Crystal structures of the catalytic domain of human stromelysin-1 (MMP-3) and collagenase-3 (MMP-13) with a hydroxamic acid inhibitor SM-25453

Crystal structures of the catalytic domain of human stromelysin-1 (MMP-3) and collagenase-3 (MMP-13) with a hydroxamic acid inhibitor SM-25453 have been solved at 2.01 and 2.37A resolutions, respectively. The results revealed that the binding modes for this inhibitor to MMP-3 and -13 were quite similar. However, subtle comparative differences were observed at the bottom of S1' pockets, which were occupied with the guanidinomethyl moiety of the inhibitor. A remarkable feature of the inhibitor was the deep penetration of its long aliphatic chain into the S1' pocket and exposure of the guanidinomethyl moiety to the solvent.     

慢性睡眠限制状态下大鼠髁突软骨MMP-1,MMP-13表达的变化

目的:探讨MMP-1,MMP-13在慢性睡眠限制引起大鼠髁突软骨结构变化中的表达变化及作用。方法:180只雄性Wistar大鼠随机分为3组(n=60):慢性睡眠限制组(CSR)、大平台组(LC)、笼养组(CON)。每组根据试验时间不同分别分为3个亚组(n=20):7天(7D)、14天(14D)、21天(21D)组。参考改良多平台法(MMPM)建立大鼠的慢性睡眠限制模型。通过HE染色观察大鼠髁突软骨的结构变化。通过免疫组化和实时定量PCR分别检测MMP-1和MMP-13的蛋白水平及m RNA水平的表达变化。结果:HE染色和扫描电镜结果显示,CSR组的大鼠髁突软骨出现了病理性的改变。与CON和LC组比较,CSR组MMP-1和MMP-13的m RNA转录和蛋白表达水平明显升高(P0.05)。结论:慢性睡眠限制能够引起大鼠颞下颌关节髁突软骨的病理性变化。MMP-1和MMP-13的表达水平的变化可能在大鼠髁突软骨病理性改变中起关键作用。     

The Recognition of Collagen and Triple-helical Toolkit Peptides by MMP-13: SEQUENCE SPECIFICITY FOR BINDING AND CLEAVAGE*

Remodeling of collagen by matrix metalloproteinases (MMPs) is crucial to tissue homeostasis and repair. MMP-13 is a collagenase with a substrate preference for collagen II over collagens I and III. It recognizes a specific, well-known site in the tropocollagen molecule where its binding locally perturbs the triple helix, allowing the catalytic domain of the active enzyme to cleave the collagen α chains sequentially, at Gly⁷⁷⁵–Leu⁷⁷⁶ in collagen II. However, the specific residues upon which collagen recognition depends within and surrounding this locus have not been systematically mapped. Using our triple-helical peptide Collagen Toolkit libraries in solid-phase binding assays, we found that MMP-13 shows little affinity for Collagen Toolkit III, but binds selectively to two triple-helical peptides of Toolkit II. We have identified the residues required for the adhesion of both proMMP-13 and MMP-13 to one of these, Toolkit peptide II-44, which contains the canonical collagenase cleavage site. MMP-13 was unable to bind to a linear peptide of the same sequence as II-44. We also discovered a second binding site near the N terminus of collagen II (starting at helix residue 127) in Toolkit peptide II-8. The pattern of binding of the free hemopexin domain of MMP-13 was similar to that of the full-length enzyme, but the free catalytic subunit bound none of our peptides. The susceptibility of Toolkit peptides to proteolysis in solution was independent of the very specific recognition of immobilized peptides by MMP-13; the enzyme proved able to cleave a range of dissolved collagen peptides.     

A systematic computational analysis of human matrix metalloproteinase 13 (MMP-13) crystal structures and structure-based identification of prospective drug candidates as MMP-13 inhibitors repurposable for osteoarthritis

Abstract

Osteoarthritis (OA) is the most common form of arthritis with no available disease-modifying treatments, and is a major cause of disability. Matrix metalloproteinase 13 (MMP-13) is vital for OA progression and thus, inhibition of MMP-13 is an effective strategy to treat OA. Since the past few decades, drug repurposing has gained substantial popularity worldwide as a time- and cost-effective approach to find new indications for the existing drugs. Therefore, more than 40 X-ray co-crystal structures of the human MMP-13 with bound inhibitors are investigated to gain the structural insights such as conserved direct interactions with binding site residues, namely Ala-238, Thr-245 and Thr-247. Afterwards, enrichment study using active and decoy set of ligands revealed three MMP-13 structures (PDB-IDs: 1XUC, 3WV1 and 5BPA) with optimal enrichment performance. Docking-based screening of existing drugs against the three crystal structures followed by binding free-energy calculation suggested drugs namely eltrombopag, cilostazol and domperidone as potential MMP-13 inhibitors that need further experimental validation. These insights may serve as a potential starting point of further experimental validation and structure-based drug design/repurposing of MMP-13 inhibitors for the treatment of OA. Abbreviations 2D two-dimensional

