Comparison of the Sulfonamide Inhibition Profiles of the ?-Carbonic Anhydrase Isoforms (SpiCA1, SpiCA2 and SpiCA3) Encoded by the Genome of the Scleractinian Coral Stylophora pistillata.
ABSTRACT: The ubiquitous metalloenzymes carbonic anhydrases (CAs, EC 188.8.131.52) are responsible for the reversible hydration of CO? to bicarbonate (HCO?-) and protons (H?). Bicarbonate may subsequently generate carbonate used in many functional activities by marine organisms. CAs play a crucial role in several physiological processes, e.g., respiration, inorganic carbon transport, intra and extra-cellular pH regulation, and bio-mineralization. Multiple transcript variants and protein isoforms exist in the organisms. Recently, 16 ?-CA isoforms have been identified in the coral Stylophora pistillata. Here, we focalized the interest on three coral isoforms: SpiCA1 and SpiCA2, localized in the coral-calcifying cells; and SpiCA3, expressed in the cytoplasm of the coral cell layers. The three recombinant enzymes were heterologously expressed and investigated for their inhibition profiles with sulfonamides and sulfamates. The three coral CA isoforms differ significantly in their susceptibility to inhibition with sulfonamides. This study provides new insights into the coral physiology and the comprehension of molecular mechanisms involved in the bio-mineralization processes, since CAs interact with bicarbonate transporters, accelerating the trans-membrane bicarbonate movement and modulating the pH at both sides of the plasma membranes.
Project description:Carbonic anhydrases (CAs, EC 184.108.40.206) are widespread metalloenzymes used by living organisms to accelerate the CO₂ hydration/dehydration reaction at rates dramatically high compared to the uncatalyzed reaction. These enzymes have different isoforms and homologues and can be found in the form of cytoplasmic, secreted or membrane-bound proteins. CAs play a role in numerous physiological processes including biomineralization and symbiosis, as is the case in reef-building corals. Previously, molecular and biochemical data have been obtained at the molecular level in the branching coral Stylophora pistillata for two coral isoforms which differ significantly in their catalytic activity and susceptibility to inhibition with anions and sulfonamides. More recently it has been determined that the genome of S. pistillata encodes for 16 CAs. Here, we cloned, expressed, purified and characterized a novel α-CA, named SpiCA3, which is cytoplasmic and ubiquitously expressed in all the cell layers including the calcifying cells. SpiCA3 is the most effective CA among the coral isoforms investigated and the most efficient catalyst known up to date in Metazoa. We also investigated the inhibition profiles of SpiCA3 and compared it with those obtained for the two other isoforms in the presence of inorganic anions and other small molecules known to interfere with metalloenzymes. These results suggest that S. pistillata has adapted its CA isoforms to achieve the physiological functions in different physicochemical microenvironments.
Project description:Carbonic anhydrases (CAs, EC 220.127.116.11) are metalloenzymes which catalyze the hydration of carbon dioxide to bicarbonate and protons. Many pathogenic bacteria encode such enzymes belonging to the ?-, ?-, and/or ?-CA families. In the last decade, enzymes from some of these pathogens, including Legionella pneumophila, have been cloned and characterized in detail. These enzymes were shown to be efficient catalysts for CO? hydration, with kcat values in the range of (3.4-8.3) × 10? s(-1) and kcat/KM values of (4.7-8.5) × 10? M(-1)·s(-1). In vitro inhibition studies with various classes of inhibitors, such as anions, sulfonamides and sulfamates, were also reported for the two ?-CAs from this pathogen, LpCA1 and LpCA2. Inorganic anions were millimolar inhibitors, whereas diethyldithiocarbamate, sulfamate, sulfamide, phenylboronic acid, and phenylarsonic acid were micromolar ones. The best LpCA1 inhibitors were aminobenzolamide and structurally similar sulfonylated aromatic sulfonamides, as well as acetazolamide and ethoxzolamide (KIs in the range of 40.3-90.5 nM). The best LpCA2 inhibitors belonged to the same class of sulfonylated sulfonamides, together with acetazolamide, methazolamide, and dichlorophenamide (KIs in the range of 25.2-88.5 nM). Considering such preliminary results, the two bacterial CAs from this pathogen represent promising yet underexplored targets for obtaining antibacterials devoid of the resistance problems common to most of the clinically used antibiotics, but further studies are needed to validate them in vivo as drug targets.
Project description:Carbonic anhydrases (CAs, EC 18.104.22.168) are metalloenzymes which catalyze the hydration of carbon dioxide to bicarbonate and protons. Many pathogenic bacteria encode such enzymes belonging to the ?-, ?-, and/or ?-CA families. In the last decade, the ?-CAs from Neisseria spp. and Helicobacter pylori as well as the ?-class enzymes from Escherichia coli, H. pylori,Mycobacterium tuberculosis, Brucella spp., Streptococcus pneumoniae, Salmonella enterica, and Haemophilus influenzae have been cloned and characterized in detail. For some of these enzymes the X-ray crystal structures were determined, and in vitro and in vivo inhibition studies with various classes of inhibitors, such as anions, sulfonamides and sulfamates reported. Although efficient inhibitors have been reported for many such enzymes, only for Neisseria spp., H. pylori, B. suis, and S. pneumoniae enzymes it has been possible to evidence inhibition of bacterial growth in vivo. Thus, bacterial CAs represent promising targets for obtaining antibacterials devoid of the resistance problems of the clinically used such agents but further studies are needed to validate these and other less investigated enzymes as novel drug targets.
