Zinc-binding Group Research Articles

Rapid Discovery of Highly Potent and Selective Inhibitors of Histone Deacetylase 8 Using Click Chemistry to Generate Candidate Libraries

To find HDAC8-selective inhibitors, we designed a library of HDAC inhibitor candidates, each containing a zinc-binding group that coordinates with the active-site zinc ion, linked via a triazole moiety to a capping structure that interacts with residues on the rim of the active site. These compounds were synthesized by using click chemistry. Screening identified HDAC8-selective inhibitors including C149 (IC(50) = 0.070 μM), which was more potent than PCI-34058 (6) (IC(50) = 0.31 μM), a known HDAC8 inhibitor. Molecular modeling suggested that the phenylthiomethyl group of C149 binds to a unique hydrophobic pocket of HDAC8, and the orientation of the phenylthiomethyl and hydroxamate moieties (fixed by the triazole moiety) is important for the potency and selectivity. The inhibitors caused selective acetylation of cohesin in cells and exerted growth-inhibitory effects on T-cell lymphoma and neuroblastoma cells (GI(50) = 3-80 μM). These findings suggest that HDAC8-selective inhibitors have potential as anticancer agents.

Glycosidic carbonic anhydrase IX inhibitors: A sweet approach against cancer

Targeting tumour associated carbonic anhydrase (CA, EC 4.2.1.1) isoforms IX and XII is now considered as a pertinent approach for the development of new cancer therapeutics against hypoxic tumours. In the last period, with the help of X-ray crystallography, much progress has been achieved for the drug-design of selective CA IX inhibitors, by considering the three main structural elements that govern both potency and selectivity, that is, a zinc binding group (ZBG), an organic scaffold, and one or more side chains substituting the scaffold. The use of sugar moiety in the structure of sulfonamide-based CA inhibitors (CAIs), has allowed the discovery of very potent CA IX inhibitors able to impair the growth of both primary tumors and metastases. The search for specific CA IX inhibitors by using the sugar approach has become an important research field, leading to sulfonamides, sulfamates, sulfamides and coumarins with excellent in vitro activity and relevant potency in vivo, in animal models of cancer. This paper will review the latest development in this hot topic.

An Integrated Computational Approach to Rationalize the Activity of Non-Zinc-Binding MMP-2 Inhibitors

Matrix metalloproteinases are a family of Zn-proteases involved in tissue remodeling and in many pathological conditions. Among them MMP-2 is one of the most relevant target in anticancer therapy. Commonly, MMP inhibitors contain a functional group able to bind the zinc ion and responsible for undesired side effects. The discovery of potent and selective MMP inhibitors not bearing a zinc-binding group is arising for some MMP family members and represents a new opportunity to find selective and non toxic inhibitors.In this work we attempted to get more insight on the inhibition process of MMP-2 by two non-zinc-binding inhibitors, applying a general protocol that combines several computational tools (docking, Molecular Dynamics and Quantum Chemical calculations), that all together contribute to rationalize experimental inhibition data. Molecular Dynamics studies showed both structural and mechanical-dynamical effects produced by the ligands not disclosed by docking analysis. Thermodynamic Integration provided relative binding free energies consistent with experimentally observed activity data. Quantum Chemical calculations of the tautomeric equilibrium involving the most active ligand completed the picture of the binding process. Our study highlights the crucial role of the specificity loop and suggests that enthalpic effect predominates over the entropic one.

Salicylaldoxime derivatives as new leads for the development of carbonic anhydrase inhibitors

New compounds containing a novel zinc binding group (salicylaldoxime system) were identified as effective inhibitors of carbonic anhydrases (CAs). This structural motif seems to bind the catalytic zinc ion of CAs, revealing itself as a new valid alternative to the sulfonamide group. Computational procedures were used to investigate the binding mode of this class of compounds, within the active site of CAII. This study suggests that the salicylaldoxime moiety binds the zinc ion through the oxime oxygen atom that also forms an H-bond with T199. The results herein obtained will allow the development of new CA-inhibitors bearing the salicylaldoxime moiety.

