Active Site Of Carbonic Anhydrase Research Articles

Synthesis, Antibacterial Activity and Molecular Modelling of Benzyl Acetate Derivatives

Benzyl alcohol derivatives are known for their antibacterial efficacy. In this work five known benzyl acetate derivatives were synthesized by the acetylation of their corresponding benzyl alcohol derivatives and their structures confirmed using 1H, 13C NMR and FTIR spectroscopic techniques. The synthesized compounds were tested for antibacterial activity against Staphylococcus aureus and Shigella spp using disc diffusion method. Also the activity of amoxicillin disc (0.5 g/L ) was measured as a positive control. Furthermore, the drug-likeness as well as the interactions of the compounds against the active site of E. coli carbonic anhydrase which share >98% similarity to that of S. spp were studied using molecular modelling method. The antibacterial activity showed that all the five compounds 3a-e inhibited the two organisms at 100 µg/ml compared to the positive control. The largest inhibition zones of Staphylococcus aureus and Shigella spp were found to be 16.5 mm and 17.5 mm for compound 3d and 3e, respectively. Molecular modelling predicted the compounds to be water soluble, highly absorbed through GI tract, not Pgp substrates and not CYP3A4 inhibitors. Molecular docking studies showed that the compounds showed affinity to E. coli carbonic anhydrase active site, blocking access to the Zn2+ cofactor.

NO Generation from Nitrite at Zinc(II): Role of Thiol Persulfidation in the Presence of Sulfane Sulfur.

Nitrite-to-NO transformation is of prime importance due to its relevance in mammalian physiology. Although such a one-electron reductive transformation at various redox-active metal sites (e.g., Cu and Fe) has been illustrated previously, the reaction at the [ZnII] site in the presence of a sacrificial reductant like thiol has been reported to be sluggish and poorly understood. Reactivity of [(Bn3Tren)ZnII-ONO](ClO4) (1), a nitrite-bound model of the tripodal active site of carbonic anhydrase (CA), toward various organic probes, such as 4-tert-butylbenzylthiol (tBuBnSH), 2,4-di-tert-butylphenol (2,4-DTBP), and 1-fluoro-2,4-dinitrobenzene (F-DNB), reveals that the ONO-moiety in the [ZnII]-nitrite coordination motif of complex 1 acts as a mild electrophile. tBuBnSH reacts mildly with nitrite at a [ZnII] site to provide S-nitrosothiol tBuBnSNO prior to the release of NO in 10% yield, whereas the phenolic substrate 2,4-DTBP does not yield the analogous O-nitrite compound (ArONO). The presence of sulfane sulfur (S0) species such as elemental sulfur (S8) and organic polysulfides (tBuBnSnBntBu) during the reaction of tBuBnSH and [ZnII]-nitrite (1) assists the nitrite-to-NO conversion to provide NO yields of 65% (for S8) and 76% (for tBuBnSnBntBu). High-resolution mass spectrometry (HRMS) analyses on the reaction of [ZnII]-nitrite (1), tBuBnSH, and S8 depict the formation of zinc(II)-persulfide species [(Bn3Tren)ZnII-Sn-BntBu]+ (where n = 2, 3, 4, 5, and 6). Trapping of the persulfide species (tBuBnSS-) with 1-fluoro-2,4-dinitrobenzene (F-DNB) confirms its intermediacy. The significantly higher nucleophilicity of persulfide species (relative to thiol/thiolate) is proposed to facilitate the reaction with the mildly electrophilic [ZnII]-nitrite (1) complex. Complementary analyses, including multinuclear NMR, electrospray ionization-MS, UV-vis, and trapping of reactive S-species, provide mechanistic insights into the sulfane sulfur-assisted reactions between thiol and nitrite at the tripodal [ZnII]-site. These findings suggest the critical influential roles of various reactive sulfur species, such as sulfane sulfur and persulfides, in the nitrite-to-NO conversion.

