Osteogenic Peptide Research Articles

Novel osteogenic peptide from bovine bone collagen hydrolysate: Targeted screening, molecular mechanism, and stability analysis

This study aimed to screen for a novel osteogenic peptide based on the calcium-sensing receptor (CaSR) and explore its molecular mechanism and gastrointestinal stability. In this study, a novel osteogenic peptide (Phe-Ser-Gly-Leu, FSGL) derived from bovine bone collagen hydrolysate was successfully screened by molecular docking and synthesised by solid phase peptide synthesis for further analysis. Cell experiments showed that FSGL significantly enhanced the osteogenic activity of MC3T3-E1 cells by acting on CaSR, including proliferation (152.53%), differentiation, and mineralization. Molecular docking and molecular dynamics further demonstrated that FSGL was a potential allosteric activator of CaSR, that turned on the activation switch of CaSR by closing the Venus flytrap (VFT) domain and driving the two protein chains in the VFT domain to easily form dimers. In addition, 96.03% of the novel osteogenic peptide FSGL was stable during gastrointestinal digestion. Therefore, FSGL showed substantial potential for enhancing the osteogenic activity of osteoblasts. This study provided new insights for the application of CaSR in the targeted screening of osteogenic peptides to improve bone health.

The Osteogenic Peptide P-15 for Bone Regeneration: A Narrative Review of the Evidence for a Mechanism of Action.

Bone regeneration is a complex multicellular process involving the recruitment and attachment of osteoprogenitors and their subsequent differentiation into osteoblasts that deposit extracellular matrixes. There is a growing demand for synthetic bone graft materials that can be used to augment these processes to enhance the healing of bone defects resulting from trauma, disease or surgery. P-15 is a small synthetic peptide that is identical in sequence to the cell-binding domain of type I collagen and has been extensively demonstrated in vitro and in vivo to enhance the adhesion, differentiation and proliferation of stem cells involved in bone formation. These events can be categorized into three phases: attachment, activation and amplification. This narrative review summarizes the large body of preclinical research on P-15 in terms of these phases to describe the mechanism of action by which P-15 improves bone formation. Knowledge of this mechanism of action will help to inform the use of P-15 in clinical practice as well as the development of methods of delivering P-15 that optimize clinical outcomes.

Purification and identification of novel osteogenic peptides from sea cucumber intestine hydrolysates and their pro-osteogenesis effects on MC3T3-E1 cells

The present study aimed to purify and identify osteogenic peptides from sea cucumber intestine hydrolysates (Mw＜3 kDa) and further evaluate their pro-osteogenesis properties on MC3T3-E1 cells. Two novel osteogenic peptides, RPQYPQYPS and LSFSPY, were purified and identified from the alcalase hydrolysate of sea cucumber intestine by gel filtration chromatography, ion exchange column chromatography, RP-HPLC, which were further sequenced by LC-MS/MS and synthesized with solid-phase synthesis method. Peptides RPQYPQYPS and LSFSPY significantly increased the proliferation of MC3T3-E1 cells by 38.7% and 32.8%. Alizarin red staining also confirmed that peptides RPQYPQYPS and LSFSPY significantly promoted MC3T3-E1 cell mineralization in a dose-dependent manner. Further, molecular docking results showed that RPQYPQYPS and LSFSPY had a higher affinity with integrin α5β1. Western blot results confirmed that RPQYPQYPS and LSFSPY significantly upregulated the expression of integrin downstream signals PI3K, ERK and Wnt10b. These findings provided evidence for the osteogenic peptides from sea cucumber intestines as a functional food ingredient.