3D three-dimensional

FDA Food and Drug Administration

MM-GBSA Molecular Mechanics Generalized Born Surface Area

MMPs matrix metalloproteinases

MMP-13 matrix metalloproteinase 13

NMR nuclear magnetic resonance

OA osteoarthritis

PDB Protein Data Bank

PDB-ID Protein Data Bank ID

PLIP protein–ligand interaction profiler

ROC receiver operating characteristic,

RMSD root mean square deviation

Communicated by Ramaswamy H. Sarma     

Isolation and characterization of human monoclonal antibodies specific to MMP-1A, MMP-2 and MMP-3

Matrix metalloproteinases (MMPs), a group of more than 20 zinc-containing endopeptidases, are up-regulated in many diseases, but the use of MMP inhibitors for therapeutic purposes has often been disappointing, possibly for the limited specificity of the drugs used in clinical trials. In principle, individual MMPs could be specifically drugged by monoclonal antibodies, either by inhibition of their catalytic activity or by antibody-based pharmacodelivery strategies. In this article we describe the isolation and affinity maturation of recombinant antibodies (SP1, SP2, SP3) specific to the murine catalytic domains of MMP-1A, MMP-2 and MMP-3. These novel reagents allowed a systematic comparative immunofluorescence analysis of the expression patterns of their cognate antigens in a variety of healthy, cancerous and arthritic murine tissues. While all three MMPs were strongly expressed in tumor and arthritis specimens, MMP-1A was completely undetectable in the normal tissues tested, while MMP-2 and MMP-3 exhibited a weak expression in certain normal tissues (e.g., liver). The new antibodies may serve as building blocks for the development of antibody-based therapy strategies in mouse models of pathology.     

Improving potency and selectivity of a new class of non-Zn-chelating MMP-13 inhibitors

Discovery and optimization of potency and selectivity of a non-Zn-chelating MMP-13 inhibitor with the aid of protein co-crystal structural information is reported. This inhibitor was observed to have a binding mode distinct from previously published MMP-13 inhibitors. Potency and selectivity were improved by extending the hit structure out from the active site into the S1′ pocket.     

The expression of gelatinase A (MMP-2) is required for normal development of zebrafish embryos

Gelatinase A, also called matrix metalloproteinase 2 (MMP-2), belongs to the matrix metalloproteinase (MMP) family. MMP-2 cleaves type IV collagen, denatured collagen (gelatin), and other extracellular matrix (ECM) components. MMP-2 has been reported to be involved in a number of biological and pathological processes, but previous studies have not indicated that its expression is essential for early embryogenesis. In the current study, we have utilized zebrafish as a developmental model to study the role of MMP-2 during embryogenesis. We have successfully isolated a zebrafish MMP-2 (zMMP-2) homologue showing over 80% identity and over 90% similarity to its human counterpart. In situ analysis showed that zMMP-2 was expressed as early as the one-cell stage implying a maternal origin during oogenesis, and embryos continued to express zMMP-2 through at least the 72-h stage of development. RT-PCR analysis confirmed the in situ expression pattern and gelatin zymography indicated that a metalloproteinase with the same gel mobility as vertebrate MMP-2 was present in zebrafish embryos. Injection of zMMP-2 antisense morpholino oligonucleotides into 1- to 4-cell embryos resulted in a truncated axis, monitored through 72 h of development indicating that this metalloproteinase plays an important role in zebrafish embryogenesis. Monpholino-induced alterations in development began to be observed at 12 h of embryogenesis based on morphological and axis marker studies. The results obtained in zebrafish are in contrast to murine knockout studies that indicate that MMP-2 does not have a major role in mouse embryogenesis.Edited by D. Tautz     

Benzofused hydroxamic acids: Useful fragments for the preparation of histone deacetylase inhibitors. Part 1: Hit identification

In the search for a new class of histone deacetylase inhibitors, we prepared a series of simple benzofused hydroxamic acids to find an anchoring fragment of minimal molecular weight. These initial hits, all belonging to the benzothiophene class, showed very good ligand efficiencies. Following these findings, a classical fragment growing approach was performed to increase binding affinity and cytotoxicity.     

