Targeting collagen strands by photo-triggered triple-helix hybridization

Abstract

Collagen remodeling is an integral part of tissue development, maintenance, and regeneration, but excessive remodeling is associated with various pathologic conditions. The ability to target collagens undergoing remodeling could lead to new diagnostics and therapeutics as well as applications in regenerative medicine; however, such collagens are often degraded and denatured, making them difficult to target with conventional approaches. Here, we present caged collagen mimetic peptides (CMPs) that can be phototriggered to fold into triple helix and bind to collagens denatured by heat or by matrix metalloproteinase (MMP) digestion. Peptidebinding assays indicate that the binding is primarily driven by stereo-selective triple-helical hybridization between monomeric CMPs of high triple-helical propensity and denatured collagen strands. Photo-triggered hybridization allows specific staining of collagen chains in protein gels as well as photo-patterning of collagen and gelatin substrates. In vivo experiments demonstrate that systemically delivered CMPs can bind to collagens in bones, as well as prominently in articular cartilages and tumors characterized by high MMP activity. We further show that CMP-based probes can detect abnormal bone growth activity in a mouse model of Marfan syndrome. This is an entirely new way to target the microenvironment of abnormal tissues and could lead to new opportunities for management of numerous pathologic conditions associated with collagen remodeling and high MMP activity.A s the most abundant protein in mammals, collagens play a crucial role in tissue development and regeneration, and their structural or metabolic abnormalities are associated with debilitating genetic diseases and various pathologic conditions. Although collagen remodeling occurs during development and normal tissue maintenance, particularly for renewing tissues (e.g., bones), excess remodeling activity is commonly seen in tumors, arthritis, and many other chronic wounds. During collagen remodeling, large portions of collagens are degraded and denatured by proteolytic enzymes, which can be explored for diagnostic and therapeutic purposes. Since unstructured proteins are not ideal targets for rational drug design, library approaches have been employed to develop monoclonal antibody (1, 2) and peptide probes (3) that specifically bind to cryptic sites in collagen strands that become exposed when denatured. However, these probes suffer from poor pharmacokinetics (4), and/or low specificity, and binding affinity (5).We envisioned that triple helix, the hallmark structural feature of collagen, could provide a unique targeting mechanism for the denatured collagens. The triple helix is nearly exclusively seen in collagens except as small subdomains in a few noncollagen proteins (6). Considering its striking structural similarity to the DNA double helix in terms of multiplex formation by periodic interchain hydrogen bonds along the polymer backbone (6), we thought that a small peptide sequence with strong triple-helix prope...

Imaging Denatured Collagen Strands <em>In vivo</em> and <em>Ex vivo</em> via Photo-triggered Hybridization of Caged Collagen Mimetic Peptides

“…When staining tissue sections with fluorescently labeled CMPs, we found it beneficial to incubate the samples in the UV-activated peptide solutions at 4 °C, because the triple helical hybridization is facilitated at lower temperature 1,20 . It was also found that immersing the slides in fresh PBS buffer in a staining jar is the most effective way to wash off unbound CMPs, which works much better than merely pipetting washing buffers over the samples.…”

“…This protocol focuses on the imaging application of CMPs as related to collagen remodeling. Methods for CMP synthesis can be found in previous reports 1,7,[9][10][11][12][13][14][15] . In this video report, imaging skeletal tissues in normal mice and tissue sections of mouse cornea were chosen for demonstration purpose; however the methods presented here can be readily applied to many pathologies and biological models involving collagen remodeling (e.g.…”

“…Collagen remodeling involves both collagen degradation (by proteases) and synthesis (promoted by growth factors). Although collagen remodeling, for example in bone, is part of the normal tissue renewal process, excess remodeling activity, or its occurrence in abnormal locations, typically indicates wound healing response to an injury or chronic pathological conditions such as cancer, osteoporosis, arthritis, and fibrosis [1][2][3][4] . The ability to directly image collagens undergoing remodeling in vivo could lead to understanding of the progression of these diseases, as well as new diagnostics and therapeutics.…”

“…The cleaved collagen fragments lose their triple helical structure and become unfolded strands (gelatin), which are further digested by nonspecific proteases 1 . It was recently discovered that the collagen mimetic peptide (CMP) which has the propensity to fold into triple helical structure can specifically target collagen strands which are dissociated from its triple helical state by either heat denaturation or by enzymatic degradation 1,7 . The binding is primarily driven by triple-helix hybridization between monomeric CMPs and the denatured collagen strands.…”

Theranostic Probes for Targeting Tumor Microenvironment: An Overview

“…Collagen mimetic peptides are photo-triggered and have a tendency to form a triple helix with the denatured collagen. The CMPs were tagged with Near Infrared Fluorophore (NIRF) (IR-Ahx-(GPO)9 ) and fluorescence revealed the accumulation of the triple helical structure in the tumor tissue [77].…”