HeLa, and it demonstrated one-digit IC 50 values range from 2.93 μM (MDA-MB-231) to 5.86 μM (A549). Furthermore, compound APBS - 5m was evaluated for its influence on hypoxia-inducible factor (HIF-1α) production, apoptosis induction, and colony formation in MDA-MB-231 cancer cells. The in vivo efficacy of APBS - 5m as an antitumor agent was additionally investigated in an animal model of Solid Ehrlich Carcinoma (SEC). In order to offer perceptions into the conveyed hCA IX inhibitory efficacy and selectivity in silico , a molecular docking investigation was also carried out. [Display omitted] • Aryl pyrimidine benzene sulfonamides (APBSs) were synthesized and characterized. • APBS - 5m showed top inhibitory activity towards CA IX (K I = 2.7 nM). • The anticancer activity of APBS - 5m was superior against MDA-MB-231 cancer cells. • APBS - 5m displayed potent proapoptotic activity on hypoxia sensitized MDA-MB-231. • X-ray crystal structure of CA I- APBS - 5b adduct was solved (PDB code: 8QUN). [ABSTRACT FROM AUTHOR]

Functionalised aliphatic selenols, straightforwardly obtained through ring-opening reaction of strained heterocycles, represent a new chemotype acting as carbonic anhydrase inhibitors (CAIs). These compounds showed pronounced selectivity towards the cytosolic human (h) isoforms such as the hCA I, II and VII rather than the membrane tumor associate hCA IX. In addition, we reported for the first time the X-ray crystal structure of an aliphatic selenol bound to the hCA I zinc ion, and that afforded the opportunity to decipher in detail the inhibition mechanism underpinning such a new class of CAIs. In this context selenols are interesting leads worth developing for the obtainment of novel and efficient selective CAIs potentially useful for the management of a variety of diseases including glaucoma, retinitis pigmentosa, epilepsy and arthritis. Image 1 • Direct and Straightforward synthesis of stable alkyl selenols. • Novel human carbonic anhydrases inhibitors. • Selectivity against cytosolic human carbonic anhydrase isoforms. • X-ray co-crystallographic studies. [ABSTRACT FROM AUTHOR]

• Human neutrophil elastase (HNE) • Porcine Pancreatic Elastase (PPE) • Study of inhibition mechanism of HNE inhibitors through X-ray crystallography • Formation of acyl-enzyme complex involving a nucleophilic attack by Ser195 Our purpose was to structurally investigate through X-ray crystallography the inhibition mechanism of small molecule inhibitors of human neutrophil elastase (HNE), a serine protease associated with some serious chronic inflammatory diseases, especially pulmonary pathologies. In particular, we selected some of the compounds previously synthesized by our research group, such as 5/4-azaindole, azaindazole and isoxazolone derivatives, which are promising therapeutic agents with an inhibition potency in the nanomolar range. In this work, we adopted Porcine Pancreatic Elastase (PPE) as a structural model for its high homology with the human enzyme, especially in the active site. The crystallographic study allowed us to gain information about the binding mode and inhibition mechanism of these new HNE inhibitors. In fact, we obtained the structures of the complexes of PPE with a fragment of the inhibitors resulted from the nucleophilic attack by the catalytic residue Ser195. In this way we confirmed an acyl enzyme inhibition mechanism for all the inhibitor scaffolds used. Furthermore, for the isoxazolone derivatives which possess two carbonyl groups we identified the exocyclic CO group as the one susceptible to the Ser attack. [Display omitted] [ABSTRACT FROM AUTHOR]

Multi-target compounds have become increasingly important for the development of safer and more effective drug candidates. In this work, we devised a combined ligand-based and structure-based multi-target repurposing strategy and applied it to a series of hexahydrocyclopenta[ c ]quinoline compounds synthesized previously. The in silico analyses identified human Carbonic Anhydrases (h CA) and Estrogen Receptors (ER) as top scoring candidates for dual modulation. h CA isoforms IX and XII, and ER subtypes ER⍺ and/or ERβ are co-expressed in various cancer cell types, including breast and prostate cancer cells. ER⍺ is the primary target of anti-estrogen therapy in breast cancer, and the h CA IX isoform is a therapeutic target in triple-negative breast cancer. ER⍺-mediated transcriptional programs and h CA activity in cancer cells promote favorable microenvironments for cell proliferation. Interestingly, several lines of evidence indicate that the combined modulation of these two targets may provide significant therapeutic benefits. Moving from these first results, two additional hexahydrocyclopenta[ c ]quinoline derivatives bearing a sulfonamide zinc binding group (h CA) and a phenolic hydroxyl (ER) pharmacophoric group placed at the appropriate locations were designed and synthesized. Interestingly, these compounds were able to directly modulate the activities of both h CA and ER targets. In cell-based assays, they inhibited proliferation of breast and prostate cancer cells with micromolar potency and cell type-selective efficacy. The compounds inhibited h CA activity with nanomolar potency and isoform-selectivity. In transactivation assays, they reduced estrogen-driven ER activity with micro-molar potency. Finally, crystal structures of the synthesized ligands in complex with the two targets revealed that the compounds bind directly to the h CA active site, as well as to the ER ligand-binding domain, providing structural explanation to the observed activity and a rationale for optimization of their dual activity. To the best of our knowledge, this work describes the design, synthesis and biological characterization of the first dual modulators of h CA and ER, laying the ground for the structure-based optimization of their multi-target activity. [Display omitted] • In silico screening allowed to repurpose a series of compounds on h CA and ER. • Three hexahydrocyclopenta[ c ]quinoline compounds showed activity on h CA and ER. • The designed compounds were crystallized in complex with the targets. • The compounds inhibited the proliferation of breast and prostate cancer cells. [ABSTRACT FROM AUTHOR]