Dimerization of matrix metalloproteinase-2 (MMP-2): functional implication in MMP-2 activation

Matrix metalloproteinase-2 (MMP-2) functions in diverse biological processes through the degradation of extracellular and non-extracellular matrix molecules. Because of its potential for tissue damage, there are several ways to regulate MMP-2 activity, including gene expression, compartmentalization, zymogen activation, and enzyme inactivation by extracellular inhibitors. Enzyme regulation through zymogen activation is important for the regulation of MMP-2 activity. In our previous studies, we showed that thrombin directly cleaved the propeptide of MMP-2 at specific sites for enzyme activation. We also demonstrated that heparan sulfate was required for thrombin-mediated activation of pro-MMP-2 by binding to thrombin, presumably through conformational changes at the active site of the enzyme. This suggests a regulatory mechanism for thrombin-mediated activation of pro-MMP-2. In this study, we found that MMP-2 formed a reduction-sensitive homodimer in a controlled manner and that Ca(2+) ion was essential for homodimerization of MMP-2. Homodimerization was not associated with protein kinase C-mediated phosphorylation of MMP-2. MMP-2 formed a homodimer through an intermolecular disulfide bond between Cys(102) and the neighboring Cys(102). Homodimerization of MMP-2 enhanced thrombin-mediated activation of pro-MMP-2. Moreover, the MMP-2 homodimer could cleave a small peptide substrate without removal of the propeptide. Taken together, our experimental data suggest a novel regulatory mechanism for pro-MMP-2 activation that is modulated through homodimerization of MMP-2.     

曲普瑞林对子宫肌瘤组织中MMP-13和TIMP-3表达的影响

目的:探讨曲普瑞林对子宫肌瘤患者肿瘤组织中MMP-13和TIMP-3表达影响。方法:选取我院收治的子宫肌瘤患者40例,随机分为两组,每组各20例。实验组予曲普瑞林后,行子宫肌瘤切除术,对照组直接行子宫肌瘤切除术,观察比较超声测量子宫和子宫肌瘤的体积,实时定量PCR法与免疫印迹法检测子宫肌层和肌瘤组织中MMP-13和TIMP-3的表达。结果:1与曲普瑞林治疗前以及对照组相比较,曲普瑞林治疗后临床症状显著改善,子宫和肌瘤体积均明显缩小,差异有统计学意义(P0.05)。2与对照组和治疗前相比较,血清雌二醇水平显著降低,差异有统计学意义(P0.05)。3与对照组相比较,肌层和肌瘤组织中MMP-13m RNA水平降低,肌层和肌瘤组织中TIMP-3m RNA水平增加,差异有统计学意义(P0.05)。结论:曲普瑞林能够通过降低血清雌二醇水平,降低肌层和肌瘤组织中MMP-3的表达,以及增加肌层和肌瘤组织中TIMP-13的表达,来缩小子宫和子宫肌瘤的体积,从而缓解患者的症状。     

Phage display of tissue inhibitor of metalloproteinases-2 (TIMP-2): identification of selective inhibitors of collagenase-1 (metalloproteinase 1 (MMP-1))

Tissue inhibitor of metalloproteinases-2 (TIMP-2) is a broad spectrum inhibitor of the matrix metalloproteinases (MMPs), which function in extracellular matrix catabolism. Here, phage display was used to identify variants of human TIMP-2 that are selective inhibitors of human MMP-1, a collagenase whose unregulated action is linked to cancer, arthritis, and fibrosis. Using hard randomization of residues 2, 4, 5, and 6 (L1) and soft randomization of residues 34-40 (L2) and 67-70 (L3), a library was generated containing 2 × 10(10) variants of TIMP-2. Five clones were isolated after five rounds of selection with MMP-1, using MMP-3 as a competitor. The enriched phages selectively bound MMP-1 relative to MMP-3 and contained mutations only in L1. The most selective variant (TM8) was used to generate a second library in which residues Cys(1)-Gln(9) were soft-randomized. Four additional clones, selected from this library, showed a similar affinity for MMP-1 as wild-type TIMP-2 but reduced affinity for MMP-3. Variants of the N-terminal domain of TIMP-2 (N-TIMP-2) with the sequences of the most selective clones were expressed and characterized for inhibitory activity against eight MMPs. All were effective inhibitors of MMP-1 with nanomolar K(i) values, but TM8, containing Ser(2) to Asp and Ser(4) to Ala substitutions, was the most selective having a nanomolar K(i) value for MMP-1 but no detectable inhibitory activity toward MMP-3 and MMP-14 up to 10 μM. This study suggests that phage display and selection with other MMPs may be an effective method for discovering tissue inhibitor of metalloproteinase variants that discriminate between specified MMPs as targets.     

