Post-translational modifications in tumor-associated carbonic anhydrases.

Human carbonic anhydrases IX (hCA IX) and XII (hCA XII) are two proteins associated with tumor formation and development. These enzymes have been largely investigated both from a biochemical and a functional point of view. However, limited data are currently available on the characterization of their post-translational modifications (PTMs) and the functional implication of these structural changes in the tumor environment. In this review, we summarize existing literature data on PTMs of hCA IX and hCA XII, such as disulphide bond formation, phosphorylation, O-/N-linked glycosylation, acetylation and ubiquitination, highlighting, when possible, their specific role in cancer pathological processes.

Biochemical and Structural Insights into the Winged Helix Domain of P150, the Largest Subunit of the Chromatin Assembly Factor 1.

The Chromatin Assembly Factor 1 is a heterotrimeric complex responsible for the nucleosome assembly during DNA replication and DNA repair. In humans, the largest subunit P150 is the major actor of this process. It has been recently considered as a tumor-associated protein due to its overexpression in many malignancies. Structural and functional studies targeting P150 are still limited and only scarce information about this subunit is currently available. Literature data and bioinformatics analysis assisted the identification of a stable DNA binding domain, encompassing residues from 721 to 860 of P150 within the full-length protein. This domain was recombinantly produced and in vitro investigated. An acidic region modulating its DNA binding ability was also identified and characterized. Results showed similarities and differences between the P150 and its yeast homologue, namely Cac-1, suggesting that, although sharing a common biological function, the two proteins may also possess different features.

α-CAs from Photosynthetic Organisms.

Carbonic anhydrases (CAs) are ubiquitous enzymes that catalyze the reversible carbon dioxide hydration reaction. Among the eight different CA classes existing in nature, the α-class is the largest one being present in animals, bacteria, protozoa, fungi, and photosynthetic organisms. Although many studies have been reported on these enzymes, few functional, biochemical, and structural data are currently available on α-CAs isolated from photosynthetic organisms. Here, we give an overview of the most recent literature on the topic. In higher plants, these enzymes are engaged in both supplying CO2 at the Rubisco and determining proton concentration in PSII membranes, while in algae and cyanobacteria they are involved in carbon-concentrating mechanism (CCM), photosynthetic reactions and in detecting or signaling changes in the CO2 level in the environment. Crystal structures are only available for three algal α-CAs, thus not allowing to associate specific structural features to cellular localizations or physiological roles. Therefore, further studies on α-CAs from photosynthetic organisms are strongly needed to provide insights into their structure-function relationship.

Design, synthesis and biochemical evaluation of novel carbonic anhydrase inhibitors triggered by structural knowledge on hCA VII.

To tackle the challenge of isoform selectivity, we explored the entrance of the cavity for selected druggable human Carbonic Anhydrases (hCAs). Based on X-ray crystallographic studies on the 4-(4-(2-chlorobenzoyl)piperazine-1-carbonyl)benzenesulfonamide in complex with the brain expressed hCA VII (PDB code: 7NC4), a series of 4-(4(hetero)aroylpiperazine-1-carbonyl)benzene-1-sulfonamides has been developed. To evaluate their capability to fit the hCA VII catalytic cavity, the newer benzenesulfonamides were preliminary investigated by means of docking simulations. Then, this series of thirteen benzenesulfonamides was synthesized and tested against selected druggable hCAs. Among them, the 4-(4-(furan-2-carbonyl)piperazine-1-carbonyl)benzenesulfonamide showed remarkable affinity towards hCA VII (Ki: 4.3 nM) and good selectivity over the physiologically widespread hCA I when compared to Topiramate (TPM).

Inhibition of the ß-carbonic anhydrase from the protozoan pathogen Trichomonas vaginalis with sulphonamides.

Sulphonamides and their isosteres are classical inhibitors of the carbonic anhydrase (CAs, EC 4.2.1.1) metalloenzymes. The protozoan pathogen Trichomonas vaginalis encodes two such enzymes belonging to the ß-class, TvaCA1 and TvaCA2. Here we report the first sulphonamide inhibition study of TvaCA1, with a series of simple aromatic/heterocyclic primary sulphonamides as well as with clinically approved/investigational drugs for a range of pathologies (diuretics, antiglaucoma, antiepileptic, antiobesity, and antitumor drugs). TvaCA1 was effectively inhibited by acetazolamide and ethoxzolamide, with KIs of 391 and 283 nM, respectively, whereas many other simple or clinically used sulphonamides were micromolar inhibitors or did not efficiently inhibit the enzyme. Finding more effective TvaCA1 inhibitors may constitute an innovative approach for fighting trichomoniasis, a sexually transmitted infection, caused by T. vaginalis.

