Catalytic Zinc Ion Research Articles

The C-terminal region of human plasma fetuin-B is dispensable for the raised-elephant-trunk mechanism of inhibition of astacin metallopeptidases

Human fetuin-B plays a key physiological role in human fertility through its inhibitory action on ovastacin, a member of the astacin family of metallopeptidases. The inhibitor consists of tandem cystatin-like domains (CY1 and CY2), which are connected by a linker containing a “CPDCP-trunk” and followed by a C-terminal region (CTR) void of regular secondary structure. Here, we solved the crystal structure of the complex of the inhibitor with archetypal astacin from crayfish, which is a useful model of human ovastacin. Two hairpins from CY2, the linker, and the tip of the “legumain-binding loop” of CY1 inhibit crayfish astacin following the “raised-elephant-trunk mechanism” recently reported for mouse fetuin-B. This inhibition is exerted by blocking active-site cleft sub-sites upstream and downstream of the catalytic zinc ion, but not those flanking the scissile bond. However, contrary to the mouse complex, which was obtained with fetuin-B nicked at a single site but otherwise intact, most of the CTR was proteolytically removed during crystallization of the human complex. Moreover, the two complexes present in the crystallographic asymmetric unit diverged in the relative arrangement of CY1 and CY2, while the two complexes found for the mouse complex crystal structure were equivalent. Biochemical studies in vitro confirmed the differential cleavage susceptibility of human and mouse fetuin-B in front of crayfish astacin and revealed that the cleaved human inhibitor blocks crayfish astacin and human meprin α and β only slightly less potently than the intact variant. Therefore, the CTR of animal fetuin-B orthologs may have a function in maintaining a particular relative orientation of CY1 and CY2 that nonetheless is dispensable for peptidase inhibition.

Synthesis and structure activity relationship of 1, 3-benzo-thiazine-2-thiones as selective HDAC8 inhibitors

Human histone deacetylase 8 (HDAC8) is a highly promising target for neuroblastoma and other types of cancer. Several HDAC inhibitors are approved for the treatment of special cancer subtypes or are evaluated in clinical trials. By far the most drugs or drug candidates contain a hydroxamate group that chelates the catalytic zinc ion within HDACs. Most hydroxamate inhibitors are more or less unselective, although there are considerable exceptions demonstrating the general feasibility to develop at least HDAC isoenzyme selective inhibitors. In addition, hydroxamates have recently come under discussion regarding their potential for mutagenicity. Recently, PD-404,182 was discovered as a selective and potent non-hydroxamate inhibitor of HDAC8. However, this active compound turned out to be decomposed in the presence of glutathion (GSH).Here, we describe the synthesis of significantly improved analogs of PD-404,182 that demonstrate both, great selectivity for HDAC8 and also chemical stability in the presence of GSH. The compounds are characterized with respect to structure-activity relationship, binding mode and target engagement in neuroblastoma cells by combining biochemical and biophysical methods with chemoinformatics.

Molecular determinants of the mechanism and substrate specificity of Clostridium difficile proline-proline endopeptidase-1

Pro-Pro endopeptidase-1 (PPEP-1) is a secreted metalloprotease from the bacterial pathogen Clostridium difficile that cleaves two endogenous adhesion proteins. PPEP-1 is therefore important for bacterial motility and hence for efficient gut colonization during infection. PPEP-1 exhibits a unique specificity for Pro-Pro peptide bonds within the consensus sequence VNP↓PVP. In this study, we combined information from crystal and NMR structures with mutagenesis and enzyme kinetics to investigate the mechanism and substrate specificity of PPEP-1. Our analyses revealed that the substrate-binding cleft of PPEP-1 is shaped complementarily to the major conformation of the substrate in solution. We found that it possesses features that accept a tertiary amide and help discriminate P1' residues by their amide hydrogen bond-donating potential. We also noted that residues Lys-101, Trp-103, and Glu-184 are crucial for proteolytic activity. Upon substrate binding, these residues position a flexible loop over the substrate-binding cleft and modulate the second coordination sphere of the catalytic zinc ion. On the basis of these findings, we propose an induced-fit model in which prestructured substrates are recognized followed by substrate positioning within the active-site cleft and a concomitant increase in the Lewis acidity of the catalytic Zn2+ ion. In conclusion, our findings provide detailed structural and mechanistic insights into the substrate recognition and specificity of PPEP-1 from the common gut pathogen C. difficile.