Project description:Aragonite skeletons in corals are key contributors to the storage of atmospheric CO2 worldwide. Hence, understanding coral biomineralization/calcification processes is crucial for evaluating and predicting the effect of environmental factors on this process. While coral biomineralization studies have focused on adult corals, the exact stage at which corals initiate mineralization remains enigmatic. Here, we show that minerals are first precipitated as amorphous calcium carbonate and small aragonite crystallites, in the pre-settled larva, which then evolve into the more mature aragonitic fibers characteristic of the stony coral skeleton. The process is accompanied by modulation of proteins and ions within these minerals. These findings may indicate an underlying bimodal regulation tactic adopted by the animal, with important ramification to its resilience or vulnerability toward a changing environment.
Project description:A series of modified saccharin sulfonamides have been designed as carbonic anhydrase (CA) inhibitors and synthesized. Their binding to CA isoforms I, II, VII, XII, and XIII was measured by the fluorescent thermal shift assay (FTSA) and isothermal titration calorimetry (ITC). Saccharin bound the CAs weakly, exhibiting the affinities of 1-10 mM for four CAs except CA I where binding could not be detected. Several sulfonamide-bearing saccharines exhibited strong affinities of 1-10 nM towards particular CA isoforms. The functional group binding Gibbs free energy additivity maps are presented which may provide insights into the design of compounds with increased affinity towards selected CAs.
Project description:Specific isoforms from the carbonic anhydrase (CA) family of zinc metalloenzymes have been associated with a variety of diseases. Isoform-specific carbonic anhydrase inhibitors (CAIs) are therefore a major focus of attention for specific disease treatments. Classical CAIs, primarily sulfonamide-based compounds and their bioisosteres, are examined as antiglaucoma, antiepileptic, antiobesity, antineuropathic pain and anticancer compounds. However, many sulfonamide compounds inhibit all CA isoforms nonspecifically, diluting drug effectiveness and causing undesired side effects due to off-target inhibition. In addition, a small but significant percentage of the general population cannot be treated with sulfonamide-based compounds due to a sulfa allergy. Therefore, CAIs must be developed that are not only isoform specific, but also non-classical, i.e. not based on sulfonamides, sulfamates, or sulfamides. This review covers the classes of non-classical CAIs and the recent advances in the development of isoform-specific inhibitors based on phenols, polyamines, coumarins and their derivatives.
Project description:By using N-?-acetyl-l-lysine or GABA scaffolds and the conversion of the terminal amino group to the guanidine one, benzenesulfonamides incorporating water solubilizing moieties were synthesized. The new compounds were medium potency inhibitors of the cytosolic carbonic anhydrase (CA, EC 22.214.171.124) isoforms I and II, and highly effective, nanomolar inhibitors of the pathogenic bacterial ?-CA from Vibrio cholerae. These sulfonamides possess good selectivity for inhibiting the bacterial over the mammalian isoforms and may be used as tools to understand the role of bacterial CAs in pathogenesis.
Project description:A method to activate sulfamoyl fluorides, fluorosulfates, and sulfonyl fluorides with calcium triflimide and DABCO for SuFEx with amines is described. The reaction was applied to a diverse set of sulfamides, sulfamates, and sulfonamides at room temperature under mild conditions. Additionally, we highlight this transformation to parallel medicinal chemistry to generate a broad array of nitrogen-based S(VI) compounds.
Project description:Carbonic anhydrases (CAs) are ubiquitous metallo-enzymes that catalyse the reversible hydration of carbon dioxide to bicarbonate and proton. In humans there are 15 isoforms among which only 12 are catalytically active. Since active human (h) CAs show different efficiency, the understanding of the molecular determinants affecting it is a matter of debate. Here we investigated, by a site-specific mutagenesis approach, residues modulating the catalytic features of one of the least investigated cytosolic isoform, i.e. hCA XIII. Results showed that residues assisting the formation of an ordered solvent network within the catalytic site as well as those forming a histidine cluster on the protein surface are important to guarantee an efficient proton transfer.
Project description:Carbonic anhydrases (CAs) are a family of enzymes involved in the pH regulation of metabolically active cells/tissues. Upregulation of the CAIX/XII isoforms is associated with hypoxic tumours and clinically linked with malignant progression, treatment resistance and poor prognosis. The elucidation of the crystal structure of the catalytic domains of CAIX/XII provided the basis for the generation of CAIX/XII selective inhibitors based on the sulfonamide, sulfamate and coumarins chemical structures. Ureido-substituted benzenesulfonamide CAIX/XII inhibitors have shown significant potential, with U-104 (SLC-0111) currently present in clinical Phase I/II. Ureido-substituted sulfamate CAIX/XII inhibitors have received less attention despite encouraging preclinical test results. In triple-negative breast cancer (TNBC), ureidosulfamates revealed a significant antitumour (FC9-398A) and antimetastatic potential (S4). In small cell lung cancer (SCLC), a cancer cell type very sensitive to a dysregulation in CAIX signaling, S4 treatment was particularly effective when combined with cisplatin with no evidence of acquired cisplatin-resistance. These successful anticancer strategies should provide a solid basis for future studies on ureido-substituted sulfamates.