Cobalt (III) Complexes as Novel Matrix Metalloproteinase-9 Inhibitors

Matrix metalloproteinases (MMPs) are a family of zinc binding endopeptidases that play crucial roles in various physiological processes and diseases such as embryogenic growth, angiogenesis, arthritis, skin ulceration, liver fibrosis and tumor metastasis. Because of their implications in a wide range of diseases, MMPs are considered as intriguing drug targets. The majority of MMP inhibitors are organic small molecules containing a hydroxamate functionality for the zinc binding group. This hydroxamate group binds to a zinc(II) center in a bidentate fashion and creates a distorted trigonal bipyramidal geometry. Although the hydroxamate group is the most effective zinc binding moiety reported, it has shown two major limitations in clinical trials: low bioavailability and lack of specificity. Because the hydroxamate group is prone to rapid metabolism and has a high binding affinity towards various transition metals, many efforts have been made to develop novel zinc binding groups, including reverse hydroxamate, phosphate, and pyridinone. However, the in vitro activity of these newly developed inhibitors are not as high as the hydroxamate derivatives. It was previously reported that cobalt(III) acacen complexes could interact with histidine residues of proteins and model peptides. The cobalt(III) complexes bind histidine residues in active sites and on enzyme surfaces in a random fashion. Spectroscopic and chromatographic data suggested that the complexes bind to a histidine residue by axial ligand substitution. When an appropriate targeting group is attached, the cobalt(III) complexes can selectively inhibit the histidinecontaining enzymes such as thermolysin, human α-thrombin, and carbonic anhydrase. Since histidine is the most commonly found residue in the active sites of zinc enzymes and zinc-binding proteins, it has been demonstrated that the cobalt(III) complexes can interact with zinc finger proteins, such as HIV-1 nucleocapsid protein NCp7 and human zinc finger transcription factor Sp1. It was also reported that a Co(III) complex with a oligonucleotide targeting Snail family zinc finger transcription factor resulted in a selective inactivation of a transcriptional activity implicated in embryonic development and breast cancer. In this work, a series of the cobalt(III) complexes are prepared as novel MMP inhibitors. Since cobalt(III) complexes have high affinity towards histidine residues and zinc binding proteins, they can be engineered to disrupt the zinc binding active site of MMP. Furthermore, the cobalt(III) complexes may be able to provide better solubility and higher binding affinity than the known organic inhibitors. To test this hypothesis, a series of cobalt(III) complexes were designed and synthesized. For active site directing groups, biphenyl sulfonate and biphenyl amide group were chosen based on the structures of well known MMP inhibitors. In vitro activity of these complexes was evaluated for MMP-9 (gelatinase B), because MMP-9 is one of the most highly expressed MMPs in tumors and has been implicated in tumor aggressiveness. The syntheses of cobalt(III) complexes are shown in Schemes 1-2. The synthesis of 1 and 2 begins with the methylation of commercially available Cbz-Lys(Boc)-OH as

A novel aminopeptidase N inhibitor developed by virtual screening approach

A virtual screening was performed to discover novel lead structures as potent aminopeptidase N(APN) inhibitors. A commercial database containing about 1,60,000 molecules in SPECS was filtered by rule of five, zinc binding groups, pharmacophore models and binding pattern analysis. At last, 24 molecules were selected for enzyme inhibition assay and compound 2 exhibited the inhibition constant (Ki) of 2.79±0.32μM against APN compared with Bestatin (Ki= 3.37±0.24μM). Our results indicated that compound 2 exhibited good antiproliferative activities against a broad spectrum of human cancer cell lines, and induced cell cycle arrest at G1 phase and eventual apoptosis. Moreover, compound 2 can inhibit the invasion of MDA-MB-231 cells. In summary, our results suggest that compound 2, a potent APN inhibitor, is worthy of further development.

Molecular architecture of zinc chelating small molecules that inhibit spliceosome assembly at an early stage

The removal of intervening sequences (introns) from a primary RNA transcript is catalyzed by the spliceosome, a large ribonucleoprotein complex. At the start of each splicing cycle, the spliceosome assembles anew in a sequentially ordered manner on the pre-mRNA intron to be removed. We describe here the identification of a series of naphthalen-2-yl hydroxamate compounds that inhibit pre-mRNA splicing in vitro with mid- to high-micromolar values of IC(50). These hydroxamates stall spliceosome assembly at the A complex stage. A structure-activity analysis of lead compounds revealed three pharmacophores that are essential for splicing inhibition. Specifically, a hydroxamate as a zinc-binding group and a 6-methoxynaphthalene cap group are both critical, and a linker chain comprising eight to nine methylene groups is also important, for the specific binding to the docking site of a target protein molecule and precise positioning of the zinc binding group. As we found no correlation between the inhibition patterns of known histone deacetylases on the one hand and pre-mRNA splicing on the other, we conclude that these compounds may function through the inhibition of the activities of other, at present, unknown spliceosome-associated zinc metalloprotein(s).

