Trivalent metal ions based on inorganic compounds with in vitro inhibitory activity of matrix metalloproteinase 13
Introduction
Matrix metalloproteinases (matrixins or MMPs) represent a family of zinc metallproteinases that is capable of degrading and remodeling structural proteins in the extracellular matrix such as gelatin, elastins, collagens, matrix glycoproteins and proteoglycan. MMPs play a major role in almost all aspects of cell behavior and belong to the metzincin superfamily of metalloproteinases. The latter is characterized by the presence of a divalent zinc atom (Zn2+) in the active center of the enzyme. The typical structure of MMPs consists of a signal peptide (SP), a catalytic domain and a haemopexin-like domain [1]. The Zn2+ binding site in the catalytic core domain of all MMPs features a HEXXHXXGXXH amino acid, and the individual sequences bear high similarity in their 3D structures. The pocket in the core domain is hydrophobic in nature, but variable in depth depending on the MMPs, representing a determining factor of substrate specificity of MMPs. In fact, the S1′ pocket in the catalytic domains of MMPs has been reported to be particularly important. The depth and the length of the S1′ pocket and amino acids all represent critical foundations for the design and synthesis of matrix metalloproteinase inhibitors (MMPIs) [2], [3], [4], [5], [6].
MMPs mainly regulate the synthesis and secretion of cytokines, growth factors, hormone receptors, and cell adhesion molecules. Moreover, MMPs are also found to be critically involved in processes including tissue remodeling, angiogenesis, arthritis, cardiovascular disease, stroke, multiple sclerosis, neurodegenerative diseases, and allergies [7], [8]. In tumor progression, MMPs play a key role not only in invasion processes, angiogenesis, and metastasis, but also in cancer cell transformation, growth, apoptosis, signal transduction and immune regulation [9], [10]. It has been shown before that the secretion of MMPs by microvascular endothelial cells represents a critical step in the formation of new blood vessels and most of the MMPs may initiate and promote angiogenesis [11], [12]. In recent years, research groups around the globe have focussed on finding suitable inhibitors for MMPs to treat cancer or other related serious health conditions [13], [14], [15], [16], [17], [18]. The activities of MMPs can be regulated by endogenous inhibitors, such as tissue inhibitors of metalloproteinase (MMPIs) [19], [20], [21]. In general, MMPIs can be classified as follows: natural MMPIs secreted by tissues, synthetic MMPIs, MMPIs screened from natural products and MMPIs screened from phage. Originally, the first generation of MMPIs, used as peptides or derivatives, has been designed to simulate the substrate of MMPs. Inhibition is achieved by competing chelation of the central zinc ion (Zn2+) in MMP by ligands present in the inhibitors, e.g. hydroxylamine, carboxyl, thiols, etc [22]. In recent years, synthetic MMPIs have been used in clinical trials for MMP inhibition in the treatment of progressive lung cancer. However, due to a variety of side effects caused by the use of synthetic small molecule inhibitors in clinical applications, research has now focussed on finding MMPIs derived from natural products [23], [24], [25]. Anti-MMPs properties have been reported from the components of various natural products. For example, epigallocatechin gallate (EGCG) extracted from green tea exhibits a potent MMPI activity [26], [27]. The Korean monkshood root (Guanbaifu in Chinese), is commonly used in the treatment of many diseases such as arthralgia, headache, convulsive epilepsy, coronary heart disease, ischemic arrhythmia, pyocutaneous disease and anemogenous phlegm. It has been demonstrated to exhibit a strong inhibitory effect on different MMPs, such as MMP-2 and MMP-9 [28], [29], [30]. The active compound featuring an inhibitory effect on MMPs extracted from the Korean monkshood root was identified as aluminum ammonium sulfate dodecahydrate (NH4Al(SO4)2·12H2O). Further experiments have confirmed that the aluminum(III) ion (Al3+) is responsible for the inhibitory activity. This was the first time that an inorganic substrate, i.e. aluminum ammonium sulfate, has been demonstrated to exhibit MMP inhibitory activity, potent enough to interfere with MMP related cellular processes [31].