Human carbonic anhydrases and post-translational modifications: a hidden world possibly affecting protein properties and functions.

Human carbonic anhydrases (CAs) have become a well-recognized target for the design of inhibitors and activators with biomedical applications. Accordingly, an enormous amount of literature is available on their biochemical, functional and structural aspects. Nevertheless post-translational modifications (PTMs) occurring on these enzymes and their functional implications have been poorly investigated so far. To fill this gap, in this review we have analysed all PTMs occurring on human CAs, as deriving from the search in dedicated databases, showing a widespread occurrence of modification events in this enzyme family. By combining these data with sequence alignments, inspection of 3 D structures and available literature, we have summarised the possible functional implications of these PTMs. Although in some cases a clear correlation between a specific PTM and the CA function has been highlighted, many modification events still deserve further dedicated studies.

Biochemical and structural characterisation of a protozoan beta-carbonic anhydrase from Trichomonas vaginalis.

We report the biochemical and structural characterisation of a beta-carbonic anhydrase (ß-CA) from Trichomonas vaginalis, a unicellular parasite responsible for one of the world's leading sexually transmitted infections, trichomoniasis. CAs are ubiquitous metalloenzymes belonging to eight evolutionarily divergent groups (α, ß, γ, δ, ζ, η, θ, and ι); humans express only α-CAs, whereas many clinically significant pathogens express only ß- and/or γ-CAs. For this reason, the latter two groups of CAs are promising biomedical targets for novel antiinfective agents. The ß-CA from T. vaginalis (TvaCA1) was recombinantly produced and biochemically characterised. The crystal structure was determined, revealing the canonical dimeric fold of ß-CAs and the main features of the enzyme active site. The comparison with the active site of human CA enzymes revealed significant differences that can be exploited for the design of inhibitors selective for the protozoan enzyme with respect to the human ones.

Exploration of the residues modulating the catalytic features of human carbonic anhydrase XIII by a site-specific mutagenesis approach.

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.

Exploring benzoxaborole derivatives as carbonic anhydrase inhibitors: a structural and computational analysis reveals their conformational variability as a tool to increase enzyme selectivity.

Recent studies identified the benzoxaborole moiety as a new zinc-binding group able to interact with carbonic anhydrase (CA) active site. Here, we report a structural analysis of benzoxaboroles containing urea/thiourea groups, showing that these molecules are very versatile since they can bind the enzyme assuming different binding conformations and coordination geometries of the catalytic zinc ion. In addition, theoretical calculations of binding free energy were performed highlighting the key role of specific residues for protein-inhibitor recognition. Overall, these data are very useful for the development of new inhibitors with higher selectivity and efficacy for various CAs.

Inhibition of carbonic anhydrase IX targets primary tumors, metastases, and cancer stem cells: Three for the price of one.

Human carbonic anhydrase (CA) IX is a tumor-associated protein, since it is scarcely present in normal tissues, but highly overexpressed in a large number of solid tumors, where it actively contributes to survival and metastatic spread of tumor cells. Due to these features, the characterization of its biochemical, structural, and functional features for drug design purposes has been extensively carried out, with consequent development of several highly selective small molecule inhibitors and monoclonal antibodies to be used for different purposes. Aim of this review is to provide a comprehensive state-of-the-art of studies performed on this enzyme, regarding structural, functional, and biomedical aspects, as well as the development of molecules with diagnostic and therapeutic applications for cancer treatment. A brief description of additional pharmacologic applications for CA IX inhibition in other diseases, such as arthritis and ischemia, is also provided.

The Crystal Structure of a hCA VII Variant Provides Insights into the Molecular Determinants Responsible for Its Catalytic Behavior.

Although important progress has been achieved in understanding the catalytic mechanism of Carbonic Anhydrases, a detailed picture of all factors influencing the catalytic efficiency of the various human isoforms is still missing. In this paper we report a detailed structural study and theoretical pKa calculations on a hCA VII variant. The obtained data were compared with those already known for another thoroughly investigated cytosolic isoform, hCA II. Our structural studies show that in hCA VII the network of ordered water molecules, which connects the zinc bound solvent molecule to the proton shuttle His64, is altered compared to hCA II, causing a reduction of the catalytic efficiency. Theoretical calculations suggest that changes in solvent network are related to the difference in pKa of the proton shuttle in the two enzymes. The residue that plays a major role in determining the diverse pKa values of the proton shuttle is the one in position four, namely His for hCA II and Gly for hCA VII. This residue is located on the protein surface, outside of the active site cavity. These findings are in agreement with our previous studies that highlighted the importance of histidines on the protein surface of hCA II (among which His4) as crucial residues for the high catalytic efficiency of this isoform.