Development of Potent Carbonic Anhydrase Inhibitors Incorporating Both Sulfonamide and Sulfamide Groups

A series of compounds incorporating both sulfonamide and sulfamide as zinc-binding groups (ZBGs) are reported as inhibitors of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1). Crystallographic studies on the complex of hCA II with the lead compound of this series, namely, 4-sulfamido-benzenesulfonamide, revealed the binding of two molecules in the enzyme active site cavity, the first one canonically coordinated to the zinc ion by means of the sulfonamide group and the second one located at the entrance of the cavity. This observation led to the design of elongated molecules incorporating these two ZBGs, separated by a linker of proper length, to allow the simultaneous binding to these different sites. The "long" inhibitors indeed showed around 10 times better enzyme inhibitory properties as compared to the shorter molecules against four physiologically relevant human (h) isoforms, hCA I, II, IX, and XII.

Toward a fragment-based approach to MMPs inhibitors: an expedite and efficient synthesis of N-hydroxylactams

Matrix metalloproteinases (MMPs), a class of zinc-enzymes over-activated in many pathologies, such as arthritis and cancer, can be efficiently inhibited by a variety of molecules bearing zinc-binding groups (ZBGs). The hydroxamic acid moiety represents one of the most potent and widely exploited ZBG but the poor target selectivity and in vivo toxicity have tempered the initial enthusiasm for this class of potential therapeutics. These drawbacks might be circumvented, at least in part, by increasing the structural constraints around the hydroxamic moiety. Following this strategy we designed and prepared N-hydroxylactam molecules of different size through a synthetic protocol based on a ring closing metathesis amenable to a fragment-based approach potentially leading to a large molecular diversity.

Structure-Activity Relationships of Glutamate Carboxypeptidase II (GCPII) Inhibitors

Glutamate carboxypeptidase II (GCPII, EC 3.4.17.21) is a zinc metallopeptidase that hydrolyzes N-acetylaspartylglutamate (NAAG) into N-acetylaspartate (NAA) and glutamate in the nervous system. Inhibition of GCPII has the potential to reduce extracellular glutamate and represents an opportune target for treating neurological disorders in which excess glutamate is considered pathogenic. Furthermore, GCPII was found to be identical to a tumor marker, prostate-specific membrane antigen (PSMA), and has drawn significant interest as a diagnostic and/or therapeutic target in oncology. Over the past 15 years, tremendous efforts have been made in the discovery of potent GCPII inhibitors, particularly those with phosphorus-, urea- and thiol-based zinc binding groups. In addition, significant progress has been made in understanding the three-dimensional structural characteristics of GCPII in complex with various ligands. The purpose of this review article is to analyze the structure-activity relationships (SAR) of GCPII inhibitors reported to date, which are classified on the basis of their zinc-binding group. SAR and crystallographic data are evaluated in detail for each of these series to highlight the future challenges and opportunities to identify clinically viable GCPII inhibitors.

MMP-2 Selectivity in Hydroxamate-Type Inhibitors

Extracellular matrix metalloproteinases (MMPs) are a family of zinc-dependent neutral endopeptidases involved in physiological and pathological processes, through the cleavage of extracellular matrix. MMPs are capable of degrading essentially all matrix components, which is crucial for malignant tumor growth, invasion, metastasis and angiogenesis. The vertebrates MMP family includes at least 26 enzymes (23 have been known in humans) with only MMP-1, 2, and 7 experimentally validated as targets for antitumoral drug design. However, inhibition of MMP-1 has been hypothesized to be the cause of the clinically observed musculoskeletal syndrome when broad spectrum inhibitors are used. On the other hand, MMP-9 is a tricky enzyme, since its inhibition might be useful in treating patients with early-stage cancers, but MMP-9 is an anti-target in patients with advanced disease. So, MMP-9 inhibition should also be prevented. Therefore, selective MMP-2 inhibition arises as a pursued profile for MMP binders. Among them, hydroxamates have been extensively studied as small molecule drug candidates characterized by an effective zinc-binding group plus additional side chains responsible for the selectivity. This article pays particular attention to MMP-2 selectivity on hydroxamate-type inhibitors, especially against MMP-9, and their chemical structure, SAR, general synthetic methods, and molecular modelling studies are here reviewed in order to inspire further design of new effective anticancer agents.

Metal–ligand interactions: An analysis of zinc binding groups using the Protein Data Bank

In the present study, we investigated zinc binding groups (ZBGs) using the coordinates of protein–ligand complex structures obtained from the Protein Data Bank. The distance from the zinc to the nearest ligand atom was measured to determine whether the atom was part of the ZBG. The most frequently found ZBG was carboxylate, followed by sulfonamide, hydroxamate, and phosphonate/phosphate. Because it was found that few heteroatoms, such as nitrogen, oxygen, and sulfur atoms, interacted with zinc, ideal distances between the zinc and these heteroatoms were identified. Whereas carboxylates bound to the zinc via both monodentate and bidentate interactions, the hydroxamates bound dominantly in a bidentate manner. These results will aid in the design of new inhibitors with the potential to interact with zinc in the target protein.