Effects of covalently attached chondroitin sulfate on aggrecan cleavage by ADAMTS-4 and MMP-13

Aggrecan is degraded by several aggrecanase-1 (ADAMTS-4) isoforms differing in the number of sulfated glycosaminoglycan (sGAG)-binding motifs. ADAMTS-4 and MMPs cleave aggrecan more efficiently within the chondroitin sulfate (CS)-rich region than the interglobular domain (IGD). We investigated the influence of CS on aggrecan core protein cleavage by ADAMTS-4 (p68) and (p40) as well as MMP-13, which has no recognizable GAG-binding sites. Chondroitinase ABC-treated cartilage aggrecan was cleaved with ADAMTS-4 (p68) less efficiently than CS-substituted aggrecan within the CS-2 domain. Keratanase-treated aggrecan exhibited reduced IGD cleavage, but when both CS and KS were removed, the IGD cleavage was restored. This result suggests that KS in the IGD may compete with CS for ADAMTS-4 (p68) binding. In the absence of KS, however, p68 binding was shifted to the CS-2 domain. CS-deficient full-length recombinant aggrecan (rbAgg) was produced by chondroitinase ABC treatment, or by expression in the xylosyltransferase-deficient CHO-pgsA745 cell line. When digested with the ADAMTS-4 (p68), each of these preparations exhibited reduced CS-2 domain cleavage compared to CS-substituted CHO-K1 cell-derived aggrecan. Additionally, CS-deficient rbAgg showed increased IGD scission prior to cleavage within the CS-2 domain. ADAMTS-4 (p40) readily cleaved both rbAggs within the IGD, but cleaved poorly within the CS-2 domain, indicating little CS dependence. MMP-13, in contrast, cleaved the CS region and the IGD of both CS-substituted and CS-deficient rbAgg equally well. These data indicate that covalently bound CS enhances ADAMTS-4-mediated cleavage within the CS-rich region. MMP-13 also cleaves preferentially within the CS-region, but by an apparently CS-independent mechanism.     

Pro-MMP-9 activation by the MT1-MMP/MMP-2 axis and MMP-3: role of TIMP-2 and plasma membranes

MMP-9 (gelatinase B) is produced in a latent form (pro-MMP-9) that requires activation to achieve catalytic activity. Previously, we showed that MMP-2 (gelatinase A) is an activator of pro-MMP-9 in solution. However, in cultured cells pro-MMP-9 remains in a latent form even in the presence of MMP-2. Since pro-MMP-2 is activated on the cell surface by MT1-MMP in a process that requires TIMP-2, we investigated the role of the MT1-MMP/MMP-2 axis and TIMPs in mediating pro-MMP-9 activation. Full pro-MMP-9 activation was accomplished via a cascade of zymogen activation initiated by MT1-MMP and mediated by MMP-2 in a process that is tightly regulated by TIMPs. We show that TIMP-2 by regulating pro-MMP-2 activation can also act as a positive regulator of pro-MMP-9 activation. Also, activation of pro-MMP-9 by MMP-2 or MMP-3 was more efficient in the presence of purified plasma membrane fractions than activation in a soluble phase or in live cells, suggesting that concentration of pro-MMP-9 in the pericellular space may favor activation and catalytic competence.     