A combined DFT and molecular docking study on novel tricarbonylrhenium(I) complexes bearing mono- and bivalent benzenesulfonamide scaffolds as human carbonic anhydrase IX and XII inhibitors

The aim of this study was to determine the reactivity and stability properties of a novel series of tricarbonylrhenium(I) complexes with 1,2,3-triazole-pyridine based on monovalent and bivalent benzenesulfonamide ligands as well as analyze their binding affinity within the active site of carbonic anhydrases IX and XII, using DFT and molecular docking. In the first setup, the DFT method was used to investigate the molecular geometries and vibrational frequencies of ligands and their Re(I) complexes. The basis set used was B3LYP/LanL2DZ-ECP/6-31G(d). The optimized geometric parameters (bond lengths and bond angles) and vibrational frequencies agree well with the experimental results. TD-DFT calculations were also performed to determine the influence of the methylene linker (-CH2-) on the bands found in the electronic spectra of these compounds. Global reactivity indices calculated from the energies of frontier molecular orbitals were used to assess their reactivity and stability properties (FMOs). Molecular electrostatic potential maps of the molecules were calculated to provide information about their chemical reactivity and to explain their intermolecular interactions. The computational data showed that bivalent ligands and their bivalent Re(I) complexes are more reactive than their monovalent counterparts. The second setup involves docking studies of the compounds within the active sites of the receptors (6QN2 for hCA IX and 1JD0 for hCA XII), which are performed using the Molegro Virtual Docker and AutoDock 4.2 programs, respectively. Our results indicate that bivalent ligands and their associated Re(I) complexes have a greater binding affinity than monovalent ligands and their associated Re(I) complexes. Compounds bis-L1 and bis-L2 exhibited the best affinity potential for hCA IX and hCA XII inhibition, making them the most promising candidates among those examined.

Novel benzenesulfonamide-thiouracil conjugates with a flexible N-ethyl acetamide linker as selective CA IX and CA XII inhibitors.

Novel benzenesulfonamide derivatives linked to diverse functionalized thiouracils through a flexible N-ethyl acetamide linker were designed and synthesized as carbonic anhydrase (CA) inhibitors. The synthesized candidates demonstrated a potent inhibitory activity against four different CA isoforms in the nanomolar range. Compound 10d showed more than twofold higher potency than the reference AAZ against CA II with Ki of 5.65 and 12 nM, respectively. Moreover, compounds 10d and 20 revealed potent activity against CA IX with Ki of 18.1 and 14.2 nM, respectively. In addition, 10c, 10d, 11b, 11c, and 20 demonstrated high potency against the CA XII isozyme with a Ki range of 4.18-4.8 nM. Most of the synthesized derivatives displayed preferential selectivity towardthe CA IX and CA XII isoforms over CA I and CA II. Compounds 11a and 20 exhibited favorable selectivity toward CA IX over CA II with a selectivity index (SI) of 14.36 and 16.62, respectively, and toward CA XII over CA II with SI of 71.01 and 51.19, respectively. Molecular docking simulations showed that the synthesized conjugates adopted comparable binding modes in the CA I, CA II, CA IX, and CA XII isoforms, involving the deep fitting of the sulfonamide moiety in the base of the CA active site via chelation of the Zn2+ ion and H-bond interaction with the key amino acids Thr199 and/or Thr200. Moreover, the N-ethyl acetamide flexible linker enables the substituted thiouracils and fused thiouracil tail to achieve multiple interactions with the surrounding hydrophobic and hydrophilic regions.

Insights into the effect of elaborating coumarin-based aryl enaminones with sulfonamide or carboxylic acid functionality on carbonic anhydrase inhibitory potency and selectivity

Recently, different mechanisms for inhibition of carbonic anhydrases (CAs) have been reported, such as the classical zinc-binding (exerted by sulfonamides and carboxylic acids) as well as occluding the entrance of the CA active site (exerted by coumarins). In this manuscript, we studied the effect of combining the pharmacopheric parts responsible for these two mechanisms on CA inhibitory potency and selectivity through the design and synthesis of novel coumarins tethered with the zinc-binding sulfonamide (5a-f, 11a-b and 13a-b) or carboxylic acid (7a-f) groups. In addition, another set of coumarin derivatives (9a-b) with no zinc-binding group (ZBG) was designed to act as non-classical CA inhibitors. The synthesized coumarins were examined for their inhibitory activities towards four hCA isoforms I, II, IX and XII. Coumarin sulfonamides (5a-f, 11a-b and 13a-b) effectively inhibited both tumor-associated hCA IX (KIs: 8.9–133.5 nM) and hCA XII (KIs: 3.4–42.9 nM) isoforms, whereas coumarin carboxylic acids (7a-f) weakly affected hCA IX (KIs: 0.49–11.2 μM) and hCA XII (KIs: 0.51–10.1 μM) isoforms. The coumarin based inhibitors featuring zinc-binding sulfonamide or carboxylic acid group achieved low to moderate hCA IX/XII selectivity. Interestingly, the ZBG-free coumarin derivatives (9a-b) emerged not only as effective hCA IX (KIs = 93.3 and 63.8 nM, respectively) and hCA XII (KIs = 85.7 and 72.1 nM, respectively) inhibitors, but also as a highly hCA IX/XII selective inhibitors over the off-target hCA I/II isoforms (SIs > 1000). Coumarin 9a was further evaluated for its anti-proliferative effect on MCF-7 and PANC-1 cancer cell lines, as well as its effect on the cell cycle and apoptosis towards MCF-7 cell line.