Inhibitors Incorporating Zinc‐Binding Groups Target the GlcNAc‐PI de‐N‐acetylase in Trypanosoma brucei, the Causative Agent of African Sleeping Sickness

Disruption of glycosylphosphatidylinositol biosynthesis is genetically and chemically validated as a drug target against the protozoan parasite Trypanosoma brucei, the causative agent of African sleeping sickness. The N-acetylglucosamine-phosphatidylinositol de-N-acetylase (deNAc) is a zinc metalloenzyme responsible for the second step of glycosylphosphatidylinositol biosynthesis. We recently reported the synthesis of eight deoxy-2-C-branched monosaccharides containing carboxylic acid, hydroxamic acid, or N-hydroxyurea substituents at the C2 position that may act as zinc-binding groups. Here, we describe the synthesis of a glucocyclitol-phospholipid incorporating a hydroxamic acid moiety and report the biochemical evaluation of the monosaccharides and the glucocyclitol-phospholipid as inhibitors of the trypanosome deNAc in the cell-free system and against recombinant enzyme. Monosaccharides with carboxylic acid or hydroxamic acid substituents were found to be the inhibitors of the trypanosome deNAc with IC50 values 0.1–1.5 mm, and the glucocyclitol-phospholipid was found to be a dual inhibitor of the deNAc and the α1-4-mannose transferase with an apparent IC50 = 19 ± 0.5 μm.

Chiral Mercaptoacetamides Display Enantioselective Inhibition of Histone Deacetylase 6 and Exhibit Neuroprotection in Cortical Neuron Models of Oxidative Stress

Mercaptoacetamide-based ligands have been designed as a new class of histone deacetylase (HDAC) inhibitors for possible use in the treatment of neurodegenerative diseases. The thiol group of these compounds provides a key binding element for interaction with the catalytic zinc ion, and thus differs from the more typically employed hydroxamic acid based zinc binding groups. Herein we disclose the chemistry and biology of some substituted mercaptoacetamides with the intention of increasing HDAC6 isoform selectivity while maintaining potency similar to their hydroxamic acid analogues. The introduction of a stereocenter α to the thiol group was found to have a considerable impact on HDAC inhibitor potency. These new compounds were also profiled for their therapeutic potential in an in vitro model of stress-induced neuronal injury and were found to act as nontoxic neuroprotective agents.

Investigating chelating sulfonamides and their use in metalloproteinase inhibitors

Matrix metalloproteinase inhibitors (MMPi) utilize zinc-binding groups (ZBGs) to chelate the catalytic Zn(II) ion resulting in enzyme inhibition. Adapting findings from the literature of Zn(II) ion sensors, we previously reported chelating sulfonamide inhibitors of MMP-2, some of which showed excellent selectivity over other gelatinases (MMP-9). Herein, we greatly expand our investigation of chelating sulfonamides as MMP inhibitors (MMPi) with the synthesis and screening of several new libraries consisting of 2-phenyl-7-sulfonamidobenzimidazole, 2-phenyl-7-sulfonamidobenzoxazole, 7-sulfonamidobenzimidazole, 7-sulfonamidobenzoxazole, and 2-(2-sulfonamidophenyl)-quinoline ZBG derivatives. A novel microwave irradiation synthetic procedure was utilized to rapidly and efficiently prepare these molecules. To better understand the coordination chemistry underlying these ZBGs, crystal structures of representative molecules with several first row transition metals were determined and differences in coordination preferences were considered. Surprisingly, only compounds with the 2-phenyl-7-sulfonamidobenzimidazole ZBG showed inhibition of MMP-2, suggesting that the specific structure of the ZBG can have a pronounced effect of inhibitory activity.

Hydroxamate represents a versatile zinc binding group for the development of new carbonic anhydrase inhibitors

Hydroxamates (R-CONHOH) have been scarcely investigated as carbonic anhydrase (CA, EC 4.2.1.1) inhibitors (CAIs). An inhibition/structural study of PhCONHOH is reported against all human isoforms. Comparing aliphatic (R = Me and CF(3)) and aromatic (R = Ph) hydroxamates as CAIs, we prove that CONHOH is a versatile zinc binding group. Depending on the nature of the R moiety, it can adopt different coordination modes to the catalytic ion within the CA active site.