[Display omitted] • Synthesis of benzoselenoates as novel chemotype of hCA inhibitors. • In vitro studies against different hCA isoforms. • X-ray structure of hCA I and hCA II of them to obtain ligand−protein interaction of novel chemotype. A series of benzoselenoates has been prepared and their inhibitory properties against the most relevant human Carbonic Anhydrases (CAs) isoforms, among which hCA I, II, IV, VII, IX, and XII were investigated. These inhibitors were designed considering the carboxylates and mono-/dithiocarbamates as lead and led to the observation that the COSe- is a new zinc-binding group (ZBG) for metalloenzymes possessing zinc ions at their active site. The substitution pattern on aromatic ring of the benzoselenoates is the crucial structural element influencing selectivity towards various isoforms. We elucidated the binding mode of benzoselenoates to hCA I and hCA II by using X-ray crystallography. The negatively charged selenium atom from the new ZBG was observed coordinated to the zinc ion from the CA active site at a distance of 2.30–2.40 Å from it. Overall, these data might be useful for the development of new inhibitors with higher selectivity and efficacy for various hCAs. [ABSTRACT FROM AUTHOR]

Platinum-based chemotherapy is widely used for the treatment of different tumors but is associated with serious side effects, among which neuropathic pain. Carbonic anhydrase (CA, EC 4.2.1.1) inhibitors have recently been validated as therapeutic agents in neuropathic pain and as antitumor agents. We report the synthesis of new organochalcogenides bearing the benzensulfonamide moiety acting as potent inhibitors of several human CA isoforms and, in particular, against hCA II and VII endowed with potent neuropathic pain attenuating effects. Moreover, in combination with cisplatin or doxorubicin, some of the new CA inhibitors enhanced the effects of the anticancer drugs capability in counteracting breast cancer MCF7 cell viability. The concomitant anti-neuropathic pain and antiproliferative effects of the new chalcogenide-based CA inhibitors represent an innovative approach for the counteraction and management of side effects associated with clinically platinum drugs as antitumor agents. [Display omitted] • New chalcogen-containing potent hCA inhibitors. • X-rays investigations. • In vitro and in vivo studies. • Neuropathic pain attenuation. • Enhanced action of anticancer drugs. [ABSTRACT FROM AUTHOR]

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

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

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

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

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

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., Competing Interests: The authors declare no competing financial interest., (© 2024 American Chemical Society.)

A novel series of sulfonamide-incorporated bis(α-aminophosphonates) acting as effective carbonic anhydrase (CA, EC 4.2.1.1) inhibitors is reported. The synthesized bivalent ligands were tested against five human (h) isoforms, hCA I, hCA II, hCA VII, hCA IX, and hCA XIII. Such derivatives showed high activity and selectivity against the cancer-related, membrane-bound isoform hCA IX, and among them, compound 5h, tetraisopropyl (1,3-phenylenebis{[(4-sulfamoylphenyl)amino]methylene})bis(phosphonate) showed a K I of 15.1 nM, being highly selective against this isoform over all other investigated ones (hCA I/IX = 42; hCA II/IX = 6, hCA VII/IX = 3, hCA XIII/IX = 5). Therefore, compound 5h could be a potential lead for the development of selective anticancer agents. The newly developed sulfonamides were also found effective inhibitors against the cytosolic hCA XIII isoform. Compound 5i displayed the best inhibition against this isoform with a K I of 17.2 nM, equal to that of the well-known inhibitor acetazolamide (AAZ), but significantly more selective over all other tested isoforms (hCA I/XIII = 239; hCA II/XIII = 23, hCA VII/XIII = 2, hCA IX/XIII = 3) compared to AAZ., (© 2024 Deutsche Pharmazeutische Gesellschaft.)

The inhibitory effects of veralipride, a benzamide-class antipsychotic acting as dopamine D 2 receptors antagonist incorporates a primary sulfonamide moiety and was investigated for its interactions with carbonic anhydrase (CA) isoforms. In vitro profiling using the stopped-flow technique revealed that veralipride exhibited potent inhibitory activity across all tested hCA isoforms, with exception of hCA III. Comparative analysis with standard inhibitors, acetazolamide (AAZ), and sulpiride, provided insights for understanding the relative efficacy of veralipride as CA inhibitor. The study reports the X-ray crystal structure analysis of the veralipride adduct with three human (h) isoforms, hCA I, II, and CA XII mimic, allowing the understanding of the molecular interactions rationalizing its inhibitory effects against each isoform. These findings contribute to our understanding of veralipride pharmacological properties and for the design of structural analogs endowed with polypharmacological properties., (© 2024 Wiley‐VCH GmbH.)