Benzoselenoates: A novel class of carbonic anhydrase inhibitors

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.

Application of the dual-tail approach for the design and synthesis of novel Thiopyrimidine–Benzenesulfonamide hybrids as selective carbonic anhydrase inhibitors

A dual-tail approach was applied to the design of a novel series of 2-thiopyrimidine–benzenesulfonamides as carbonic anhydrase (CA) inhibitors. The design strategy is based on the hybridization between a benzenesulfonamide moiety as Zn2+ binding group and 2,4-disubstituted thiopyridimidine as a tail. Among the synthesized compounds, 14h displayed the highest potency (Ki = 1.72 nM) and selectivity for CA II over the isoforms CA IX and CA XII with selectivity indexes of 50 and 5.26, respectively. Meanwhile, compounds 14a and 14l displayed a potent inhibitory activity against CA IX (Ki = 7.4 and 7.0 nM, respectively) compared with the reference drug acetazolamide (AAZ) (Ki = 25 nM), and compound 14l showed higher potency (Ki = 4.67 nM) than AAZ (Ki = 5.7 nM) against the tumor-associated isoform CA XII. Evaluation of the antiproliferative activity in NCI single-dose testing of selected hybrids revealed a pronounced potency of the selective CA II inhibitor 14h against most of the tested NCI cancer cell lines. Moreover, compound 14h demonstrated an IC50 values ranging from 2.40 to 4.50 μM against MCF-7, T-47D, MDA-MB-231, HCT-116, HT29 and SW-620. These results demonstrate that CA II inhibition can be an alternative therapeutic target for cancer treatment. A cell cycle analysis of MCF-7 and MDA-MB-231 showed that treatment with 14h arrested both cell lines at the G2/M phase with significant accumulation of cells in the pre-G1 phase. Moreover, compound 14h showed a noticeable induction of late apoptosis and necrotic cell death of both cell lines compared with untreated cells as a control. A molecular docking study suggested that the sulfonamide moiety accommodates deeply in the CA active site and interacts with the Zn2+ ion while the dual-tail extension interacts with the surrounding amino acids via several hydrophilic and hydrophobic interactions, which affects the potency and selectivity of the hybrids.

Structure and mechanism of secondary sulfonamide binding to carbonic anhydrases.

Zinc-containing metalloenzyme carbonic anhydrase (CA) binds primary sulfonamides with extremely high, up to picomolar, affinity by forming a coordination bond between the negatively charged amino group and the zinc ion and making hydrogen bonds and hydrophobic contacts with other parts of the inhibitor molecule. However, N-methyl-substituted, secondary or tertiary sulfonamides bind CA with much lower affinity. In search for an explanation for this diminished affinity, a series of secondary sulfonamides were synthesized and, together with analogous primary sulfonamides, the affinities for 12 recombinant catalytically active human CA isoforms were determined by the fluorescent thermal shift assay, stopped-flow assay of the inhibition of enzymatic activity and isothermal titration calorimetry. The binding profile of secondary sulfonamides as a function of pH showed the same U-shape dependence seen for primary sulfonamides. This dependence demonstrated that there were protein binding-linked protonation reactions that should be dissected for the estimation of the intrinsic binding constants to perform structure-thermodynamics analysis. X-ray crystallographic structures of secondary sulfonamides and computational modeling dissected the atomic contributions to the binding energetics. Secondary sulfonamides bind to carbonic anhydrases via coordination bond between the negatively charged nitrogen of alkylated amino group and Zn(II) in the active site of CA. The binding reaction is linked to deprotonation of the amino group and protonation of the Zn(II)-bound hydroxide. To perform the structure-thermodynamics analysis, contributions of these linked reactions must be subtracted to determine the intrinsic energetics. In this aspect, the secondary sulfonamides are similar to primary sulfonamides as CA inhibitors.