Human matrix metallopeptidase 13 (MMP-13), also known as collagenase-3, represents a key member of the MMP family and has been identified as an important target for the treatment of osteoarthritis (OA) [32], [33], [34], [35]. Until now, MMP-13 inhibitors have been identified mainly viahigh-throughput screening (HTS) in the synthetic compound library. Hydroxamic acid is a typical inhibitor for MMP-13 and the reaction mechanism for this activity has been suggested to be due to a functional group that is able to coordinate the central zinc(II) ion to two oxygen atoms via a bidentate ligand interaction [36], [37], [38], [39]. In our previous work, Al3+ has been shown to exhibit strong inhibitory effects on both MMP-2 and MMP-9 using a HT1080 cell line [31]. We are now interested to determine whether Al3+ or other metal ions would exhibit similar effects on the catalytic domain in MMP-13 (cdMMP-13) in vitro. Therefore, we further characterized the cdMMP-13 inhibitory activity using different metal ions and analyzed the corresponding active components. In this work, we obtained the target protein by refolding the recombinant histidine-tagged catalytic domain of MMP-13 [40]. Circular dichroism (CD) analysis confirmed that cdMMP-13, desalted with the metal ions, increases the stability of the secondary structure of the protein. By comparison of the inhibitory activity of compounds containing different valence metal ions with a positive control inhibitor (i.e. CL-82198) on cdMMP-13, the half maximal inhibitory concentration value (IC50) showed that trivalent metal compounds in particular exhibit a strong cdMMP-13 inhibition. Compared to other compounds, potassium hexacyanoferrate(III) (K3[Fe(CN)6]) has been shown to exhibit the best inhibitory effect [36]. Furthermore, the binding event between K3[Fe(CN)6] and cdMMP-13 has been confirmed by isothermal titration calorimetry (ITC) and an endothelial cell tube formation test provided evidence for the notion that this interaction may have a potential effect on anti-angiogenesis.
Section snippets
Materials
Yeast extract and tryptone were purchased from OXOID (Basingstoke, UK). Isopropyl β-d-1-thiogalactopyranoside (IPTG), kanamycin sulfate, urea, imidazole, acrylamide, tris(hydroxymethyl)aminomethane (Tris), sodium dodecyl sulfate (SDS), N,N,-methylenebisacrylamide and glycine were purchased from Sangon Biotech (Shanghai, China). The markers for SDS-polyacrylamide gel electrophoresis (SDS-PAGE) were purchased from Thermo Scientific (Waltham, MA, USA). Brij-35 was purchased from Amresco (Solon,
Expression and purification of cdMMP-13
The recombinant cdMMP-13 has been expressed and purified using the optimized conditions. The denatured his-cdMMP-13 was eluted (peak 4, cf. Fig. S1) and was analyzed by SDS-PAGE to give a single band with a protein size of about 23 kDa (Fig. S2). The target protein was obtained in 98% purity and was used for the refolding studies.
Optimization of refolding buffer
In the refolding buffer without metal ions (pH 7.5, 20 mM Tris), the recombinant cdMMP-13 could be obtained with a purity of about 90%. It has been reported that ionic
Conclusions
Potassium hexacyanoferrate(III), K3[Fe(CN)6], as a new MMP-13 specific inhibitor has been studied. Moreover, we refoldedthe target protein, exhibiting a higher bioactivity,using size exclusion chromatography (SEC). The refolding buffer with metal ions results in a higher α-helix than those without metal ions, did not affect the secondary structures of the enzymes and provided the target protein in a higher purity. Further studies show that trivalent metal ions, present in K3[Fe(CN)6], pose a
Acknowledgments
This study was supported financially by the National Science Foundation of China (21006077 and 31200641)and Science & Technology Department of Shaanxi Province of China (Grant No. 2011K16-05-08).
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