Insights into the role of reactive sulfhydryl groups of Carbonic Anhydrase III and VII during oxidative damage.

Carbonic anhydrases (CAs) III and VII are two cytosolic isoforms of the α-CA family which catalyze the physiological reaction of carbon dioxide hydration to bicarbonate and proton. Despite these two enzymes share a 49% sequence identity and present a very similar three-dimensional structure, they show profound differences when comparing the specific activity for CO2 hydration reaction, with CA VII being much more active than CA III. Recently, CA III and CA VII have been proposed to play a new role as scavenger enzymes in cells where oxidative damage occurs. Here, we will examine functional and structural features of these two isoforms giving insights into their newly proposed protective role against oxidative stress.

The anticonvulsant sulfamide JNJ-26990990 and its S,S-dioxide analog strongly inhibit carbonic anhydrases: solution and X-ray crystallographic studies.

JNJ-26990990 ((benzo[b]thien-3-yl)methyl)sulfamide, a sulfamide derivative structurally related to the antiepileptic drug zonisamide, was reported to be devoid of carbonic anhydrase (CA, EC 4.2.1.1) inhibitory properties. Here we report that JNJ-26990990 and its S,S-dioxide analog significantly inhibit six human (h) isoforms, hCA I, II, VII, IX, XII and XIV, involved in crucial physiological processes. Inhibition and X-ray crystallographic data for the binding of the two compounds to these enzymes show significant similarity with the zonisamide inhibitory pattern. These findings prompted us to reconsider the structural/pharmacological requirements for designing effective antiepileptics possessing zinc-binding groups of the sulfamide, sulfamate or sulfonamide type in their molecules.

The zinc coordination pattern in the η-carbonic anhydrase from Plasmodium falciparum is different from all other carbonic anhydrase genetic families.

In this Letter we reinvestigate the sequence analysis and report a homology model of the carbonic anhydrase (CA, EC 4.2.1.1) from the protozoan parasite Plasmodium falciparum, recently reported by us to belong to a new genetic family, the η-CA class. Our findings show that the metal ion coordination pattern of this CA is unique among all five other genetic families encoding for such enzymes, comprising two His and one Gln residues, in addition to the water molecule/hydroxide ion acting as nucleophile in the catalytic cycle. Although the η- and α-CAs present the same 3D fold, strongly suggesting the first ones to be evolutionary derived from the last, there are significant differences between the two families to allow optimism for the drug design of selective inhibitors for the parasite over the host enzymes. The preliminary studies reported here are relevant for drug design campaigns of anti-plasmodium CA inhibitors but further work by X-ray crystallography should validate the proposed model.

Crystal structure of the most catalytically effective carbonic anhydrase enzyme known, SazCA from the thermophilic bacterium Sulfurihydrogenibium azorense.

Two thermostable α-carbonic anhydrases (α-CAs) isolated from thermophilic Sulfurihydrogenibium spp., namely SspCA (from S. yellowstonensis) and SazCA (from S. azorense), were shown in a previous work to possess interesting complementary properties. SspCA was shown to have an exceptional thermal stability, whereas SazCA demonstrated to be the most active α-CA known to date for the CO2 hydration reaction. Here we report the crystallographic structure of SazCA and the identification of the structural features responsible for its high catalytic activity, by comparing it with SspCA structure. These data are of relevance for the design of engineered proteins showing higher stability and catalytic activity than other α-CAs known to date.

Thermostable Carbonic Anhydrases in Biotechnological Applications.

Carbonic anhydrases are ubiquitous metallo-enzymes which catalyze the reversible hydration of carbon dioxide in bicarbonate ions and protons. Recent years have seen an increasing interest in the utilization of these enzymes in CO2 capture and storage processes. However, since this use is greatly limited by the harsh conditions required in these processes, the employment of thermostable enzymes, both those isolated by thermophilic organisms and those obtained by protein engineering techniques, represents an interesting possibility. In this review we will provide an extensive description of the thermostable carbonic anhydrases so far reported and the main processes in which these enzymes have found an application.

Faox enzymes inhibited Maillard reaction development during storage both in protein glucose model system and low lactose UHT milk.