Halogenated and di-substituted benzenesulfonamides as selective inhibitors of carbonic anhydrase isoforms

By applying an approach of a “ring with two tails”, a series of novel inhibitors possessing high-affinity and significant selectivity towards selected carbonic anhydrase (CA) isoforms has been designed. The “ring” consists of 2-chloro/bromo-benzenesulfonamide, where the sulfonamide group is as an anchor coordinating the Zn(II) in the active site of CAs, and halogen atom orients the ring affecting the affinity and selectivity. First “tail” is a substituent containing carbonyl, carboxyl, hydroxyl, ether groups or hydrophilic amide linkage. The second “tail” contains aryl- or alkyl-substituents attached through a sulfanyl or sulfonyl group. Both “tails” are connected to the benzene ring and play a crucial role in selectivity. Varying the substituents, we designed compounds selective for CA VII, CA IX, CA XII, or CA XIV.Since due to binding-linked protonation reactions the binding-ready fractions of the compound and protein are much lower than one, the “intrinsic” affinities were calculated that should be used to study correlations between crystal structures and the thermodynamics of binding for rational drug design. The “intrinsic” affinities together with the intrinsic enthalpies and entropies of binding together with co-crystal structures were used demonstrate structural factors determining major contributions for compound affinity and selectivity.

Application of hydrazino and hydrazido linkers to connect benzenesulfonamides with hydrophilic/phobic tails for targeting the middle region of human carbonic anhydrases active site: Selective inhibitors of hCA IX

Herein we report the design and synthesis of three different sets of novel benzenesulfonamides (5a-e, 7a-e and 10a-d) incorporating hydrophilic/hydrophobic tails by hydrazido or hydrazino linkers. The newly synthesized benzenesulfonamides were examined in vitro for their inhibitory activity towards four human (h) carbonic anhydrase (hCA, EC 4.2.1.1) isoforms, hCA I, II, IX and XII using a stopped-flow CO2 hydrase assay. All these isoforms were inhibited by the sulfonamides (5a-e, 7a-e and 10a-d) with variable degrees in the following KI ranges: 76.8–357.4 nM for hCA I, 8.2–94.6 nM for hCA II, 2.0–46.3 nM for hCA XI, and 8.3–88.3 nM for hCA XII. The sulfonamide 7d exhibited potent anti-proliferative activity against breast MCF-7 cancer cell line under both normoxic and hypoxic conditions with IC50 values equal 3.32 ± 0.06 and 8.53 ± 0.32 μM, respectively, which are comparable to the reference drug doxorubicin (IC50 = 2.36 ± 0.04 and 8.39 ± 0.25 μM, respectively). Furthermore, 7d was screened for cell cycle disturbance and apoptosis induction in MCF-7 cells. It was found to persuade cell cycle arrest at G2-M stage as well as to alter the Sub-G1 phase, also, 7d resulted in a significant increase in the percent of annexinV-FITC positive apoptotic cells from 1.03 to 18.54%. Molecular docking study was carried out for 7d within the hCA IX and hCA XII active sites to rationalize the obtained inhibition results.

Reparametrization of the COSMO Solvent Model for Semiempirical Methods PM6 and PM7.

An accurate description of solvation effects is of high importance in modeling biomolecular systems. Our main interest is to find an accurate yet efficient solvation model for semiempirical quantum-mechanical methods applicable to large protein-ligand complexes in the context of computer-aided drug design. We present a survey of readily available methods and a new reparametrization of the COSMO solvent model for PM6 and PM7 calculations in MOPAC. We have tested the reparametrized method on validation data sets of small drug-like molecules for which experimental solvation free energies are available as well as on a set of large model systems of the active site of carbonic anhydrase II interacting with a series of ligands for which experimental affinity values are known. In both cases, there is a significant improvement in accuracy after the reparametrization and the addition of a nonpolar term to the COSMO solvent model.