Crystal Structure of a Highly Thermostable α-Carbonic Anhydrase from Persephonella marina EX-H1.

Bacterial α-type carbonic anhydrase (α-CA) is a zinc metalloenzyme that catalyzes the reversible and extremely rapid interconversion of carbon dioxide to bicarbonate. In this study, we report the first crystal structure of a hyperthermostable α-CA from Persephonella marina EX-H1 (pmCA) in the absence and presence of competitive inhibitor, acetazolamide. The structure reveals a compactly folded pmCA homodimer in which each monomer consists of a 10-stranded β-sheet in the center. The catalytic zinc ion is coordinated by three highly conserved histidine residues with an exchangeable fourth ligand (a water molecule, a bicarbonate anion, or the sulfonamide group of acetazolamide). Together with an intramolecular disulfide bond, extensive interfacial networks of hydrogen bonds, ionic and hydrophobic interactions stabilize the dimeric structure and are likely responsible for the high thermal stability. We also identified novel binding sites for calcium ions at the crystallographic interface, which serve as molecular glue linking negatively charged and otherwise repulsive surfaces. Furthermore, this large negatively charged patch appears to further increase the thermostability at alkaline pH range via favorable charge-charge interactions between pmCA and solvent molecules. These findings may assist development of novel α-CAs with improved thermal and/or alkaline stability for applications such as CO2 capture and sequestration.

Identification of adenine-N9-(methoxy)ethyl-β-bisphosphonate as NPP1 inhibitor attenuates NPPase activity in human osteoarthritic chondrocytes.

Overproduction of extracellular diphosphate due to hydrolysis of ATP by NPP1 leads to pathological calcium diphosphate (pyrophosphate)dihydrate deposition (CPPD) in cartilage, resulting in a degenerative joint disease that today lacks a cure. Here, we targeted the identification of novel NPP1 inhibitors as potential therapeutic agents for CPPD deposition disease. Specifically, we synthesized novel analogs of AMP (NPP1 reaction product) and ADP (NPP1 inhibitor). These derivatives incorporate several chemical modifications of the natural nucleotides including (1) a methylene group replacing the Pα,β-bridging oxygen atom to provide metabolic resistance, (2) sulfonate group(s) replacing phosphonate(s) to improve binding to NPP1's catalytic zinc ions, (3) an acyclic nucleotide analog to allow flexible binding in the NPP1 catalytic site, and (4) a benzimidazole base replacing adenine. Among the investigated compounds, adenine-N9-(methoxy)ethyl-β-bisphosphonate, 10, was identified as an NPP1 inhibitor (Ki 16.3μM vs. the artificial substrate p-nitrophenyl thymidine-5'-monophosphate (p-Nph-5'-TMP), and 9.60μM vs. the natural substrate, ATP). Compound 10 was selective for NPP1 vs. human NPP3, human CD39, and tissue non-specific alkaline phosphatase (TNAP), but also inhibited human CD73 (Ki 12.6μM). Thus, 10 is a dual NPP1/CD73 inhibitor, which could not only be of interest for treating CPPD deposition disease and calcific aortic valve disease but may also be considered for the immunotherapy of cancer. Compound 10 proved to be a promising inhibitor, which almost completely reduces NPPase activity in human osteoarthritic chondrocytes at a concentration of 100μM.

Oxaprozin: A new hope in the modulation of matrix metalloproteinase 9 activity.

