RESUMEN
There is a critical need for the development of more potent inhibitors for osteoarthritis (OA) therapy given the poor life quality of arthritis patients. Aggrecanase ADAMTS-5 (a disintegrin and metalloproteinase with thrombospondin motifs 5) is an established drug target identified for osteoarthritis. In this study, we evolved and characterized two new DNA aptamer inhibitors of ADAMTS-5, namely apt21 and apt25. The aptamers exhibited nanomolar binding affinity and high specificity against ADAMTS-5. KD values of apt21 and apt25 were determined by the Enzyme-linked Oligonucleotide Assay (ELONA) at 1.54 ± 0.16 nM and 1.79 ± 0.08 nM, respectively. Circular Dichroism (CD) analysis demonstrated that both aptamers formed monovalent cation dependent G-quadruplex structures. Calcium ions did not affect the binding of the aptamers to ADAMTS-5. The inhibitory effects of apt21 and apt25 on ADAMTS-5 were evaluated by the Förster Resonance Energy Transfer (FRET) assay, in which IC50 values of apt21 and apt25 were estimated at 52.76 ± 6.70 µM and 61.14 ± 9.67 µM, respectively. These two aptamers are the first DNA G-quadruplex aptamers demonstrated to inhibit ADAMTS-5 and could have value for OA therapy.
Asunto(s)
Aptámeros de Nucleótidos , Osteoartritis , Proteína ADAMTS4/química , Proteína ADAMTS4/genética , Proteína ADAMTS4/metabolismo , Proteína ADAMTS5/genética , Proteína ADAMTS5/metabolismo , Aptámeros de Nucleótidos/farmacología , Calcio , Cationes Monovalentes , ADN , Desintegrinas , Humanos , Osteoartritis/tratamiento farmacológico , TrombospondinasRESUMEN
A disintegrin-like and metalloproteinase with thrombospondin type-1 motifs-4 (ADAMTS-4) and ADAMTS-5 are zinc-dependent metalloproteinases that are involved in the maintenance of cartilage extracellular matrix (ECM) and are currently considered the major aggrecanases in the development of osteoarthritis. In this chapter we describe the establishment and cultivation of cell lines expressing ADAMTS-4,-5 and their domain deletion mutants; the collection of medium containing expressed ADAMTS-4,-5; the subsequent purification of this medium through anti-FLAG affinity chromatography; and the characterization of ADAMTS-4,-5 activity using synthetic Förster resonance energy transfer (FRET) peptide substrates.
Asunto(s)
Proteína ADAMTS4/química , Proteína ADAMTS4/metabolismo , Proteína ADAMTS5/química , Proteína ADAMTS5/metabolismo , Mutación , Proteína ADAMTS4/genética , Proteína ADAMTS5/genética , Cartílago/metabolismo , Dominio Catalítico , Técnicas de Cultivo de Célula/métodos , Cromatografía de Afinidad , Medios de Cultivo/química , Matriz Extracelular/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Células HEK293 , Humanos , Dominios ProteicosRESUMEN
ADAMTS (A Disintegrin-like and Metalloproteinase domain with Thrombospondin type 1 Motif)-1, -4 and -5 share the abilities to cleave large aggregating proteoglycans including versican and aggrecan. These activities are highly relevant to cardiovascular disease and osteoarthritis and during development. Here, using purified recombinant ADAMTS-1, -4 and -5, we quantify, compare, and define the molecular basis of their versicanase activity. A novel sandwich-ELISA detecting the major versican cleavage fragment was used to determine, for the first time, kinetic constants for versican proteolysis. ADAMTS-5 (kcat/Km 35 × 105 M-1 s-1) is a more potent (~18-fold) versicanase than ADAMTS-4 (kcat/Km 1.86 × 105 M-1 sec-1), whereas ADAMTS-1 versicanase activity is comparatively low. Deletion of the spacer domain reduced versicanase activity of ADAMTS-5 19-fold and that of ADAMTS-4 167-fold. Co-deletion of the ADAMTS-5 cysteine-rich domain further reduced versicanase activity to a total 153-fold reduction. Substitution of two hypervariable loops in the spacer domain of ADAMTS-5 (residues 739-744 and 837-844) and ADAMTS-4 (residues 717-724 and 788-795) with those of ADAMTS-13, which does not cleave proteoglycans, caused spacer-dependent reductions in versicanase activities. Our results demonstrate that these loops contain exosites critical for interaction with and processing of versican. The hypervariable loops of ADAMTS-5 are shown to be important also for its aggrecanase activity. Together with previous work on ADAMTS-13 our results suggest that the spacer domain hypervariable loops may exercise significant control of ADAMTS proteolytic activity as a general principle. Identification of specific exosites also provides targets for selective inhibitors.