Metalloenzyme Mimicry at the Nodes of Metal-Organic Frameworks

In this issue of Chem, Dincă and co-workers show that the node of the metal-organic framework MFU-4l can be modified by anion exchange to serve as a biomimetic model of carbonic anhydrase given the similarity in their ligand fields. The resulting material, which has reactivity characteristics of the enzyme, gives great promise for CO2-related applications. In this issue of Chem, Dincă and co-workers show that the node of the metal-organic framework MFU-4l can be modified by anion exchange to serve as a biomimetic model of carbonic anhydrase given the similarity in their ligand fields. The resulting material, which has reactivity characteristics of the enzyme, gives great promise for CO2-related applications. Enzymes, nature’s creation of catalysts, show extreme activity and selectivity for a range of chemical conversions. However, even when an enzyme is identified as being useful for a given reaction, its extensive application is often hampered by its lack of long-term operational stability and also by difficulties in recovery and recycling.1Schmid A. Dordick J.S. Hauer B. Kiener A. Wubbolts M. Witholt B. Industrial biocatalysis today and tomorrow.Nature. 2001; 409: 258-268Crossref PubMed Scopus (2017) Google Scholar Advances have been made in biotechnology to allow for enzyme production at commercially acceptable prices and manipulation of them such that they exhibit the desired properties with regard to substrate specificity, activity, selectivity, and stability. Nevertheless, only a handful of successful paradigms that function properly have been synthesized. To overcome this ambiguity, biomimicry provides rational solutions through the incorporation of active-site synthetic analogs of enzymes in suitable matrices by taking advantage of the versatility of synthetic chemstry.2Sanchez C. Arribart H. Guille M.M.G. Biomimetism and bioinspiration as tools for the design of innovative materials and systems.Nat. Mater. 2005; 4: 277-288Crossref PubMed Scopus (1167) Google Scholar Metal-organic frameworks (MOFs) are envisioned as the most promising candidate for this purpose.3Chen Y. Ma S. Biomimetic catalysis of metal-organic frameworks.Dalton Trans. 2016; 45: 9744-9753Crossref PubMed Google Scholar, 4Nath I. Chakraborty J. Verpoort F. Metal organic frameworks mimicking natural enzymes: a structural and functional analogy.Chem. Soc. Rev. 2016; 45: 4127-4170Crossref PubMed Google Scholar, 5Doonan C. Riccò R. Liang K. Bradshaw D. Falcaro P. Metal−organic frameworks at the biointerface: synthetic strategies and applications.Acc. Chem. Res. 2017; 50: 1423-1432Crossref PubMed Scopus (391) Google Scholar MOFs are highly tunable nanoporous network compounds composed of inorganic nodes connected by organic linkers, and they offer tremendous potential for rational material design.6Zhou H.-C. Long J.R. Yaghi O.M. Introduction to metal-organic frameworks.Chem. Rev. 2012; 112: 673-674Crossref PubMed Scopus (5091) Google Scholar Their crystalline structures trace out topological backbones onto which desired functionalities can be introduced, either as functional moieties on the organic linkers or through the choice of metal nodes. The molecular-level structural control and modularity possible with MOFs allow for a hydrophobic or hydrophilic confined environment akin to natural enzymes, as well as offer an ordered distribution of highly dense substrate-accessible active catalytic sites throughout the architecture. In addition, the higher stability of MOFs than of enzymes potentially leads to the broader use of biomimetic chemistry in industrially relevant applications. Carbonic anhydrase (CA) is a ubiquitous zinc metalloenzyme that catalyzes the reversible hydration of CO2 with an exceptional turnover rate of 106 per second, one of the fastest rates in enzyme catalysis.7Parkin G. Synthetic analogues relevant to the structure and function of zinc enzymes.Chem. Rev. 2004; 104: 699-767Crossref PubMed Scopus (828) Google Scholar To expand their utilization, researchers have therefore employed a large variety of tridentate ligands in efforts to obtain synthetic analogs of CA. Among them, the hydroxide complex TptBu,MeZnOH (Tp = tris(pyrazolyl)hydroborate) has also been shown to be a functional analog of CA. Unfortunately, these systems lack heterogeneous analogs with similar activities, significantly hindering their industrial use. Given the similar ligand fields, the nodes of MOFs are attractive sites for mimicking metalloenzymes. In this issue of Chem, Dincă and co-workers took the lessons learned from these homogeneous systems and targeted the material MFU-4l (Zn5Cl4(BTDD)3, where H2BTDD = bis(1H-1,2,3-triazolo[4,5-b],[4’,5′-i ])dibenzo[1,4]dioxin).8Wright A.M. Wu Z. Zhang G. Mancuso J.L. Comito R.J. Day R.W. Hendon C.H. Miller J.T. Dincă M. A structural mimic of carbonic anhydrase in a metal-organic framework.Chem. 2018; 4: 2894-2901Abstract Full Text Full Text PDF Scopus (58) Google Scholar The secondary building unit (SBU) of this triazole-based MOF features facial coordination of Zn2+ by three nitrogen atoms and is a scorpionate unit shown to structurally and functionally emulate the Tp-like environments within this MOF (Figures 1A and 1B ). In this paper, the benefits of using MOFs as a biomimetic platform are at the forefront: pore environments and structural tunability, site isolation, high surface area, and periodicity are all highly sought-after attributes for any