Oxaprozin (4,5-diphenyl-2-oxazolepropionic acid) is a non-steroidal, analgesic and antipyretic propionic acid derivative, whose activity in treating inflammatory disorders is well known. The aim of this study was to investigate the ability of oxaprozin to modulate the activity of matrix metalloproteinase 9 (MMP-9), a zinc-dependent endopeptidase involved in a wide range of physiological and pathological events associated with extracellular matrix (ECM) remodelling. The interaction between oxaprozin and MMP-9 was firstly investigated in silico by molecular docking and analysis with LIGPLOT software. Subsequently, the potential inhibitory activity of oxaprozin against MMP-9 and the possible mechanism of the ligand-enzyme interaction were investigated in vitro. Taking into account the in silico findings, MMP-9 can be considered a potential target of oxaprozin, which seems to be able to chelate the catalytic zinc ion through the nitrogen of the oxazole ring and the carboxylate moiety. Moreover, one of the phenyl rings interact with the S1' inhibitor-binding pocket through hydrophobic interaction. Gelatin zymography and enzymatic inhibition assay confirmed the potential role of oxaprozin as a competitive inhibitor of MMP-9. These observations sound particularly interesting if we consider the pathological role of MMP-9, especially evident in inflammatory conditions and cancer. This work may represent a starting point to improve the understanding of the role of oxaprozin, as well as its structural analogues, in modulating the MMP-9 function.

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.

Hydroxamic Acid Inhibitors Provide Cross-Species Inhibition of Plasmodium M1 and M17 Aminopeptidases.

There is an urgent clinical need for antimalarial compounds that target malaria caused by both Plasmodium falciparum and Plasmodium vivax. The M1 and M17 metalloexopeptidases play key roles in Plasmodium hemoglobin digestion and are validated drug targets. We used a multitarget strategy to rationally design inhibitors capable of potent inhibition of the M1 and M17 aminopeptidases from both P. falciparum ( Pf-M1 and Pf-M17) and P. vivax ( Pv-M1 and Pv-M17). The novel chemical series contains a hydroxamic acid zinc binding group to coordinate catalytic zinc ion/s, and a variety of hydrophobic groups to probe the S1' pockets of the four target enzymes. Structural characterization by cocrystallization showed that selected compounds utilize new and unexpected binding modes; most notably, compounds substituted with bulky hydrophobic substituents displace the Pf-M17 catalytic zinc ion. Excitingly, key compounds of the series potently inhibit all four molecular targets and show antimalarial activity comparable to current clinical candidates.

Stereo- and Regioselectivity in Catalyzed Transformation of a 1,2-Disubstituted Vicinal Diol and the Corresponding Diketone by Wild Type and Laboratory Evolved Alcohol Dehydrogenases

ADH-A from Rhodococcus ruber DSM 44541 catalyzes the oxidation of (S)-1-phenylethanol 3000-fold more efficiently as compared with the 2-hydroxylated derivative (R)-phenylethane-1,2-diol. The enzyme is also highly selective for sec-alcohols with comparably low activities with the corresponding primary alcohols. When challenged with a substrate containing two secondary alcohols, such as 1-phenylpropane-(1R,2S)-diol, ADH-A favors the oxidation of the benzylic carbon of this alcohol. The catalytic efficiency, however, is modest in comparison to the activity with (S)-1-phenylethanol. To investigate the structural requirements for improved oxidation of vicinal diols, we conducted iterative saturation mutagenesis combined with activity screening. A first-generation variant, B1 (Y54G, L119Y) displays a 2-fold higher kcat value with 1-phenylpropane-(1R,2S)-diol and a shift in the cooperative behavior in alcohol binding, from negative in the wild type, to positive in B1, suggesting a shift from a less active enzyme form (T) in the wild type to a more active form (R) in the B1 variant. Also, the regiopreference changed to favor oxidation of C-2. A second-generation variant, B1F4 (F43T, Y54G, L119Y, F282W), shows further improvement in the turnover and regioselectivity in oxidation of 1-phenylpropane-(1R,2S)-diol. The crystal structures of the B1 and B1F4 variants describe the structural alterations to the active site, the most significant of which is a repositioning of a Tyr side-chain located distal to the coenzyme and the catalytic zinc ion. The links between the changes in structures and stereoselectivities are rationalized by molecular dynamics simulations of substrate binding at the respective active sites.

