Zinc-binding Function Research Articles

Atypical interactions between GCPII and hydroxamate-based inhibitors

Human glutamate carboxypeptidase II (GCPII) is a zinc-dependent metallopeptidase implicated in diverse pathologies. GCPII-specific ligands can be used in diagnostic and therapeutic applications in prostate cancer and various neurological disorders [1]. Such ligands typically consist of a glutamate moiety linked to a zinc-binding group to ensure high specificity and affinity, respectively [2]. Hydroxamate functionality is one of the prominent zinc-binding functions used in the field, however, there are no structural data describing interactions between hydroxamates and GCPII. Here we report X-ray structures of six complexes between GCPII and hydroxamate-based inhibitors. Our structures reveal unexpected positioning of hydroxamates in the internal GCPII pocket that differs markedly from binding modes of matching prototypical GCPII inhibitors featuring different zinc-binding groups. They provide mechanistic explanation for prior structure-activity relationship studies and can be exploited for the structure-assisted design of novel GCPII-specific compounds.

Design, synthesis and evaluation of N-substituted saccharin derivatives as selective inhibitors of tumor-associated carbonic anhydrase XII

A series of N-alkylated saccharin derivatives were synthesized and tested for the inhibition of four different isoforms of human carbonic anhydrase (CA, EC 4. 2.1.1): the transmembrane tumor-associated CA IX and XII, and the cytosolic CA I and II. Most of the reported derivatives inhibited CA XII in the nanomolar/low micromolar range, hCA IX with KIs ranging between 11 and 390nM, whereas they were inactive against both CA I (KIs >50μM) and II (KIs ranging between 39.1nM and 50μM). Since CA I and II are off-targets of antitumor carbonic anhydrase inhibitors (CAIs), the obtained results represent an encouraging achievement for the development of new anticancer candidates without the common side effects of non-selective CAIs. Moreover, the lack of an explicit zinc binding function on these inhibitors opens the way towards the exploration of novel mechanisms of inhibition that could explain the high selectivity of these compounds for the inhibition of the transmembrane, tumor-associated isoforms over the cytosolic ones.

A Novel Hepacivirus with an Unusually Long and Intrinsically Disordered NS5A Protein in a Wild Old World Primate

GB virus B (GBV-B; family Flaviviridae, genus Hepacivirus) has been studied in New World primates as a model for human hepatitis C virus infection, but the distribution of GBV-B and its relatives in nature has remained obscure. Here, we report the discovery of a novel and highly divergent GBV-B-like virus in an Old World monkey, the black-and-white colobus (Colobus guereza), in Uganda. The new virus, guereza hepacivirus (GHV), clusters phylogenetically with GBV-B and recently described hepaciviruses infecting African bats and North American rodents, and it shows evidence of ancient recombination with these other hepaciviruses. Direct sequencing of reverse-transcribed RNA from blood plasma from three of nine colobus monkeys yielded near-complete GHV genomes, comprising two distinct viral variants. The viruses contain an exceptionally long nonstructural 5A (NS5A) gene, approximately half of which codes for a protein with no discernible homology to known proteins. Computational structure-based analyses indicate that the amino terminus of the GHV NS5A protein may serve a zinc-binding function, similar to the NS5A of other viruses within the family Flaviviridae. However, the 521-amino-acid carboxy terminus is intrinsically disordered, reflecting an unusual degree of structural plasticity and polyfunctionality. These findings shed new light on the natural history and evolution of the hepaciviruses and on the extent of structural variation within the Flaviviridae.

Dithiocarbamates Strongly Inhibit Carbonic Anhydrases and Show Antiglaucoma Action in Vivo

A series of dithiocarbamates were prepared by reaction of primary/secondary amines with carbon disulfide in the presence of bases. These compounds were tested for the inhibition of four human (h) isoforms of the zinc enzyme carbonic anhydrase, CA (EC 4.2.1.1), hCA I, II, IX, and XII, involved in pathologies such as glaucoma (CA II and XII) or cancer (CA IX). Several low nanomolar inhibitors targeting these CAs were detected. The X-ray crystal structure of the hCA II adduct with morpholine dithiocarbamate evidenced the inhibition mechanism of these compounds, which coordinate to the metal ion through a sulfur atom from the dithiocarbamate zinc-binding function. Some dithiocarbamates showed an effective intraocular pressure lowering activity in an animal model of glucoma.