efficient heterogeneous catalyst. The crystalline nature of MOFs offers opportunities to use their nodes as structurally characterizable active sites where mechanistic details of a catalytic process can be teased out, allowing for rational design and thereby improved overall efficiency. Herein, the structure of MFU-4l lends itself perfectly as the platform for mimicking CA for two primary reasons: (1) the SBUs in this material feature four peripheral zinc chloride sites that are coordinated by three triazoles in C3 symmetry analogous to the Tp ligand and, accordingly, CA, and (2) the metal node of MFU-4l can be further modified by anion exchange to serve as a biomimetic model of CA. The original terminal N3Zn–Cl centers are quantitatively transformed to terminal N3Zn–OH units by treatment with tetrabutylammonium hydroxide to yield MFU-4l-(OH) with a conserved framework structure. After confirming the structure, Dincă and co-workers proceeded to probe the enzymatic reactivity of MFU-4l-(OH). Considering that the essential function of CA is to catalyze the reversible hydration of carbon dioxide and a critical step in this process is CO2 insertion into the Zn–OH bond, the authors thus studied its CO2 sorption behavior by collecting CO2 sorption isotherms. To gain more insight into the framework-adsorbate interaction, they calculated the isosteric heat of adsorption (Qst) as a function of the amount of CO2 adsorbed on the basis of the adsorption isotherms measured at three different temperatures (288, 298, and 308 K). Calculated with the virial equation, the Qst at zero CO2 coverage was 81 kJ mol−1, reflecting a strong chemisorption mechanism and agreeing with the reaction enthalpies determined for CO2 fixation by transition-metal hydroxide complexes (49–61 kJ mol−1). With increasing coverage, the Qst stayed relatively constant up to 0.5 equiv of CO2 per SBU and then steadily decreased from 62 to 35 kJ mol−1 for the second CO2 equivalent and from 35 to 20 kJ mol−1 for the third CO2 equivalent. To reveal the underlying mechanism, the authors carried out quantum density functional theory computations to understand the enthalpies required for CO2 insertion into different Zn–OH units. The trend of decreasing binding enthalpy along with the CO2 adsorption is consistent with the experimental data. These results suggest that with the conversion of the highly basic –OH to weakly basic –OCO2H (Figure 1C), the nucleophilicity of the remaining Zn–OH sites within a given metal node are weakened, thereby resulting in decreased Qst values. Given the significant uptake capacity (3.41 mmol g−1 at 298 K) and limited energy cost required for regeneration, this material gives great promise for CO2 capture and sequestration. To further prove the ability of MFU-4l-(OH) to perform CA-like reactivity, the authors investigated the reversible hydration of CO2 by studying the exchange of 18O-labeled H2O with gaseous CO2. The time to reach an equilibrium mixture of the three possible CO2 isotopologues (C16O2, C16,18O2, and C18O2) was significantly reduced from 10 to 5 hr after MFU-4l-(OH) was introduced. Also, MFU-4l-(OH) exhibited activity in the hydrolysis of 4-nitrophenyl acetate (4-NPA) with a 15% ± 2% conversion after 24 hr, in contrast with 1.7% ± 0.2% conversion of the blank control. These results thereby prove that the metal nodes of MFU-4l can be functionalized to serve as a high-fidelity biomimetic model of the CA active site. This contribution demonstrates an elegant example of functionalizing a MOF metal node to mimic the activity of an enzyme. Facilitated by the modularity of MOFs and the diverse toolbox of synthetic modification techniques that they offer, it is conceivable that MOF-based materials show great potential in the field of biomimetic chemistry.9Jin C. Zhang S. Zhang Z. Chen Y. Mimic carbonic anhydrase using metalorganic frameworks for CO2 capture and conversion.Inorg. Chem. 2018; 57: 2169-2174Crossref PubMed Scopus (46) Google Scholar, 10Bien C.E. Chen K.K. Chien S.-C. Reiner B.R. Lin L.-C. Wade C.R. Ho W.S.W. Bioinspired metal-organic framework for trace CO2 capture.J. Am. Chem. Soc. 2018; 140: 12662-12666Crossref PubMed Scopus (90) Google Scholar Taking inspiration from enzymes, it will be intriguing to see the advantages that MOFs can offer in advanced material design. As this paper shows, MOFs with enzyme-like active sites afford great opportunities to push the boundaries of numerous challenges faced nowadays, such as CO2 capture and sequestration. With high structural fidelity and reactivity, MOFs hold potential for future understanding of enzyme chemistry and also the potential to perform enzyme-inspired reactions in stable frameworks. As more and more stable MOFs are discovered, researchers are beginning to tap into the potential of this class of material for industrially viable applications. A Structural Mimic of Carbonic Anhydrase in a Metal-Organic FrameworkWright et al.ChemOctober 4, 2018In BriefThe nodes of metal-organic frameworks are attractive sites for mimicking metalloenzymes, primarily through their site isolation and similar ligand fields. In this article, the metal-organic framework MFU-4l is shown to mimic the active site of carbonic anhydrase with high structural fidelity and reactivity. The material adsorbs high quantities of carbon dioxide at low pressures and mimics critical features of carbonic anhydrase, such as isotopic exchange of oxygen atoms from water and carbon dioxide. Full-Text PDF Open Archive