A Novel Class of Schistosoma mansoni Histone Deacetylase 8 (HDAC8) Inhibitors Identified by Structure-Based Virtual Screening and In Vitro Testing.

A promising means in the search of new small molecules for the treatment of schistosomiasis (amongst other parasitic ailments) is by targeting the parasitic epigenome. In the present study, a docking based virtual screening procedure using the crystal structure of histone deacetylase 8 from Schistosoma mansoni (smHDAC8) was designed. From the developed screening protocol, we were able to identify eight novel N-(2,5-dioxopyrrolidin-3-yl)-n-alkylhydroxamate derivatives as smHDAC8 inhibitors with IC50 values ranging from 4.4–20.3 µM against smHDAC8. These newly identified inhibitors were further tested against human histone deacetylases (hsHDAC1, 6 and 8), and were found also to be exerting interesting activity against them. In silico prediction of the docking pose of the compounds was confirmed by the resolved crystal structure of one of the identified hits. This confirmed these compounds were able to chelate the catalytic zinc ion in a bidentate fashion, whilst showing an inverted binding mode of the hydroxamate group when compared to the reported smHDAC8/hydroxamates crystal structures. Therefore, they can be considered as new potential scaffold for the development of new smHDAC8 inhibitors by further investigation of their structure–activity relationship.

The epitope-mediated MMP activation assay: detection and quantification of the activation of Mmp2 in vivo in the zebrafish embryo.

Matrix remodeling is a consequence of tightly regulated matrix metalloproteinase (MMP) activity. MMPs are synthesized as inactive precursors with auto-inhibitory N-terminal propeptides, the proteolytic removal of which exposes the catalytic zinc ion, rendering the protease active. The regulation of MMP activation has been investigated primarily in tissue culture and biochemical assays that lack important biological context. Here we present the epitope-mediated MMP activation (EMMA) assay and use it to observe the activation of Mmp2 (gelatinase A) by endogenous mechanisms in the intact zebrafish embryo. The hemagglutinin (HA) and GFP-tagged reporter construct becomes activated on the surface of specific cells and this activation is abolished by broad-spectrum inhibition of metalloproteinase activity, consistent with existing models of gelatinase A activation. The mechanism(s) acting on the construct are spatially restricted, metalloproteinase-dependent and replacing the HA tag with mCherry abolishes activation, showing that the mechanism(s) are sensitive to the structure of the N-terminal domain. The construct is activated strongly in maturing myotome boundaries, but also intracellularly within myofibrils, consistent with reports implicating this protease in muscle development and function. In addition to general-purpose tools for the production of "EMMAed" MMPs and other proteins, we have established a transgenic line of zebrafish expressing EMMAedMmp2 under control of an inducible promoter to facilitate further investigation into the regulation of this ubiquitous ECM-remodeling protease in vivo.

