Synthesis and Enzymatic Evaluation of a Small Library of Substituted Phenylsulfonamido-Alkyl Sulfamates towards Carbonic Anhydrase II
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SUBMITTER: Denner T
PROVIDER: S-EPMC11243685 | biostudies-literature | 2024 Jun
REPOSITORIES: biostudies-literature
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Project description:The visible absorption of crystals of Co(II)-substituted human carbonic anhydrase II (Co(II)-HCA II) were measured over a pH range of 6.0-11.0 giving an estimate of pK(a) 8.4 for the ionization of the metal-bound water in the crystal. This is higher by about 1.2 pK(a) units than the pK(a) near 7.2 for Co(II)-CA II in solution. This effect is attributed to a nonspecific ionic strength effect of 1.4M citrate in the precipitant solution used in the crystal growth. A pK(a) of 8.3 for the aqueous ligand of the cobalt was measured for Co(II)-HCA II in solution containing 0.8M citrate. Citrate is not an inhibitor of the catalytic activity of Co(II)-HCA II and was not observed in crystal structures. The X-ray structures at 1.5-1.6A resolution of Co(II)-HCA II were determined for crystals prepared at pH 6.0, 8.5 and 11.0 and revealed no conformational changes of amino-acid side chains as a result of the use of citrate. However, the studies of Co(II)-HCA II did reveal a change in metal coordination from tetrahedral at pH 11 to a coordination consistent with a mixed population of both tetrahedral and penta-coordinate at pH 8.5 to an octahedral geometry characteristic of the oxidized enzyme Co(III)-HCA II at pH 6.0.
| S-EPMC2937836 | biostudies-literature
Project description:Carbonic anhydrase IX (CA IX) is an extracellular transmembrane homodimeric zinc metalloenzyme that has been validated as a prognostic marker and therapeutic target for several types of aggressive cancers. CA IX shares a close homology with other CA isoforms, making the design of CA IX isoform selective inhibitors challenging. In this paper, we describe the development of a new class of CA IX inhibitors that comprise a sulfamate as the zinc binding group, a variable linker, and a carbohydrate "tail" moiety. Seven compounds inhibited CA IX with low nM Ki values of 1-2 nM and also exhibited permeability profiles to preferentially target the binding of extracellular CA IX over cytosolic CAs. The crystal structures of two of these compounds in complex with a CA IX-mimic (a variant of CA II, with active site residues that mimic CA IX) and one compound in complex with CA II have been determined to 1.7 Å resolution or better and demonstrate a selective mechanism of binding between the hydrophilic and hydrophobic pockets of CA IX versus CA II. These compounds present promising candidates for anti-CA IX drugs and the treatment for several aggressive cancer types.
| S-EPMC4207533 | biostudies-literature
Project description:Small molecule rescue of mutant forms of human carbonic anhydrase II (HCA II) occurs by participation of exogenous donors/acceptors in the proton transfer pathway between the zinc-bound water and solution. To examine more thoroughly the energetics of this activation, we have constructed a mutant, H64W HCA II, which we have shown is activated by 4-methylimidazole (4-MI) by a mechanism involving the binding of 4-MI to the side chain of Trp-64 approximately 8 A from the zinc. A series of experiments are consistent with the activation of H64W HCA II by the interaction of imidazole and pyridine derivatives as exogenous proton donors with the indole ring of Trp-64; these experiments include pH profiles and H/D solvent isotope effects consistent with proton transfer, observation of approximately fourfold greater activation with the mutant containing Trp-64 compared with Gly-64, and the observation by x-ray crystallography of the binding of 4-MI associated with the indole side chain of Trp-64 in W5A-H64W HCA II. Proton donors bound at the less flexible side chain of Trp-64 in W5A-H64W HCA II do not show activation, but such donors bound at the more flexible Trp-64 of H64W HCA II do show activation, supporting suggestions that conformational mobility of the binding site is associated with more efficient proton transfer. Evaluation using Marcus theory showed that the activation of H64W HCA II by these proton donors was reflected in the work functions w(r) and w(p) rather than in the intrinsic Marcus barrier itself, consistent with the role of solvent reorganization in catalysis.