Selective inhibition of carbonic anhydrase IX over carbonic anhydrase XII in breast cancer cells using benzene sulfonamides: Disconnect between activity and growth inhibition.

Carbonic anhydrases (CAs) have been linked to tumor progression, particularly membrane-bound CA isoform IX (CA IX). The role of CA IX in the context of breast cancer is to regulate the pH of the tumor microenvironment. In contrast to CA IX, expression of CA XII, specifically in breast cancer, is associated with better outcome despite performing the same catalytic function. In this study, we have structurally modeled the orientation of bound ureido-substituted benzene sulfonamides (USBs) within the active site of CA XII, in comparison to CA IX and cytosolic off-target CA II, to understand isoform specific inhibition. This has identified specific residues within the CA active site, which differ between isoforms that are important for inhibitor binding and isoform specificity. The ability of these sulfonamides to block CA IX activity in breast cancer cells is less effective than their ability to block activity of the recombinant protein (by one to two orders of magnitude depending on the inhibitor). The same is true for CA XII activity but now they are two to three orders of magnitude less effective. Thus, there is significantly greater specificity for CA IX activity over CA XII. While the inhibitors block cell growth, without inducing cell death, this again occurs at two orders of magnitude above the Ki values for inhibition of CA IX and CA XII activity in their respective cell types. Surprisingly, the USBs inhibited cell growth even in cells where CA IX and CA XII expression was ablated. Despite the potential for these sulfonamides as chemotherapeutic agents, these data suggest that we reconsider the role of CA activity on growth potentiation.

In Situ Photoregulation of Carbonic Anhydrase Activity Using Azobenzenesulfonamides.

We report two small molecule azobenzenesulfonamide probes, CAP1 and CAP2, capable of photomodulating the activity of carbonic anhydrase (CA) on demand. In the trans form, CAP azobenzene probes adopt a linear shape, making them suitable for occupying the CA active site and interacting with Zn2+, thereby inhibiting enzyme activity. Following irradiation with either 365 or 410 nm light, the CAP probes isomerize to their cis form. Because of the change in steric profile, the probe exits the active site, and the activity of the enzyme is restored. The cis isomer can revert back to the trans isomer through thermal relaxation or via photoirradiation with 460 nm light and thereby inhibit protein activity again. This process can be repeated multiple times without any photodegradation and thus can be used to inhibit or activate the protein reversibly. Importantly, we demonstrate our ability to apply CAP azobenzene probes to regulate CA activity both in an isolated protein solution and in live cells, where the two isomers of CAP1 differentially regulate the intracellular cytosolic pH.

Detachment of HCO3– from the Active Site of Carbonic Anhydrase: Molecular Dynamics Simulation and Machine Learning

While tremendous efforts have been devoted, for many years both experimentally and theoretically, to explore the potential of carbonic anhydrase (CA) as an industrial catalyst for CO2 sequestration...

Design of two-tail compounds with rotationally fixed benzenesulfonamide ring as inhibitors of carbonic anhydrases

Rational design of compounds that would bind specific pockets of the target proteins is a difficult task in drug design. The 12 isoforms of catalytically active human carbonic anhydrases (CAs) have highly similar active sites that make it difficult to design inhibitors selective for one or several CA isoforms. A series of CA inhibitors based on 2-chloro/bromo-benzenesulfonamide that is largely fixed in the CA active site together with one or two tails yielded compounds that were synthesized and evaluated as inhibitors of CA isoforms. Introduction of a second tail had significant influence on the binding affinity and two-tailed compounds in most cases provided high affinity and selectivity for CA IX and CA XIV. The contacts between several compounds and CA amino acids were determined by X-ray crystallography. Together with the intrinsic enthalpy and entropy of binding they provided the structure-thermodynamics correlations for this series of compounds with the insight how to rationally build compounds with desired CA isoform as a target.