Dithiocarbamates are strong inhibitors of the beta-class fungal carbonic anhydrases from Cryptococcus neoformans, Candida albicans and Candida glabrata

A series of N-mono- and N,N-disubstituted dithiocarbamates have been investigated as inhibitors of three β-carbonic anhydrases (CAs, EC 4.2.1.1) from the fungal pathogens Cryptococcus neoformans, Candida albicans and Candida glabrata, that is, Can2, CaNce103 and CgNce103, respectively. These enzymes were inhibited with efficacies between the subnanomolar to the micromolar range, depending on the substitution pattern at the nitrogen atom from the dithiocarbamate zinc-binding group. This new class of β-CA inhibitors may have the potential for developing antifungal agents with a diverse mechanism of action compared to the clinically used drugs for which drug resistance was reported, and may also explain the efficacy of dithiocarbamates as agricultural antifungal agents.

Novel 1-Hydroxypiperazine-2,6-diones as New Leads in the Inhibition of Metalloproteinases

New compounds containing a novel zinc-binding group (1-hydroxypiperazine-2,6-dione, HPD) have been identified as effective inhibitors of matrix metalloproteinases (MMPs), with activities in the nanomolar concentration range. That moiety seemed to bind the catalytic zinc ion of MMPs, revealing itself as a new potential substitute for the hydroxamate group in the next generation of metalloproteinase inhibitors. The X-ray crystal structure of 1b elucidated its 3D conformation and supramolecular packing in solid state. Theoretical procedures were used to investigate the binding mode of this class of compounds, within the active site of MMP13. A computational method involving docking and hybrid quantum mechanical and molecular mechanical (QM/MM) dynamic simulations was developed and applied. This study suggested that the HPD moiety binds bidentately to the catalytic zinc through its oxygen atoms. The final structure obtained will allow straightforward drug design approaches in view of further optimization and development of new MMP inhibitors bearing the HPD moiety.

Insights into the Complex Formed by Matrix Metalloproteinase-2 and Alloxan Inhibitors: Molecular Dynamics Simulations and Free Energy Calculations

Matrix metalloproteinases (MMP) are well-known biological targets implicated in tumour progression, homeostatic regulation, innate immunity, impaired delivery of pro-apoptotic ligands, and the release and cleavage of cell-surface receptors. Hence, the development of potent and selective inhibitors targeting these enzymes continues to be eagerly sought. In this paper, a number of alloxan-based compounds, initially conceived to bias other therapeutically relevant enzymes, were rationally modified and successfully repurposed to inhibit MMP-2 (also named gelatinase A) in the nanomolar range. Importantly, the alloxan core makes its debut as zinc binding group since it ensures a stable tetrahedral coordination of the catalytic zinc ion in concert with the three histidines of the HExxHxxGxxH metzincin signature motif, further stabilized by a hydrogen bond with the glutamate residue belonging to the same motif. The molecular decoration of the alloxan core with a biphenyl privileged structure allowed to sample the deep S1′ specificity pocket of MMP-2 and to relate the high affinity towards this enzyme with the chance of forming a hydrogen bond network with the backbone of Leu116 and Asn147 and the side chains of Tyr144, Thr145 and Arg149 at the bottom of the pocket. The effect of even slight structural changes in determining the interaction at the S1′ subsite of MMP-2 as well as the nature and strength of the binding is elucidated via molecular dynamics simulations and free energy calculations. Among the herein presented compounds, the highest affinity (pIC50 = 7.06) is found for BAM, a compound exhibiting also selectivity (>20) towards MMP-2, as compared to MMP-9, the other member of the gelatinases.

Structure and Dynamics of a Dizinc Metalloprotein: Effect of Charge Transfer and Polarization

Structures and dynamics of a recently designed dizinc metalloprotein (DFsc) (J. Mol. Biol. 2003, 334, 1101) are studied by molecular dynamics simulation using a dynamically adapted polarized force field derived from fragment quantum calculation for protein in solvent. To properly describe the effect of charge transfer and polarization in the present approach, quantum chemistry calculation of the zinc-binding group is periodically performed (on-the-fly) to update the atomic charges of the zinc-binding group during the MD simulation. Comparison of the present result with those obtained from simulations under standard AMBER force field reveals that charge transfer and polarization are critical to maintaining the correct asymmetric metal coordination in the DFsc. Detailed analysis of the result also shows that dynamic fluctuation of the zinc-binding group facilitates solvent interaction with the zinc ions. In particular, the dynamic fluctuation of the zinc-zinc distance is shown to be an important feature of the catalytic function of the di-ion zinc-binding group. Our study demonstrates that the dynamically adapted polarization approach is computationally practical and can be used to study other metalloprotein systems.