Biomimetic Nanostructures: Creating a High-Affinity Zinc-Binding Site in a Folded Nonbiological Polymer

One of the long-term goals in developing advanced biomaterials is to generate protein-like nanostructures and functions from a completely nonnatural polymer. Toward that end, we introduced a high-affinity zinc-binding function into a peptoid (N-substituted glycine polymer) two-helix bundle. Borrowing from well-understood zinc-binding motifs in proteins, thiol and imidazole moieties were positioned within the peptoid such that both helices must align in close proximity to form a binding site. We used fluorescence resonance energy transfer (FRET) reporter groups to measure the change of the distance between the two helical segments and to probe the binding of zinc. We systematically varied the position and number of zinc-binding residues, as well as the sequence and size of the loop that connects the two helical segments. We found that certain peptoid two-helix bundles bind zinc with nanomolar affinities and high selectivity compared to other divalent metal ions. Our work is a significant step toward generating biomimetic nanostructures with enzyme-like functions.

Design of Zinc Binding Functions for Carbonic Anhydrase Inhibitors

Zinc ion plays a crucial role in the protein's functions and is linked to a variety of physiological processes. It constitutes an essential component of numerous enzymes especially carbonic anhydrase (CAs, EC 4.2.1.1), a pharmaceutically-important metalloprotein which catalyses efficiently the reversible hydration of carbon dioxide to bicarbonate with discharge of a proton. The potential therapeutic applications of selective carbonic anhydrase inhibitors has become an important challenge over the last few years, as some isoforms of this enzyme on the 16 described in higher vertebrates have been found to be involved in important pathologies such as cancer, obesity and ophthalmologic diseases. Coordination of the inhibitor with the zinc ion present in the active site is an important determinant which has to be taken into consideration for the design of isozyme-specific and organ-selective inhibitors. Besides the well known sulfonamide function, others zinc binding groups have been described constituting a new platform for the development of novel pharmacological agents. In this review, recent studies on the discovery of new zinc binding function will be discussed.

Diuretics: From Classical Carbonic Anhydrase Inhibitors to Novel Applications of the Sulfonamides

The widely clinically used benzothiadiazines and high ceiling diuretics, such as hydrochlorothiazide, hydroflumethiazide, quinethazone, metolazone, chlorthalidone, indapamide, furosemide and bumetanide, contain SO(2)NH(2) moieties acting as an effective zinc-binding function in carbonic anhydrases (CAs, EC 4.2.1.1) inhibitors. These drugs were launched in a period when only isoform CA II was known and considered physiologically/pharmacologically relevant. Although acting as moderate-weak inhibitors of CA II, all these drugs considerably inhibit other CA isozymes known nowadays to be involved in critical physiologic processes, among the 16 CAs present in vertebrates. Some low nanomolar (or even subnanomolar) inhibitors against such isoforms were recently detected, such as metholazone against CA VII, XII and XIII, chlorthalidone against CA VB, VII, IX, XII and XIII, indapamide against CA VII, IX, XII and XIII, furosemide against CA I, II and XIV, and bumethanide against CA IX and XII. The X-ray crystal structure of the CA II-indapamide adduct was also reported recently, revealing interesting aspects useful for the drug design of CA inhibitors. It has also been proposed that the recently observed beneficial effect of indapamide for the treatment of patients with hypertension and type 2 diabetes might be due to its potent inhibition of CA isoforms present in kidneys and blood vessels, which would thus explain both the blood pressure lowering effects as well as organ-protective activity of the drug. Thus, these old drugs may be useful as leads for new applications.

Metal Binding Functions in the Design of Carbonic Anhydrase Inhibitors

The carbonic anhydrases (CAs, EC 4.2.1.1) are zinc containing metalloenzymes which catalyse efficiently the reversible hydration of carbon dioxide to bicarbonate with discharge of a proton, playing important physiological and physiopathological functions. To date, 16 different carbonic anhydrase isoforms have been described in higher vertebrates, including humans, and some of them have been considered as important targets for inhibitors with therapeutic applications. The catalytic and structural role of zinc in these enzyme are understood in great detail, and this provided molecular basis for the design of potent inhibitors, some of which possessing important clinical applications mainly as topically acting anti-glaucoma drugs, anticancer or antiobesity agents. The metal binding function is a critically important factor in the development of isozyme-specific and organ-selective inhibitors. Discovery of compounds that possess zinc binding function different from that of the classical one (sulfonamide type) is in constant progress and can offer opportunities for developing novel pharmacological agents. In the present review we will discuss the different zinc binding function reported in the literature up to now in the design of carbonic anhydrase inhibitors.