CXCL10和MMP-13在妊娠滋养细胞疾病滋养细胞中的表达及其临床意义

目的检测妊娠滋养细胞疾病滋养细胞中CXCL10和MMP-13表达情况,探讨其在滋养细胞疾病浸润和转移中的作用。方法采用免疫组织化学Powervision法检测40例早孕绒毛,30例葡萄胎,18例葡萄胎恶变(随访发生恶变),35例滋养细胞肿瘤(28例绒癌,7例胎盘部位滋养细胞肿瘤)中CXCL10和MMP-13的表达。结果CXCL10和MMP-13在滋养细胞肿瘤中表达阳性率和免疫反应性强度最高;在葡萄胎恶变组中表达阳性率和免疫反应性强度较高,但是低于在滋养细胞肿瘤中的表达阳性率和免疫反应性强度;在葡萄胎组和早孕绒毛组中表达阳性率和免疫强度较低。CXCL10和MMP-13在滋养细胞肿瘤中的表达,年龄<40岁组中表达低于年龄>40岁组;在临床分期分组(Ⅰ、Ⅱ)中表达低于临床分期分组(Ⅲ、Ⅳ);在FIGO预后评分分组低危组中表达低于高危组。结论CXCL10和MMP-13在早孕绒毛组织中低表达,而在滋养细胞肿瘤中高表达,提示其可能参与滋养细胞疾病的浸润和转移过程,因此联合检测CXCL10和MMP-13的表达可能对葡萄胎恶变的早期诊断以及对滋养细胞肿瘤的预后评估提供依据。     

Potent, selective, and orally bioavailable matrix metalloproteinase-13 inhibitors for the treatment of osteoarthritis

Modification of -biphenylsulfonamidocarboxylic acids led to potent and selective MMP-13 inhibitors. Compound 16 showed 100% oral bioavailability in rats and demonstrated >50% inhibition of bovine cartilage degradation at 10 ng/mL.     

Microarray and proteomic analysis of breast cancer cell and osteoblast co-cultures: role of osteoblast matrix metalloproteinase (MMP)-13 in bone metastasis

Dynamic reciprocal interactions between a tumor and its microenvironment impact both the establishment and progression of metastases. These interactions are mediated, in part, through proteolytic sculpting of the microenvironment, particularly by the matrix metalloproteinases, with both tumors and stroma contributing to the proteolytic milieu. Because bone is one of the predominant sites of breast cancer metastases, we used a co-culture system in which a subpopulation of the highly invasive human breast cancer cell line MDA-MB-231, with increased propensity to metastasize to bone, was overlaid onto a monolayer of differentiated osteoblast MC3T3-E1 cells in a mineralized osteoid matrix. CLIP-CHIP® microarrays identified changes in the complete protease and inhibitor expression profile of the breast cancer and osteoblast cells that were induced upon co-culture. A large increase in osteoblast-derived MMP-13 mRNA and protein was observed. Affymetrix analysis and validation showed induction of MMP-13 was initiated by soluble factors produced by the breast tumor cells, including oncostatin M and the acute response apolipoprotein SAA3. Significant changes in the osteoblast secretomes upon addition of MMP-13 were identified by degradomics from which six novel MMP-13 substrates with the potential to functionally impact breast cancer metastasis to bone were identified and validated. These included inactivation of the chemokines CCL2 and CCL7, activation of platelet-derived growth factor-C, and cleavage of SAA3, osteoprotegerin, CutA, and antithrombin III. Hence, the influence of breast cancer metastases on the bone microenvironment that is executed via the induction of osteoblast MMP-13 with the potential to enhance metastases growth by generating a microenvironmental amplifying feedback loop is revealed.     

Virtual screening identification and chemical optimization of substituted 2-arylbenzimidazoles as new non-zinc-binding MMP-2 inhibitors

Matrix metalloproteinases (MMPs) are a large family of zinc-dependent endoproteases known to exert multiple regulatory roles in tumor progression and invasiveness. This encouraged over the years the approach of MMP, and particularly MMP-2, targeting for anticancer treatment. Early generations of MMP inhibitors, based on aspecific zinc binding groups (ZBGs) assembled on (pseudo)peptide scaffolds, have been discontinued due to the clinical emergence of toxicity and further drawbacks, giving the way to inhibitors with alternative zinc-chelator moieties or not binding the catalytic zinc ion.In the present paper, we continue the search for new non-zinc binding MMP-2 inhibitors: exploiting previously identified compounds, a virtual screening (VS) campaign was carried out and led to the identification of a new class of ligands. The structure-activity relationship (SAR) of the benzimidazole scaffold was explored by synthesis of several analogues whose inhibition activity was tested with enzyme inhibition assays. By performing the molecular simplification approach, we disclosed different sets of single-digit micromolar inhibitors of MMP-2, with up to a ten-fold increase in inhibitory activity and ameliorated selectivity towards off-target MMP-8, compared to selected lead compound. Molecular dynamics calculations conducted on complexes of MMP-2 with docked privileged structures confirmed that analyzed inhibitors avoid targeting the zinc ion and dip inside the S1′ pocket. Present results provide a further enrichment of our insights for the design of novel MMP-2 selective inhibitors.