Characterization and preparation of food-derived peptides on improving osteoporosis: A review

Osteoporosis is a systemic bone disease characterized by reduced bone mass and deterioration of the microstructure of bone tissue, leading to an increased risk of fragility fractures and affecting human health worldwide. Food-derived peptides are widely used in functional foods due to their low toxicity, ease of digestion and absorption, and potential to improve osteoporosis. This review summarized and discussed methods of diagnosing osteoporosis, treatment approaches, specific peptides as alternatives to conventional drugs, and the laboratory preparation and identification methods of peptides. It was found that peptides interacting with RGD (arginine-glycine-aspartic acid)-binding active sites in integrin could alleviate osteoporosis, analyzed the interaction sites between these osteogenic peptides and integrin, and further discussed their effects on improving osteoporosis. These may provide new insights for rapid screening of osteogenic peptides, and provide a theoretical basis for their application in bone materials and functional foods.

Novel Peptide Derived from Gadus morhua Stimulates Osteoblastic Differentiation and Mineralization through Wnt/β-Catenin and BMP Signaling Pathways.

Marine biodiversity offers a wide array of active ingredient resources. Gadus morhua peptides (GMPs) showed excellent osteoprotective effects in ovariectomized mice. However, the potential osteogenesis mechanisms of key osteogenic peptides in GMP were seldom reported. In this study, a novel osteogenic peptide (GETNPADSKPGSIR, P-GM-2) was screened from GMP. P-GM-2 has a high stability coefficient and a strong interaction with epidermal growth factor receptor. Cell culture experiments showed that P-GM-2 stimulated the expression of osteogenic differentiation markers to promote osteoblast proliferation, differentiation, and mineralization. Additionally, P-GM-2 phosphorylates GSK-3β, leading to the stabilization of β-catenin and its translocation to the nucleus, thus initiating the activation of the Wnt/β-catenin signaling pathway. Meanwhile, P-GM-2 could also regulate the osteogenic differentiation of preosteoblasts by triggering the BMP/Smad and mitogen-activated protein kinase signaling pathways. Further validation with specific inhibitors (ICG001 and Noggin) demonstrated that the osteogenic activity of P-GM-2 was revealed by the activation of the BMP and Wnt/β-catenin pathways. In summary, these results provide theoretical and practical insights into P-GM-2 as an effective antiosteoporosis active ingredient.

Enriched mesoporous bioactive glass scaffolds as bone substitutes in critical diaphyseal bone defects in rabbits

In the field of orthopedic surgery, there is an increasing need for the development of bone replacement materials for the treatment of bone defects. One of the main focuses of biomaterials engineering are advanced bioceramics like mesoporous bioactive glasses (MBG´s). The present study compared the new bone formation after 12 weeks of implantation of MBG scaffolds with composition 82,5SiO2–10CaO–5P2O5-x 2.5SrO alone (MBGA), enriched with osteostatin, an osteoinductive peptide, (MBGO) or enriched with bone marrow aspirate (MBGB) in a long bone critical defect in radius bone of adult New Zealand rabbits. New bone formation from the MBG scaffold groups was compared to the gold standard defect filled with iliac crest autograft and to the unfilled defect. Radiographic follow-up was performed at 2, 6, and 12 weeks, and microCT and histologic examination were performed at 12 weeks. X-Ray study showed the highest bone formation scores in the group with the defect filled with autograft, followed by the MBGB group, in addition, the microCT study showed that bone within defect scores (BV/TV) were higher in the MBGO group. This difference could be explained by the higher density of newly formed bone in the osteostatin enriched MBG scaffold group. Therefore, MBG scaffold alone and enriched with osteostatin or bone marrow aspirate increase bone formation compared to defect unfilled, being higher in the osteostatin group. The present results showed the potential to treat critical bone defects by combining MBGs with osteogenic peptides such as osteostatin, with good prospects for translation into clinical practice. Statement of significanceTreatment of bone defects without the capacity for self-repair is a global problem in the field of Orthopedic Surgery, as evidenced by the fact that in the U.S alone it affects approximately 100,000 patients per year. The gold standard of treatment in these cases is the autograft, but its use has limitations both in the amount of graft to be obtained and in the morbidity produced in the donor site. In the field of materials engineering, there is a growing interest in the development of a bone substitute equivalent. Mesoporous bioactive glass (MBG´s) scaffolds with three-dimensional architecture have shown great potential for use as a bone substitutes. The osteostatin-enriched Sr-MBG used in this long bone defect in rabbit radius bone in vivo study showed an increase in bone formation close to autograft, which makes us think that it may be an option to consider as bone substitute.