Carbonic anhydrase inhibitors: The X-ray crystal structure of the adduct of N-hydroxysulfamide with isozyme II explains why this new zinc binding function is effective in the design of potent inhibitors

N-Hydroxysulfamide is a 2000-fold more potent inhibitor of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1) as compared to sulfamide. It also inhibits other physiologically relevant isoforms, such as the tumor-associated CA IX and XII ( K Is in the range of 0.865–1.34 μM). In order to understand the binding of this inhibitor to the enzyme active site, the X-ray crystal structure of the human hCA II– N-hydroxysulfamide adduct was resolved. The inhibitor coordinates to the active site zinc ion by the ionized primary amino group, participating in an extended network of hydrogen bonds with amino acid residues Thr199, Thr200 and two water molecules. The additional two hydrogen bonds in which N-hydroxysulfamide bound to hCA II is involved as compared to the corresponding adduct of sulfamide may explain its higher affinity for the enzyme, also providing hints for the design of tight-binding CA inhibitors possessing an organic moiety substituting the NH group in the N-hydroxysulfamide structure.

New Zinc Binding Motifs in the Design of Selective Carbonic Anhydrase Inhibitors

The carbonic anhydrases (CAs, EC 4.2.1.1) are ubiquitous zinc enzymes which catalyze a very simple physiological reaction, the interconversion between carbon dioxide and the bicarbonate ion, and are involved in physiological and pathological processes. The different isozymes have been considered as important targets for inhibitors with clinical applications. Several sulfonamide carbonic anhydrase inhibitors (CAIs) were used for decades as diuretics, anti-glaucoma, anti-epileptic, anti-ulcer agents, or as drugs for treating other neurological/neuromuscular disorders, whereas presently several such agents still find wide applications in therapy, mainly as topically acting anti-glaucoma drugs, anti-cancer, or anti-obesity agents. Although sulfonamides were considered the moiety par excellence to coordinate the catalytic zinc and for designing potent CAIs, in recent years related functional groups such as sulfamate, sulfamide and others have proven to be successful in the design of selective CAIs. The present review will deal with these different zinc binding functions recently reported in literature.

N-Hydroxyurea—A versatile zinc binding function in the design of metalloenzyme inhibitors

N-Hydroxyurea binds both to carbonic anhydrase (CA) and to matrix metalloproteinases (MMPs). X-ray crystallography showed N-hydroxyurea to bind in a bidentate mode by means of the oxygen and nitrogen atoms of the NHOH moiety to the Zn(II) ion of CA, participating in a network of hydrogen bonds with a water molecule and Thr199. A derivatized N-hydroxyurea showed low-micromolar affinity for several CAs. This simple zinc binding function may be exploited for obtaining potent metalloenzyme inhibitors, due to its versatility of binding to the metal ion present in the active site of such enzymes.

Carbonic Anhydrase Inhibitors: Synthesis and Inhibition of Cytosolic/Tumor‐Associated Carbonic Anhydrase Isozymes I, II, IX, and XII with N‐Hydroxysulfamides — A New Zinc‐Binding Function in the Design of Inhibitors.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Carbonic anhydrase inhibitors: synthesis and inhibition of cytosolic/tumor-associated carbonic anhydrase isozymes I, II, IX, and XII with N-hydroxysulfamides—a new zinc-binding function in the design of inhibitors

A small library of N-hydroxysulfamides was synthesized by an original approach in order to investigate whether this zinc-binding function is efficient for the design of inhibitors targeting the cytosolic (hCA I and II) and transmembrane, tumor-associated (hCA IX and XII) isozymes of carbonic anhydrase (CA, EC 4.2.1.1). The parent derivative, N-hydroxysulfamide was a more potent inhibitor as compared to sulfamide or sulfamic acid against all isozymes, with inhibition constants in the range of 473 nM–4.05 μM. Its substituted n-decyl-, n-dodecyl-, benzyl-, and biphenylmethyl-derivatives were less inhibitory against hCA I ( K Is in the range of 5.8–8.2 μM) but more inhibitory against hCA II ( K Is in the range of 50.5–473 nM). The same situation was true for the tumor-associated isozymes, with K Is in the range of 353–790 nM against hCA IX and 372–874 nM against hCA XII. Some sulfamides/sulfamates possessing similar substitution patterns have also been investigated for the inhibition of these isozymes, being shown that in some particular cases sulfamides are more efficient inhibitors as compared to the corresponding sulfamates. Potent CA inhibitors targeting the cytosolic or tumor-associated CA isozymes can thus be designed from various classes of sulfonamides, sulfamides, or sulfamates and their derivatives, considering the extensive interactions in which the inhibitor and the enzyme active site are engaged, based on recent X-ray crystallographic data.

