Collagen hybridizing peptide

Schematic showing relationship between the CMP and CHP. Triple helical CMPs can be heated (above a defined temperature) to dissociate into monomeric CHPs; upon cooling, CHP strands can re-assemble into a triple helix over time.

Collagen is the main component of the extracellular matrix (ECM).[7] The collagen superfamily consists of 28 different types of collagen.[7] Although the function and hierarchical structure of these collagens may vary, they all share the defining structural feature known as the triple helix,[1] where three left handed polyproline II-type (PPII) helices assemble to form a right-handed supercoiled helical motif.[1][8] Short synthetic peptides known as collagen mimetic peptides (CMPs) or collagen-like peptides (CLPs) have played a major role in elucidating the 3D structure of the collagen triple helix, its folding kinetics, and thermal stability as small triple helical models.[3][9][10][11] CMPs, CLPs, and CHPs are all very similar in terms of their amino acid sequences but only when CMPs or CLPs are heated above their melting temperatures, do they exist in the dissociated, single-stranded state and can be considered as CHPs.[2]

Single-stranded CHPs bind to denatured collagen chains and gelatin in a manner that is unique from other targeting mechanisms, in that they specifically recognize a unique structural motif (collagen triple helix) for folding and chain assembly, as opposed to specific epitopes binding that is seen for monoclonal antibodies (mAbs), for example.[12] Due to their unique targeting mechanism, CHPs have a high binding specificity towards denatured collagen chains but have almost no affinity for intact (triple helical) collagen.[13] CHPs can broadly target collagen chains that have been denatured by thermal,[13] chemical,[14] mechanical,[15] or enzymatic processes,[13] as well as multiple collagen types (e.g., Col I, II, IV).[5][6] Studies also showed CHPs and their fluorophore conjugates have superior stability in contact with serum.[16]

A fluorescence image of an axial cross section of a mouse heart at day 14 post myocardial infarction, stained with Hoechst 33342 (blue) and biotin-labeled CHP (detected with AlexaFluor647-streptavidin, red). Scale bar: 1 mm.

Controlled collagen turnover is crucial for embryonic development, organ morphogenesis, as well as tissue maintenance and repair.[17] However, changes of collagen homeostasis are associated with numerous diseases and pathological conditions. Excessive collagen degradation may be associated with cancer metastasis, skin ageing, arthritis, and osteoporosis.[17] CHPs can target tissues undergoing remodelling based on their ability to bind to degraded and unfolded collagen strands through triple helix formation. As a targeting moiety, CHPs offer great potential in histopathology, diagnostics, and drug delivery for a wide range of diseases.

Most methods for the evaluation of collagen denaturation in disease states are indirect, such as detecting matrix metalloproteinase (MMP) activity or quantifying collagen peptide fragments in urine, serum, or synovial fluid.[18][19][20] Using conventional methods for directly targeting collagen, researchers have to relied on collagen binding peptides selected by phage display,[21] derived from collagen binding proteins,[22] or antibodies raised against collagens. Unfortunately, these compounds cannot target denatured collagens which are unstructured and do not present a defined 3D epitope. In addition, antibodies that were reported to distinguish specific degraded collagen fragments can only recognize one or few collagen types.[2][23] In contrast, CHPs, in principle, can bind to all types of denatured collagens.[4][5][6]