Asunto(s)
Proteína ADAMTS1/química , Proteína ADAMTS4/química , Proteína ADAMTS5/química , Versicanos/metabolismo , Proteína ADAMTS13/química , Sitios de Unión , Dominio Catalítico , Humanos , Cinética , Unión ProteicaRESUMEN
The ADAMTS4 (a disintegrin and metalloproteinase with thrombospondin motifs 4) enzyme is a matrix-associated zinc metalloendopeptidase that plays an essential role in the degradation of cartilage aggrecan in arthritic diseases and has been recognized as one of the most primary targets for therapeutic intervention in osteoarthritis (OA). Here, we reported computational modeling of the atomic-level complex structure of ADAMTS4 with its cognate inhibitory protein TIMP3 based on high-resolution crystal template. By systematically examining the modeled complex structure we successfully identified a short inhibitory loop (62EASESLC68) in TIMP3 N-terminal inhibitory domain (NID) that directly participates in blocking the enzyme's active site, which, and its extended versions, were then broken from the full-length protein to serve as the peptide inhibitor candidates of ADAMTS4. Atomistic molecular dynamics simulation, binding energetic analysis, and fluorescence-based assay revealed that the TIMP3-derived linear peptides can only bind weakly to the enzyme (Kdâ¯=â¯74⯱â¯8⯵M), which would incur a considerable entropy penalty due to the high conformational flexibility and intrinsic disorder of these linear peptides. In this respect, we proposed a cyclization strategy to improve enzyme-peptide binding affinity by, instead of traditionally maximizing enthalpy contribution, minimizing entropy cost of the binding, where a disulfide bond was added across the two terminal residues of linear peptides, resulting in a number of TIMP3-derived cyclic peptides. Our studies confirmed that the cyclization, as might be expected, can promote peptide binding capability against ADAMTS4 substantially, with affinity increase by 3-fold, 9-fold and 7-fold for cyclic peptides , and , respectively.
Asunto(s)
Proteína ADAMTS4/metabolismo , Fragmentos de Péptidos/metabolismo , Péptidos Cíclicos/metabolismo , Inhibidor Tisular de Metaloproteinasa-3/metabolismo , Proteína ADAMTS4/química , Sitios de Unión , Entropía , Humanos , Simulación de Dinámica Molecular , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/química , Péptidos Cíclicos/síntesis química , Péptidos Cíclicos/química , Unión Proteica , Dominios Proteicos , Inhibidor Tisular de Metaloproteinasa-3/síntesis química , Inhibidor Tisular de Metaloproteinasa-3/químicaRESUMEN
In the development of osteoarthritis, aggrecan degrades prior to cartilage destruction. Aggrecanase-1 (ADAMTS-4) is considered to be the major enzyme responsible for cleaving the Glu373-Ala374 bond in the interglobular domain of aggrecan in humans. Therefore, inhibitors of ADAMTS-4 have therapeutic potential in the treatment of osteoarthritis. In the present work, we developed a chemical feature based pharmacophore model of ADAMTS-4 inhibitors using the HipHop module within the Catalyst program package in order to elucidate the structure-activity relationship and to carry out in-silico screening. The Maybridge database was screened using Hypo1 as a 3D query, and the best-fit hits that followed Lipinski's rule of five were subsequently screened to select the compounds. The hit compounds were then docked into the active site of ADAMTS-4, and interactions were visualized to determine the potential lead molecules. After subjecting all of the hits to various screening and filtering processes, 13 compounds were finally evaluated for their in vitro inhibitory activities. This study resulted in the identification of two lead compounds with potent inhibitory effects on ADAMTS-4 activity, with IC50 values of 0.042 µM and 0.028 µM, respectively. These results provide insight into the pharmacophoric requirements for the development of more potent ADAMTS-4 inhibitors. Graphical Abstract The aggrecan-degrading metalloprotease ADAMTS-4 has been identified as a novel therapeutic target for osteoarthritis. In this work, we used HipHop-based pharmacophore modeling and virtual screening of the Maybridge database to identify novel ADAMTS-4 inhibitors. These novel lead compounds act as potent and specific inhibitors for the ADAMTS-4 enzyme and could have therapeutic potential in the treatment of OA.