In the last decades, carbonic anhydrases (CAs) have become the top investigated innovative pharmacological targets and, in particular, isoforms IX and XII have been widely studied due to the evidence of their overexpression in hypoxic tumors. The frantic race to find new anticancer agents places the quick preparation of large libraries of putative bioactive compounds as the basis of a successful drug discovery and development programme. In this context, multi-component and, in general, one-step reactions are becoming very popular and, among them, Biginelli's reaction gave clean and easy-to-isolate products. Thus, we synthesized a series of Biginelli's products (10-17a-b) and similar derivatives (20-21) bearing the benzenesulfonamide moiety, which is known to inhibit CA enzymes. Through the stopped-flow technique, we were able to assess their ability to inhibit the targeted CAs IX and XII in the nanomolar range with promising selectivity over the physiologically relevant isoforms I and II. Crystallography studies and docking simulations helped us to gain insight into the interaction patterns established in the enzyme-inhibitor complex. From a chemical similarity-based screening of in-house libraries of compounds, a diphenylpyrimidine (23) emerged. The surprisingly potent inhibitory activity of 23 for CAs IX and XII along with its strong antiproliferative effect on two (triple-negative breast cancer MDA-MB-231 and glioblastoma U87MG) cell lines laid the foundation for further investigation, again confirming the key role of CAs in cancer., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Glaucoma, a leading cause of irreversible vision loss worldwide, is characterized by elevated intraocular pressure (IOP), a well-established risk factor across all its forms. We present the design and synthesis of 39 novel carbonic anhydrase inhibitors by a dual-tailed approach, strategically crafted to interact with distinct hydrophobic and hydrophilic pockets of CA active sites. The series was investigated against the CA isoforms implicated in glaucoma (hCA II, hCA IV, and hCA XII), and the X-ray crystal structures of compounds 25a , 25f , and 26a with CA II, along with 14b in complex with a hCA XII mimic, were determined. Selected compounds ( 14a , 25a , and 26a ) underwent evaluation for their ability to reduce IOP in rabbits with ocular hypertension. Derivative 26a showed significant potency and sustained IOP-lowering effects, surpassing the efficacy of the drugs dorzolamide and bimatoprost. This positions compound 26a as a promising candidate for the development of a novel anti-glaucoma medication.

New dihydro-pyrrol-2-one compounds, featuring dual sulfonamide groups, were synthesized through a one-pot, three-component approach utilizing trifluoroacetic acid as a catalyst. Computational analysis using density functional theory (DFT) and condensed Fukui function explored the structure-reactivity relationship. Evaluation against human carbonic anhydrase isoforms (hCA I, II, IX, XII) revealed potent inhibition. The widely expressed cytosolic hCA I was inhibited across a range of concentrations ( K I 3.9-870.9 nM). hCA II, also cytosolic, exhibited good inhibition as well. Notably, all compounds effectively inhibited tumor-associated hCA IX ( K I 1.9-211.2 nM) and hCA XII (low nanomolar). Biological assessments on MCF7 cancer cells highlighted the compounds' ability, in conjunction with doxorubicin, to significantly impact tumor cell viability. These findings underscore the potential therapeutic relevance of the synthesized compounds in cancer treatment.

β-Carbonic anhydrases (β-CA; EC 4.2.1.1) are widespread zinc metalloenzymes which catalyze the interconversion of carbon dioxide and bicarbonate. They have been isolated in many pathogenic and non-pathogenic bacteria where they are involved in multiple roles, often related to their growth and survival. β-CAs are structurally distant from the CAs of other classes. In the active site, located at the interface of a fundamental dimer, the zinc ion is coordinated to two cysteines and one histidine. β-CAs have been divided in two subgroups depending on the nature of the fourth ligand on the zinc ion: class I have a zinc open configuration with a hydroxide ion completing the metal coordination, which is the catalytically active species in the mechanism proposed for the β-CAs similar to the well-known of α-CAs, while in class II an Asp residue substitute the hydroxide. This latter active site configuration has been showed to be typical of an inactive form at pH below 8. An Asp-Arg dyad is thought to play a key role in the pH-induced catalytic switch regulating the opening and closing of the active site in class II β-CAs, by displacing the zinc-bound solvent molecule. An allosteric site well-suited for bicarbonate stabilizes the inactive form. This bicarbonate binding site is composed by a triad of well conserved residues, strictly connected to the coordination state of the zinc ion. Moreover, the escort site is a promiscuous site for a variety of ligands, including bicarbonate, at the dimer interface, which may be the route for bicarbonate to the allosteric site., (Copyright © 2024. Published by Elsevier Inc.)