Design, synthesis, docking and biological evaluation of 4-phenyl-thiazole derivatives as autotaxin (ATX) inhibitors

The autotaxin-lysophophatidic acid (ATX-LPA) signaling pathway is involved in several human diseases such as cancer, autoimmune diseases, inflammatory diseases neurodegenerative diseases and fibrotic diseases. Herein, a series of 4-phenyl-thiazole based compounds was designed and synthesized. Compounds were evaluated for their ATX inhibitory activity using FS-3 and human plasma assays. In the FS-3 assay, compounds 20 and 21 significantly inhibited the ATX at low nanomolar level (IC50=2.99 and 2.19nM, respectively). Inhibitory activity of 21 was found to be slightly better than PF-8380 (IC50=2.80nM), which is one of the most potent ATX inhibitors reported till date. Furthermore, 21 displayed higher potency (IC50=14.99nM) than the first clinical ATX inhibitor, GLPG1690 (IC50=242.00nM) in the human plasma assay. Molecular docking studies were carried out to explore the binding pattern of newly synthesized compounds within active site of ATX. Docking studies suggested the putative binding mode of the novel compounds. Good ATX inhibitory activity of 21 was attributed to the hydrogen bonding interactions with Asn230, Trp275 and active site water molecules; electrostatic interaction with catalytic zinc ion and hydrophobic interactions with amino acids of the hydrophobic pocket.

Myroilysin Is a New Bacterial Member of the M12A Family of Metzincin Metallopeptidases and Is Activated by a Cysteine Switch Mechanism

Proteases play important roles in all living organisms and also have important industrial applications. Family M12A metalloproteases, mainly found throughout the animal kingdom, belong to the metzincin protease family and are synthesized as inactive precursors. So far, only flavastacin and myroilysin, isolated from bacteria, were reported to be M12A proteases, whereas the classification of myroilysin is still unclear due to the lack of structural information. Here, we report the crystal structures of pro-myroilysin from bacterium Myroides sp. cslb8. The catalytic zinc ion of pro-myroilysin, at the bottom of a deep active site, is coordinated by three histidine residues in the conserved motif HEXXHXXGXXH; the cysteine residue in the pro-peptide coordinates the catalytic zinc ion and inhibits myroilysin activity. Structure comparisons revealed that myroilysin shares high similarity with the members of the M12A, M10A, and M10B families of metalloproteases. However, a unique "cap" structure tops the active site cleft in the structure of pro-myroilysin, and this "cap" structure does not exist in the above structure-reported subfamilies. Further structure-based sequence analysis revealed that myroilysin appears to belong to the M12A family, but pro-myroilysin uses a "cysteine switch" activation mechanism with a unique segment, including the conserved cysteine residue, whereas other reported M12A family proteases use an "aspartate switch" activation mechanism. Thus, our results suggest that myroilysin is a new bacterial member of the M12A family with an exceptional cysteine switch activation mechanism. Our results shed new light on the classification of the M12A family and may suggest a divergent evolution of the M12 family.

New biochemical insight of conserved water molecules at catalytic and structural Zn2+ ions in human matrix metalloproteinase-I: a study by MD-simulation.

Human matrix metalloproteinase (MMP)-1 or collagenase-1 plays a significant role in embryonic development, tissue remodeling, and is also involved in several diseases like arthritis, metastasis, etc. Molecular dynamics simulation studies on hMMP-1 X-ray structures (PDB Id. 1CGE, 1CGF, 1CGL, 1HFC, and 2TCL) suggest that the three conserved water molecules (WH/1, WI, WS) are coordinated with catalytic zinc (ZnC), and one water molecule (W) is associated at structural zinc ion (ZnS). Transition of the coordination geometry around ZnC from tetrahedral to octahedral and tetrahedral to trigonal bipyramidal at ZnS are also observed during the dynamics. Recognition of two zinc ions through water mediated bridges (ZnC - WH (W1)…W2….H183 - ZnS) and stabilization of secondary coordination zone around the metal ions indicates the possibility of ZnC…ZnS coupled catalytic mechanism in hMMP-I. This study not only reveals a functionally important role of conserved water molecules in hMMP-I but also highlights the involvement of other non catalytic residues, such as S172 and D170 in the catalytic mechanism. The results obtained in this study could be relevant for importance of conserved water mediated recognition site of the sequence residue id. 202(RWTNNFREY)210, interaction of W(tryptophan)203 to zinc bound histidine, their influence on the water molecules that are involved in bridging between ZnC and ZnS, and structure-based design of specific hMMP inhibitors. Graphical abstract Water mediated recognition of structural and catalytic zinc ions of hMMP-1 structure (MD simulatated conformation).