Synthesis, structure and bioactivity of primary sulfamate-containing natural products

Here we report the synthesis of natural products (NPs) 5′-O-sulfamoyl adenosine 1 and 5′-O-sulfamoyl-2-chloroadenosine 2. As primary sulfamates these compounds represent an uncommon class of NPs, furthermore there are few NPs known that contain a NS bond. Compounds 1 and 2 were evaluated for inhibition of carbonic anhydrases (CA), a metalloenzyme family where the primary sulfamate is known to coordinate to the active site zinc and form key hydrogen bonds with adjacent CA active site residues. Both NPs were good to moderate CA inhibitors, with compound 2 a 20–50-fold stronger CA inhibitor (Ki values 65–234 nM) than compound 1. The protein X-ray crystal structures of 1 and 2 in complex with CA II show that it is not the halogen-hydrophobic interactions that give compound 2 a greater binding energy but a slight movement in orientation of the ribose ring that allows better hydrogen bonds to CA residues. Compounds 1 and 2 were further investigated for antimicrobial activity against a panel of microbes relevant to human health, including Gram-negative bacteria (4 strains), Gram-positive bacteria (1 strain) and yeast (2 strains). Antimicrobial activity and selectivity was observed. The minimum inhibitory concentration (MIC) of NP 1 was 10 µM against Gram-positive Staphylococcus aureus and NP 2 was 5 µM against Gram-negative Escherichia coli. This is the first time that NP primary sulfamates have been assessed for inhibition and binding to CAs, with systematic antimicrobial activity studies also reported.

Catalytic Water Oxidation by Iridium-Modified Carbonic Anhydrase.

Carbonic anhydrase (CA) is a ubiquitous metalloenzyme with a Zn cofactor coordinated to trigonal histidine imidazole moieties in a tetrahedral geometry. Removal of the Zn cofactor in CA and subsequent binding of Ir afforded CA[Ir]. Under mild and neutral conditions (30 °C, pH 7), CA[Ir] exhibited water-oxidizing activity with a turnover frequency (TOF) of 39.8 min-1 , which is comparable to those of other Ir-based molecular catalysts. Coordination of Ir to the apoprotein of CA is thermodynamically preferred and is associated with an exothermic energy change (ΔH) of -10.8 kcal mol-1 , which implies that the CA apoprotein is stabilized by Ir binding. The catalytic oxygen-evolving activity of CA[Ir] is displayed only if Ir is bound to CA, which functions as an effective biological scaffold that activates the Ir center for catalysis. The results of this study indicate that the histidine imidazoles at the CA active site could be exploited as beneficial biological ligands to provide unforeseen biochemical activity by coordination to a variety of transition-metal ions.

Adhesive Photoswitch: Selective Photochemical Modulation of Enzymes under Physiological Conditions

We developed a water-soluble adhesive photoswitch (Gluen-Azo-SA, average n = 5) that selectively binds to a target enzyme and photochemically modulates its enzymatic activity even in cell lysates. Its design strategy features a photochromic azobenzene unit (Azo), which carries on one side an inhibitory motif for the target enzyme and on the other a glue part (Gluen) that utilizes its multiple guanidinium ion (Gu+) pendants for adhering onto the target surface. The target of Gluen-Azo-SA is carbonic anhydrase (CA) because sulfonamide (SA) derivatives, such as SA at the terminus of Gluen-Azo-SA, are known to bind selectively to the CA active site. The SA moiety, upon docking at the CA active site, possibly guides the connecting Gluen part to an oxyanion-rich area in proximity to the active site for adhesion, so that the conjugation between Gluen-Azo-SA and CA is ensured. With this geometry, the photochemical isomerization of the Azo unit likely generates a push-pull motion of SA, resulting in its docking and undocking at the active site of CA with the help of a competing substrate. Consequently, Gluen-Azo-SA can selectively photomodulate the enzymatic action of CA even in cell lysates. Azo-SA without Gluen can likewise suppress the enzymatic activity of CA by docking SA at its active site, but the resulting CA/Azo-SA conjugate, in contrast, does not respond to light.

A bio-inspired zinc finger analogue anchored in 2D hexagonal mesoporous silica for room temperature CO2activation via a hydrogenocarbonate route

A room temperature hydrogenocarbonate intermediate in CO2activation by a carbonic anhydrase active site analog inserted in the nanopores of mesostructured porous silicas.