| S-EPMC1751391 | biostudies-literature
Project description:Carbonic anhydrase has been well studied structurally and functionally owing to its importance in respiration. A large number of X-ray crystallographic structures of carbonic anhydrase and its inhibitor complexes have been determined, some at atomic resolution. Structure determination of a sulfonamide-containing inhibitor complex has been carried out and the structure was refined at 0.9 A resolution with anisotropic atomic displacement parameters to an R value of 0.141. The structure is similar to those of other carbonic anhydrase complexes, with the inhibitor providing a fourth nonprotein ligand to the active-site zinc. Comparison of this structure with 13 other atomic resolution (higher than 1.25 A) isomorphous carbonic anhydrase structures provides a view of the structural similarity and variability in a series of crystal structures. At the center of the protein the structures superpose very well. The metal complexes superpose (with only two exceptions) with standard deviations of 0.01 A in some zinc-protein and zinc-ligand bond lengths. In contrast, regions of structural variability are found on the protein surface, possibly owing to flexibility and disorder in the individual structures, differences in the chemical and crystalline environments or the different approaches used by different investigators to model weak or complicated electron-density maps. These findings suggest that care must be taken in interpreting structural details on protein surfaces on the basis of individual X-ray structures, even if atomic resolution data are available.
| S-EPMC2865367 | biostudies-literature
Project description:Human carbonic anhydrases (hCAs) belong to a well characterized group of metalloenzymes that catalyze the conversion of carbonic dioxide into bicarbonate. There are currently 15 known human isoforms of carbonic anhydrase with different functions and distribution in the body. This links to the relevance of hCA variants to several diseases such as glaucoma, epilepsy, mountain sickness, ulcers, osteoporosis, obesity and cancer. This review will focus on two of the human isoforms, hCA I and hCA II. Both are cytosolic enzymes with similar topology and 60% sequence homology but different catalytic efficiency and stability. Proteins in general adsorb on surfaces and this is also the case for hCA I and hCA II. The adsorption process can lead to alteration of the original function of the protein. However, if the function is preserved interesting biotechnological applications can be developed. This review will cover the knowledge about the interaction between hCAs and nanomaterials. We will highlight how the interaction may lead to conformational changes that render the enzyme inactive. Moreover, the importance of different factors on the final effect on hCAs, such as protein stability, protein hydrophobic or charged patches and chemistry of the nanoparticle surface will be discussed.
| S-EPMC7582742 | biostudies-literature
Project description:Fragment screening is a powerful tool to identify and characterize binding pockets in proteins. We herein present the results of a proof-of-concept screening campaign of a versatile 96-entry fragment library from our laboratory against the drug target and model protein human carbonic anhydrase II. The screening revealed a novel chemotype for carbonic anhydrase inhibition, as well as less common non-covalent interaction types and unexpected covalent linkages. Lastly, different runs of the PanDDA tool reveal a practical hint for its application.
| S-EPMC7226012 | biostudies-literature
Project description:Cobalt(II)-substituted Haemophilus influenzae β-carbonic anhydrase (HICA) has been produced by overexpression in minimal media supplemented with CoCl(2), enabling kinetic, structural, and spectroscopic characterization. Co(II)-substituted HICA (Co-HICA) has comparable catalytic activity to that of wild-type enzyme with k(cat)=82±19 ms(-1) (120% of wild-type). The X-ray crystal structure of Co-HICA was determined to 2.5Å resolution, and is similar to the zinc enzyme. The absorption spectrum of Co-HICA is consistent with four-coordinate geometry. pH-dependent changes in the absorption spectrum of Co-HICA, including an increase in molar absorptivity and a red shift of a 580 nm peak with decreasing pH, correlate with the pH dependence of k(cat)/K(m). The absence of isosbestic points in the pH-dependent absorption spectra suggest that more than two absorbing species are present. The addition of bicarbonate ion at pH 8.0 triggers spectral changes in the metal coordination sphere that mimic that of lowering pH, supporting its hypothesized role as an allosteric inhibitor of HICA. Homogeneously (99±1% Co) and heterogeneously (52±5% Co) substituted Co-HICA have distinctly different colors and absorption spectra, suggesting that the metal ions in the active sites in the allosteric dimer of Co-HICA engage in intersubunit communication.