Carbonic anhydrase inhibitors. Inhibition of the cytosolic and tumor-associated carbonic anhydrase isozymes I, II, and IX with a series of 1,3,4-thiadiazole- and 1,2,4-triazole-thiols

A series of heterocyclic mercaptans incorporating 1,3,4-thiadiazole- and 1,2,4-triazole rings have been prepared and assayed for the inhibition of three physiologically relevant carbonic anhydrase (CA, EC 4.2.1.1) isozymes, the cytosolic human isozymes I and II, and the transmembrane, tumor-associated hCA IX. Against hCA I the investigated thiols showed inhibition constants in the range of 97 nM to 548 μM, against hCA II in the range of 7.9–618 μM, and against hCA IX in the range of 9.3–772 μM. Thiadiazoles were generally more active than triazoles against all investigated isozymes. Generally, the best inhibitors were the simple derivative 5-amino-1,3,4-thiadiazole-2-thiol and its N-acetylated derivative, which were anyhow at least two orders of magnitude less effective inhibitors when compared to the corresponding sulfonamides, acetazolamide, and its deacetylated derivative. An exception was constituted by 5-(2-pyridylcarboxamido)-1,3,4-thiadiazole-2-thiol, which is the first hCA I-selective inhibitor ever reported, possessing an inhibition constant of 97 nM against isozyme I, and being a 105 times less effective hCA II inhibitor, and 3154 times less effective hCA IX inhibitor. Thus, the thiol moiety may lead to effective CA inhibitors targeting isozyme I, whereas it is a less effective zinc-binding function for the design of CA II and CA IX inhibitors over the sulfonamide group.

A Complex Zinc Finger Controls the Enzymatic Activities of Nidovirus Helicases

Nidoviruses (Coronaviridae, Arteriviridae, and Roniviridae) encode a nonstructural protein, called nsp10 in arteriviruses and nsp13 in coronaviruses, that is comprised of a C-terminal superfamily 1 helicase domain and an N-terminal, putative zinc-binding domain (ZBD). Previously, mutations in the equine arteritis virus (EAV) nsp10 ZBD were shown to block arterivirus reproduction by disrupting RNA synthesis and possibly virion biogenesis. Here, we characterized the ATPase and helicase activities of bacterially expressed mutant forms of nsp10 and its human coronavirus 229E ortholog, nsp13, and correlated these in vitro activities with specific virus phenotypes. Replacement of conserved Cys or His residues with Ala proved to be more deleterious than Cys-for-His or His-for-Cys replacements. Furthermore, denaturation-renaturation experiments revealed that, during protein refolding, Zn2+ is essential for the rescue of the enzymatic activities of nidovirus helicases. Taken together, the data strongly support the zinc-binding function of the N-terminal domain of nidovirus helicases. nsp10 ATPase/helicase deficiency resulting from single-residue substitutions in the ZBD or deletion of the entire domain could not be complemented in trans by wild-type ZBD, suggesting a critical function of the ZBD in cis. Consistently, no viral RNA synthesis was detected after transfection of EAV full-length RNAs encoding ATPase/helicase-deficient nsp10 into susceptible cells. In contrast, diverse phenotypes were observed for mutants with enzymatically active nsp10, which in a number of cases correlated with the activities measured in vitro. Collectively, our data suggest that the ZBD is critically involved in nidovirus replication and transcription by modulating the enzymatic activities of the helicase domain and other, yet unknown, mechanisms.

Mechanism of DNA cleavage catalyzed by Mung Bean Nuclease

The interaction of zinc with different forms of DNA (λ phage DNA, ss-oligo, ds-oligo) and Mung Bean Nuclease was studied by voltammetric techniques in order to investigate the mechanism of DNA cleavage catalyzed by a zinc metalloenzyme. Stoichiometry, dissociation constant, zinc binding sites and functions were determined for these systems. Two zinc ions were found to be involved in stabilization of a 19 mer ds-oligodeoxyribonucleotide, which was synthesized by the phosphoramidite method and used as a DNA model in the studies. Three zinc ions (Zn1, Zn2, and Zn3), which have different roles in ds-oligo cleavage, were identified in the active site of Mung Bean Nuclease. A concerted S N2 mechanism, which assigns a catalytic function to Zn2 and structural functions to Zn1 and Zn3, was proposed. The hydrolysis of phosphodiester bonds proceeds with inversion of configuration at the phosphorus center, forming a pentacoordinate transition state, which is stabilized by an arginine. Zn2 supplies the nucleophile, which is oriented by an aspartic acid, and activates the ds-oligo by its coordination to the phosphate free oxygen of the phosphodiester bond. Zn1 and Zn3 ions, besides stabilizing the tertiary structure of Mung Bean Nuclease, bind to the leaving group, blocking the cleavage reverse reaction.