Fructosamines, also known as Amadori products, are formed by the condensation of glucose with the amino group of amino acids or proteins. These compounds are precursors of advanced glycation end products (AGEs) that can be formed either endogenously during aging and diabetes, and exogenously in heat-processed food. The negative effects of dietary AGEs on human health as well as their negative impact on the quality of dairy products have been widely described, therefore specific tools able to prevent the formation of glycation products are needed. Two fructosamine oxidase enzymes isolated from Aspergillus sp. namely, Faox I and Faox II catalyze the oxidative deglycation of Amadori products representing a potential tool for inhibiting the Maillard reaction in dairy products. In this paper, the ability of recombinant Faox I and II in limiting the formation of carboxy-methyl lysine (CML) and protein-bound hydroxymethyl furfurol (b-HMF) in a commercial UHT low lactose milk and a beta-lactoglobulin (ß-LG) glucose model system was investigated. Results show a consistent reduction of CML and b-HMF under all conditions. Faox effects were particularly evident on b-HMF formation in low lactose commercial milk. Peptide analysis of the ß-LG glucose system identified some peptides, derived from cyanogen bromide hydrolysis, as suitable candidates to monitor Faox action in milk-based products. All in all data suggested that non-enzymatic reactions in dairy products might be strongly reduced by implementing Faox enzymes.

X-ray structure of the first `extremo-α-carbonic anhydrase', a dimeric enzyme from the thermophilic bacterium Sulfurihydrogenibium yellowstonense YO3AOP1.

SspCA, a novel `extremo-α-carbonic anhydrase' isolated from the thermophilic bacterium Sulfurihydrogenibium yellowstonense YO3AOP1, is an efficient catalyst for the hydration of CO2 and presents exceptional thermostability. Indeed, SspCA retains a high catalytic activity even after being heated to 343-373 K for several hours. Here, the crystallographic structure of this α-carbonic anhydrase (α-CA) is reported and the factors responsible for its function at high temperature are elucidated. In particular, the study suggests that increased structural compactness, together with an increased number of charged residues on the protein surface and a greater number of ionic networks, seem to be the key factors involved in the higher thermostability of this enzyme with respect to its mesophilic homologues. These findings are of extreme importance, since they provide a structural basis for the understanding of the mechanisms responsible for thermal stability in the α-CA family for the first time. The data obtained offer a tool that can be exploited to engineer α-CAs in order to obtain enzymes with enhanced thermostability for use in the harsh conditions of the CO2 capture and sequestration processes.

Discovery of a novel series of potent carbonic anhydrase inhibitors with selective affinity for µ Opioid receptor for Safer and long-lasting analgesia.

In this study, we investigated the development of dual-targeted ligands that bind to both µ-opioid receptor (MOR) and carbonic anhydrase (CA) enzymes, using fentanyl structure as a template. We synthesized and evaluated 21 novel compounds with dual-targeted affinity identifying the lead candidate compound 8, showing selective affinity for MOR and potent inhibition of several cytosolic CA isoforms. By means of repeated treatment of 3 daily administrations for 17 days, fentanyl (0.1 mg/kg, subcutaneously) led to tolerance development, pain threshold alterations and withdrawal symptoms in CD-1 mice, as well as astrocyte and microglia activation in the dorsal horn of the lumbar spinal cord. In contrast, compound 8 (0.32 mg/kg s.c.) maintained stable during days its analgesic effect at the higher dose tested with fewer withdrawal symptoms, allodynia development and glial cells activation. Our results suggest that targeting both MOR and CA enzymes can lead to the development of new class of potent analgesic agents with fewer side effects and reduced tolerance development. Further studies are needed to explore the potential mechanisms underlying these effects and to further optimize the therapeutic potential of these compounds.

Carbonic anhydrase VII is S-glutathionylated without loss of catalytic activity and affinity for sulfonamide inhibitors.

Human carbonic anhydrase (CA, EC 4.2.1.1) VII is a cytosolic enzyme with high carbon dioxide hydration activity. Here we report an unexpected S-glutathionylation of hCA VII which has also been observed earlier in vivo for hCA III, another cytosolic isoform. Cys183 and Cys217 were found to be the residues involved in reaction with glutathione for hCA VII. The two reactive cysteines were then mutated and the corresponding variant (C183S/C217S) expressed. The native enzyme, the variant and the S-glutathionylated adduct (sgCA VII) as well as hCA III were fully characterized for their CO(2) hydration, esterase/phosphatase activities, and inhibition with sulfonamides. Our findings suggest that hCA VII could use the in vivo S-glutathionylation to function as an oxygen radical scavenger for protecting cells from oxidative damage, as the activity and affinity for inhibitors of the modified enzyme are similar to those of the wild type.