| S-EPMC3115399 | biostudies-literature
Project description:Aryl sulfonamides are a widely used drug class for the inhibition of carbonic anhydrases. In the context of our program of photochromic pharmacophores we were interested in the exploration of azobenzene-containing sulfonamides to block the catalytic activity of human carbonic anhydrase II (hCAII). Herein, we report the synthesis and in vitro evaluation of a small library of nine photochromic sulfonamides towards hCAII. All molecules are azobenzene-4-sulfonamides, which are substituted by different functional groups in the 4´-position and were characterized by X-ray crystallography. We aimed to investigate the influence of electron-donating or electron-withdrawing substituents on the inhibitory constant K i. With the aid of an hCAII crystal structure bound to one of the synthesized azobenzenes, we found that the electronic structure does not strongly affect inhibition. Taken together, all compounds are strong blockers of hCAII with K i = 25-65 nM that are potentially photochromic and thus combine studies from chemical synthesis, crystallography and enzyme kinetics.
| S-EPMC4505086 | biostudies-literature
Project description:Carbonic anhydrase II (CAII) is a metalloenzyme that catalyzes the reversible hydration/dehydration of CO2/HCO3-. In addition, CAII is attributed to other catalytic reactions, including esterase activity. Aspirin (acetyl-salicylic acid), an everyday over-the-counter drug, has both ester and carboxylic acid moieties. Recently, compounds with a carboxylic acid group have been shown to inhibit CAII. Hence, we hypothesized that Aspirin could act as a substrate for esterase activity, and the product salicylic acid (SA), an inhibitor of CAII. Here, we present the crystal structure of CAII in complex with SA, a product of CAII crystals pre-soaked with Aspirin, to 1.35Å resolution. In addition, we provide kinetic data to support the observation that CAII converts Aspirin to its deacetylated form, SA. This data may also explain the short half-life of Aspirin, with CAII so abundant in blood, and that Aspirin could act as a suicide inhibitor of CAII.
| S-EPMC7226357 | biostudies-literature
Project description:Carbonic anhydrase (CA, general abbreviation for human carbonic anhydrase II) is a well-studied, zinc-dependent metalloenzyme that catalyzes hydrolysis of carbon dioxide to the bicarbonate ion. The apo-form of CA (apoCA, CA where Zn(2+) ion has been removed) is relatively easy to generate, and reconstitution of the human erythrocyte CA has been initially investigated. In the past, these studies have continually relied on equilibrium dialysis measurements to ascertain an extremely strong association constant (K(a) ≈ 1.2 × 10(12)) for Zn(2+). However, new reactivity data and isothermal titration calorimetry (ITC) data reported herein call that number into question. As shown in the ITC experiments, the catalytic site binds a stoichiometric quantity of Zn(2+) with a strong equilibrium constant (K(a) ≈ 2 × 10(9)) that is 3 orders of magnitude lower than the previously established value. Thermodynamic parameters associated with Zn(2+) binding to apoCA are unraveled from a series of complex equilibria associated with the in vitro metal binding event. This in-depth analysis adds clarity to the complex ion chemistry associated with zinc binding to carbonic anhydrase and validates thermochemical methods that accurately measure association constants and thermodynamic parameters for complex-ion and coordination chemistry observed in vitro. Additionally, the zinc sites in both the as-isolated and the reconstituted ZnCA (active CA containing a mononuclear Zn(2+) center) were probed using X-ray absorption spectroscopy. Both X-ray absorption near edge structure (XANES) and extended X-ray absorption fine structure (EXAFS) analyses indicate the zinc center in the reconstituted carbonic anhydrase is nearly identical to that of the as-isolated protein and confirm the notion that the metal binding data reported herein is the reconstitution of the zinc active site of human CA II.
| S-EPMC4066895 | biostudies-literature