Carbonic anhydrase inhibitors: X-ray crystallographic structure of the adduct of human isozyme II with a topically acting antiglaucoma sulfonamide

The X-ray crystal structure for the adduct of human carbonic anhydrase (hCA) II with a topically acting antiglaucoma sulfonamide (the 2-N,N-diethylaminoethylamide of 5-(4-carboxybenzenesulfonamido-1,3,4-thiadiazole-2-sulfonamide), has been resolved at a resolution of 1.6Å. This compound is a very potent inhibitor of the physiologically most relevant isozyme hCA II for the secretion of aqueous humor within the eye (KI of 1.4nM), and in animal models of glaucoma showed very effective intraocular pressure (IOP) lowering after topical administration. Surprisingly, the inhibitor bound within the enzyme active site is in the sulfonylimido-4H-δ2-1,3,4-thiadiazoline tautomeric form. The inhibitor is directly bound to the Zn(II) ion of the enzyme through the deprotonated primary sulfonamide moiety, participating to the classical hydrogen bond network involving residues of the zinc-binding function and Thr 199 and Glu 106. The 1,3,4-thiadiazoline fragment of the inhibitor makes two hydrogen bonds with the active site residue Thr 200, the secondary sulfonamide moiety makes two hydrogen bonds involving a water molecule and the residue Gln 92, whereas the phenyl ring of the inhibitor participates to an edge-to-face interaction with the phenyl ring of Phe 131, the two cycles being almost perfectly perpendicular to each other. The tertiary amine fragment of the carboxamido tail and the carboxamido moiety itself make hydrogen bonds with water molecules present at the rim of the active site entrance and van der Waals contacts with His 4, Trp 5, and Phe 20. All these multiple interactions never evidenced previously in CA–sulfonamide complexes, explain the very high affinity of this inhibitor for the hCA II active site and may allow further optimization of this class of inhibitors.

Conserved cysteine and histidine residues in the putative zinc finger motif of the influenza A virus M1 protein are not critical for influenza virus replication.

The influenza virus matrix protein (M1) possesses a cysteine and histidine (CCHH) motif in the helix 9 (H9) and adjacent region ((148)CATCEQIADSQHRSH(162)). The CCHH motif has been proposed as a putative zinc finger motif and zinc-binding activity has been implicated in virus uncoating as well as transcription inhibition and mRNA regulation. The function of the CCHH motif in the influenza virus life cycle was investigated by site-directed mutagenesis (alanine replacement) and by rescuing mutant viruses by reverse genetics. Mutant viruses containing an alanine replacement of the cysteine and histidine residues, either individually or in combination, were seen to exhibit wt phenotype in multiple virus growth cycles and plaque morphology. In addition, synthetic peptides containing the putative zinc finger motif did not inhibit virus replication in MDCK cells. However, mutation of Ala(155) in H9 was lethal for rescuing infectious virus. These data show that the CCHH motif does not provide a critical function in the influenza virus life cycle in cell culture and that the zinc-binding function may not be involved in virus biology. However, the lethal phenotype of the Ala(155) mutation shows that the H9 region of M1 provides some other critical function(s) in virus replication.

Protease Inhibitors: Synthesis of Matrix Metalloproteinase and Bacterial Collagenase Inhibitors Incorporating 5‐Amino‐2‐mercapto‐1,3,4‐thiadiazole Zinc Binding Functions.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Protease inhibitors: Synthesis of matrix metalloproteinase and bacterial collagenase inhibitors incorporating 5-amino-2-mercapto-1,3,4-thiadiazole zinc binding functions

Matrix metalloproteinase (MMP)/bacterial collagenase inhibitors incorporating 5-amino-2-mercapto-1,3,4-thiadiazole zinc binding functions are reported. A series of compounds was prepared by reaction of arylsulfonyl isocyanates or arylsulfonyl halides with phenylalanyl-alanine, followed by coupling with 5-amino-2-mercapto-1,3,4-thiadiazole in the presence of carbodiimides. These new compounds were assayed as inhibitors of human MMP-1, MMP-2, MMP-8 and MMP-9, and of the collagenase isolated from the anaerobe Clostridium histolyticum (ChC). The new derivatives proved to be powerful inhibitors of these metalloproteases, with activities in the low micromolar range for some of the target enzymes, depending on the substitution pattern at the arylsulfonyl(ureido) moieties.