Experimental Dissection of Metalloproteinase Inhibition-Mediated and Toxic Effects of Phenanthroline on Zebrafish Development

Metalloproteinases are zinc-dependent endopeptidases that function as primary effectors of tissue remodeling, cell-signaling, and many other roles. Their regulation is ferociously complex, and is exquisitely sensitive to their molecular milieu, making in vivo studies challenging. Phenanthroline (PhN) is an inexpensive, broad-spectrum inhibitor of metalloproteinases that functions by chelating the catalytic zinc ion, however its use in vivo has been limited due to suspected off-target effects. PhN is very similar in structure to phenanthrene (PhE), a well-studied poly aromatic hydrocarbon (PAH) known to cause toxicity in aquatic animals by activating the aryl hydrocarbon receptor (AhR). We show that zebrafish are more sensitive to PhN than PhE, and that PhN causes a superset of the effects caused by PhE. Morpholino knock-down of the AhR rescues the effects of PhN that are shared with PhE, suggesting these are due to PAH toxicity. The effects of PhN that are not shared with PhE (specifically disruption of neural crest development and angiogenesis) involve processes known to depend on metalloproteinase activity. Furthermore these PhN-specific effects are not rescued by AhR knock-down, suggesting that these are bona fide effects of metalloproteinase inhibition, and that PhN can be used as a broad spectrum metalloproteinase inhibitor for studies with zebrafish in vivo.

High-speed atomic force microscopy reveals strongly polarized movement of clostridial collagenase along collagen fibrils.

Bacterial collagenases involved in donor infection are widely applied in many fields due to their high activity and specificity; however, little is known regarding the mechanisms by which bacterial collagenases degrade insoluble collagen in host tissues. Using high-speed atomic force microscopy, we simultaneously visualized the hierarchical structure of collagen fibrils and the movement of a representative bacterial collagenase, Clostridium histolyticum type I collagenase (ColG), to determine the relationship between collagen structure and collagenase movement. Notably, ColG moved ~14.5 nm toward the collagen N terminus in ~3.8 s in a manner dependent on a catalytic zinc ion. While ColG was engaged, collagen molecules were not only degraded but also occasionally rearranged to thicken neighboring collagen fibrils. Importantly, we found a similarity of relationship between the enzyme-substrate interface structure and enzyme migration in collagen-collagenase and DNA-nuclease systems, which share a helical substrate structure, suggesting a common strategy in enzyme evolution.

An Investigation into the Extended Coordination Network of Catalytic Zinc Ions in a Class B β‐lactamase

Antibiotics have a long history as essential tools in fighting bacterial infections. The β‐lactam subgroup of antibiotics has been widely and successfully used since the identification of penicillin in the early 20th century. In recent years, β‐lactamases have been responsible for an alarming increase of bacterial resistance to many β‐lactam antibiotics. Our study gains insight into the mechanism of action of New Delhi Metallo β‐lactamase (NDM‐1 EC: 3.5.2.6) to facilitate the development of mechanism‐based inhibitors to metallo β‐lactamases. NDM1 is a dinuclear zinc‐containing enzyme that demonstrates higher catalytic efficiency than its mono‐zinc counterparts. High‐resolution crystal structures of NDM‐1 by Zhang and Hao reveal insight into the role zinc ions play in the formation of a catalytic hydroxide as well as additional stabilization of the leaving group. Our inquiry explores the role of a key residue of NDM‐1: in particular, Asp‐223 that may play a role in the extended coordination network of the catalytic metals. Asparagine and alanine mutants have been generated, and the steady‐state kinetic parameters are being evaluated.

