The proteolytic processing of collagen (collagenolysis) is critical in development and homeostasis, but also contributes to numerous pathologies. Mammalian interstitial collagenolytic enzymes include members of the matrix metalloproteinase (MMP) family and cathepsin K. While MMPs have long been recognized for their ability to catalyze the hydrolysis of collagen, the roles of individual MMPs in physiological and pathological collagenolysis are less defined. The use of knockout and mutant animal models, which reflect human diseases, has revealed distinct collagenolytic roles for MT1-MMP and MMP-13. A better understanding of temporal and spatial collagen processing, along with the knowledge of the specific MMP involved, will ultimately lead to more effective treatments for cancer, arthritis, cardiovascular conditions, and infectious diseases.
Primary
hits that arise from screening one bead one compound (OBOC) libraries
against a target of interest rarely have high potency. However, there
has been little work focused on the development of an efficient workflow
for primary hit improvement. In this study, we show that by characterizing
the binding constants for all of the hits that arise from a screen,
structure–activity relationship (SAR) data can be obtained
to inform the design of “derivative libraries” of a
primary hit that can then be screened under more demanding conditions
to obtain improved compounds. Here, we demonstrate the rapid improvement
of a primary hit against matrix metalloproteinase-14 using this approach.
Analysis of MMP expression profiles in various pathologies correlated their presence in promoting disease progression. Drugs were designed to inhibit MMPs in an extreme manner by chelating the active site zinc ion. This approach did not distinguish between the 24 members of the MMP family and had devastating consequences during clinical trials. Subsequent knockout mouse studies showed that some MMPs are beneficial in regulating tumor growth, metastasis and indirectly stimulating the immune system. The broad-spectrum inhibitor approach was rethought and modified in order to increase specificity by taking into account the non-conserved secondary binding sites or differences in structures within MMPs and also generating antibodies. These showed interesting results in vitro and in vivo. The recent technological advances that allow us to better understand the function and structure of MMPs are aiding in the development of selective inhibitors.
Cell surface proteolysis is an integral yet poorly understood physiological process. The present study has examined how the pericellular collagenase membrane-type 1 matrix metalloproteinase (MT1-MMP) and membrane-mimicking environments interplay in substrate binding and processing. NMR derived structural models indicate that MT1-MMP transiently associates with bicelles and cells through distinct residues in blades III and IV of its hemopexin-like domain, while binding of collagen-like triple-helices occurs within blades I and II of this domain. Examination of simultaneous membrane interaction and triple-helix binding revealed a possible regulation of proteolysis due to steric effects of the membrane. At bicelle concentrations of 1%, enzymatic activity towards triple-helices was increased 1.5-fold. A single mutation in the putative membrane interaction region of MT1-MMP (Ser466Pro) resulted in lower enzyme activation by bicelles. An initial structural framework has thus been developed to define the role(s) of cell membranes in modulating proteolysis.
Remodeling of the extracellular matrix (ECM) is crucial in development and homeostasis, but also has a significant role in disease progression. Two metalloproteinase families, the matrix metalloproteinases (MMPs) and a disintegrin and metalloproteases (ADAMs), participate in the remodeling of the ECM, either directly or through the liberation of growth factors and cell surface receptors. The correlation of MMP and ADAM activity to a variety of diseases has instigated numerous drug development programs. However, broad-based and Zn -chelating MMP and ADAM inhibitors have fared poorly in the clinic. Selective MMP and ADAM inhibitors have been described recently based on (a) antibodies or antibody fragments or (b) small molecules designed to take advantage of protease secondary binding sites (exosites) or allosteric sites. Clinical trials have been undertaken with several of these inhibitors, while others are in advanced pre-clinical stages.
Nanodiamonds (NDs) have received considerable attention as potential drug delivery vehicles. NDs are small (~5 nm diameter), can be surface modified in a controllable fashion with a variety of functional groups, and have little observed toxicity in vitro and in vivo. However, most biomedical applications of NDs utilize surface adsorption of biomolecules, as opposed to covalent attachment. Covalent modification provides reliable and reproducible ND–biomolecule ratios, and alleviates concerns over biomolecule desorption prior to delivery. The present study has outlined methods for the efficient solid-phase conjugation of ND to peptides and characterization of ND–peptide conjugates. Utilizing collagen-derived peptides, the ND was found to support or even enhance the cell adhesion and viability activities of the conjugated sequence. Thus, NDs can be incorporated into peptides and proteins in a selective manner, where the presence of the ND could potentially enhance the in vivo activities of the biomolecule it is attached to.
Quantitative assessment of MT1‐MMP cell surface‐associated proteolytic activity remains undefined. Presently, MT1‐MMP was stably expressed and a cell‐based FRET assay developed to quantify activity toward synthetic collagen‐model triple‐helices. To estimate the importance of cell surface localization and specific structural domains on MT1‐MMP proteolysis, activity measurements were performed using a series of membrane‐anchored MT1‐MMP mutants and compared directly with those of soluble MT1‐MMP. MT1‐MMP activity (kcat/KM) on the cell surface was 4.8‐fold lower compared with soluble MT1‐MMP, with the effect largely manifested in kcat. Deletion of the MT1‐MMP cytoplasmic tail enhanced cell surface activity, with both kcat and KM values affected, while deletion of the hemopexin‐like domain negatively impacted KM and increased kcat. Overall, cell surface localization of MT1‐MMP restricts substrate binding and protein‐coupled motions (based on changes in both kcat and KM) for catalysis. Comparison of soluble and cell surface‐bound MT2‐MMP revealed 12.9‐fold lower activity on the cell surface. The cell‐based assay was utilized for small molecule and triple‐helical transition state analog MMP inhibitors, which were found to function similarly in solution and at the cell surface. These studies provide the first quantitative assessments of MT1‐MMP activity and inhibition in the native cellular environment of the enzyme.
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