Asunto(s)
Proteína ADAMTS4 , Descubrimiento de Drogas , Simulación del Acoplamiento Molecular , Osteoartritis , Inhibidores de Proteasas/química , Proteína ADAMTS4/antagonistas & inhibidores , Proteína ADAMTS4/química , Humanos , Osteoartritis/tratamiento farmacológico , Osteoartritis/enzimología , Inhibidores de Proteasas/uso terapéuticoRESUMEN
Tissue engineering strategies for repairing and regenerating articular cartilage face critical challenges to recapitulate the dynamic and complex biochemical microenvironment of native tissues. One approach to mimic the biochemical complexity of articular cartilage is through the use of recombinant bacterial collagens as they provide a well-defined biological 'blank template' that can be modified to incorporate bioactive and biodegradable peptide sequences within a precisely defined three-dimensional system. We customized the backbone of a Streptococcal collagen-like 2 (Scl2) protein with heparin-binding, integrin-binding, and hyaluronic acid-binding peptide sequences previously shown to modulate chondrogenesis and then cross-linked the recombinant Scl2 protein with a combination of matrix metalloproteinase 7 (MMP7)- and aggrecanase (ADAMTS4)-cleavable peptides at varying ratios to form biodegradable hydrogels with degradation characteristics matching the temporal expression pattern of these enzymes in human mesenchymal stem cells (hMSCs) during chondrogenesis. hMSCs encapsulated within the hydrogels cross-linked with both degradable peptides exhibited enhanced chondrogenic characteristics as demonstrated by gene expression and extracellular matrix deposition compared to the hydrogels cross-linked with a single peptide. Additionally, these combined peptide hydrogels displayed increased MMP7 and ADAMTS4 activities and yet increased compression moduli after 6 weeks, suggesting a positive correlation between the degradation of the hydrogels and the accumulation of matrix by hMSCs undergoing chondrogenesis. Our results suggest that including dual degradation motifs designed to respond to enzymatic activity of hMSCs going through chondrogenic differentiation led to improvements in chondrogenesis. Our hydrogel system demonstrates a bimodal enzymatically degradable biological platform that can mimic native cellular processes in a temporal manner. As such, this novel collagen-mimetic protein, cross-linked via multiple enzymatically degradable peptides, provides a highly adaptable and well defined platform to recapitulate a high degree of biological complexity, which could be applicable to numerous tissue engineering and regenerative medicine applications.
Asunto(s)
Proteínas Bacterianas/química , Materiales Biomiméticos/química , Condrogénesis , Colágeno/química , Hidrogel de Polietilenoglicol-Dimetacrilato/química , Células Madre Mesenquimatosas/citología , Proteína ADAMTS4/química , Proteínas Bacterianas/genética , Materiales Biomiméticos/metabolismo , Cartílago Articular/citología , Diferenciación Celular , Células Cultivadas , Colágeno/genética , Colágeno/metabolismo , Reactivos de Enlaces Cruzados/química , Endopeptidasas/química , Matriz Extracelular/ultraestructura , Humanos , Metaloproteinasa 7 de la Matriz/química , Péptidos/química , Proteolisis , Streptococcus , Ingeniería de Tejidos/métodosRESUMEN
ADAMTS4 (Aggrecanase-1) is an important enzyme, which belongs to ADAMTS family. Aggrecanase-1 is involved in aggrecan degradation of articular cartilage in osteoarthritis and rheumatoid arthritis. Overall variability of S1' domain of ADAMTS4 has been the main selectivity determinant to design the unique inhibitors. 34 inhibitors from Binding database and literature were used to develop the pharmacophore model. The five featured pharmacophore model AHHRR had the best survival score of 3.493 and post-hoc score of 2.545, indicating that the model is highly reliable. The 3D-QSAR acquired had excellent r(2) value of 0.99 and GH score of 0.839. The validated pharmacophore model was used for insilico screening of Asinex and ZINC database for finding the potential lead compounds. ZINC00987406 and ASN04459656 which pose high glide score i.e >7 Kcal/mol and H-bond and hydrophobic interactions in the S1'loop residues of ADAMTS4 were subjected to Molecular Dynamics Simulation studies. Molecular dynamic simulation result indicates that the RMSD and RMSF of backbone atoms for the above complexes were within the limit of 2.0 AË. These compounds can be potential candidates for osteoarthritis by inhibiting ADAMTS4.