Substrate complexes of human dipeptidyl peptidase III reveal the mechanism of enzyme inhibition.

Human dipeptidyl-peptidase III (hDPP III) is a zinc-dependent hydrolase cleaving dipeptides off the N-termini of various bioactive peptides. Thus, the enzyme is likely involved in a number of physiological processes such as nociception and is also implicated in several forms of cancer. We present high-resolution crystal structures of hDPP III in complex with opioid peptides (Met-and Leu-enkephalin, endomorphin-2) as well as with angiotensin-II and the peptide inhibitor IVYPW. These structures confirm the previously reported large conformational change of the enzyme upon ligand binding and show that the structure of the closed conformation is independent of the nature of the bound peptide. The overall peptide-binding mode is also conserved ensuring the correct positioning of the scissile peptide bond with respect to the catalytic zinc ion. The structure of the angiotensin-II complex shows, how longer peptides are accommodated in the binding cleft of hDPP III. Differences in the binding modes allow a distinction between real substrates and inhibitory peptides or “slow” substrates. The latter displace a zinc bound water molecule necessitating the energetically much less favoured anhydride mechanism as opposed to the favoured promoted-water mechanism. The structural data also form the necessary framework for the design of specific hDPP III inhibitors.

Molecular Recognition of the Catalytic Zinc(II) Ion in MMP-13: Structure-Based Evolution of an Allosteric Inhibitor to Dual Binding Mode Inhibitors with Improved Lipophilic Ligand Efficiencies.

Matrix metalloproteinases (MMPs) are a class of zinc dependent endopeptidases which play a crucial role in a multitude of severe diseases such as cancer and osteoarthritis. We employed MMP-13 as the target enzyme for the structure-based design and synthesis of inhibitors able to recognize the catalytic zinc ion in addition to an allosteric binding site in order to increase the affinity of the ligand. Guided by molecular modeling, we optimized an initial allosteric inhibitor by addition of linker fragments and weak zinc binders for recognition of the catalytic center. Furthermore we improved the lipophilic ligand efficiency (LLE) of the initial inhibitor by adding appropriate zinc binding fragments to lower the clogP values of the inhibitors, while maintaining their potency. All synthesized inhibitors showed elevated affinity compared to the initial hit, also most of the novel inhibitors displayed better LLE. Derivatives with carboxylic acids as the zinc binding fragments turned out to be the most potent inhibitors (compound 3 (ZHAWOC5077): IC50 = 134 nM) whereas acyl sulfonamides showed the best lipophilic ligand efficiencies (compound 18 (ZHAWOC5135): LLE = 2.91).

Classification of ADAMTS binding sites: The first step toward selective ADAMTS7 inhibitors

Genome-wide association studies identified ADAMTS7 as a risk locus for coronary artery disease. In carotid arteries of rats, neointima formation after balloon-mediated injury goes along with enhanced Adamts7 expression. Vice versa, Adamts7-deficient mice display reduced neointima formation following vascular injury. Although a causal link between ADAMTS7 and coronary artery disease remains to be proven, inhibition of ADAMTS7 represents a potential new target for intervention in this disease.ADAMTS7, a member of the ‘a disintegrin and metalloproteinase with thrombospondin motifs’ (ADAMTS) family of proteins, contains a catalytic zinc ion in the binding site of its metalloproteinase domain. The structure of ADAMTS7 and its inhibitors are unknown. In this study, we used in silico methods, including homology modeling and pharmacophore modeling, to analyze the ADAMTS7 metalloproteinase domain, particularly its binding site. The results revealed structural and sequence differences relative to the binding sites of the other ADAMTS proteins; these non-conserved regions represent potential binding regions for selective ADAMTS7 inhibitors. The main contribution of this study is the proposal of a pharmacophore for ADAMTS7. The characterization of the ADAMTS7 binding site and definition of a pharmacophore are the first step toward developing a new therapeutic target for coronary artery disease.