Identification and validation of fermented milk-derived osteogenic peptides via molecular docking and osteoblastic cell model

Fermented milk has abundant bioactive peptides, and there is a growing interest in seeking osteogenic peptides of dairy origin. This study employed computer-assisted screening to identify potential osteogenic peptides from fermented milk by Lactobacillus helveticus and investigated the effects of these osteogenic peptides on the proliferation, differentiation, and mineralization of MC3T3-E1 cells, along with their underlying mechanisms. Three osteogenic peptides were identified: AF10 (ARHPHPHLSF), AY17 (AAGGPGAPADPGRPTGY), and NE19 (NIPPLTQTPVVVPPFLQPE). Treatment with 0.2 μM of AF10, AY17, and NE19 promoted the proliferation of MC3T3-E1 cells by increasing the proportion of cells in the S phase and G2/M phase. Further molecular docking revealed significant differences in the interaction of the three osteogenic peptides with the critical domain (Asn12-Lys13-Leu14-Thr15-Gln16-Leu17-Gly18) of the epidermal growth factor receptor (EGFR). The three peptides exhibited different degrees of promotion on the differentiation and mineralization of MC3T3-E1 cells, as well as significant differences in the expression of genes and proteins related to ERK1/2, RUNX2, and BMP2. The AF10 peptide was selected for further validation of the effect of the mitogen-activated protein kinase (MAPK) signaling pathway on the proliferation and expression of differentiation and mineralization-associated genes in MC3T3-E1 cells by combining MAPK pathway inhibitors. Molecular dynamics simulation revealed the stability of AF10 when binding to EGFR. These findings provide valuable insights into the screening of osteogenic peptides of various origins and the development of functional dairy products accordingly.

Osteogenic Potential of a Biomaterial Enriched with Osteostatin and Mesenchymal Stem Cells in Osteoporotic Rabbits.

Mesoporous bioactive glasses (MBGs) of the SiO2-CaO-P2O5 system are biocompatible materials with a quick and effective in vitro and in vivo bioactive response. MBGs can be enhanced by including therapeutically active ions in their composition, by hosting osteogenic molecules within their mesopores, or by decorating their surfaces with mesenchymal stem cells (MSCs). In previous studies, our group showed that MBGs, ZnO-enriched and loaded with the osteogenic peptide osteostatin (OST), and MSCs exhibited osteogenic features under in vitro conditions. The aim of the present study was to evaluate bone repair capability after large bone defect treatment in distal femur osteoporotic rabbits using MBGs (76%SiO2-15%CaO-5%P2O5-4%ZnO (mol-%)) before and after loading with OST and MSCs from a donor rabbit. MSCs presence and/or OST in scaffolds significantly improved bone repair capacity at 6 and 12 weeks, as confirmed by variations observed in trabecular and cortical bone parameters obtained by micro-CT as well as histological analysis results. A greater effect was observed when OST and MSCs were combined. These findings may indicate the great potential for treating critical bone defects by combining MBGs with MSCs and osteogenic peptides such as OST, with good prospects for translation to clinical practice.

Oxidation-resistant nanoliposomes loaded with osteogenic peptides: Characteristics, stability and bioaccessibility

Phosphatidylcholine (PC) oxidation leads to the fusion of nanoliposomes and leakage of containment compounds during the storage period. This study aims to improve the oxidation resistance by partially substituting PC in the osteogenic peptides (OPs) loaded nanoliposomes with hydrogenated phosphatidylcholine (HPC). The investigation assessed the characteristics, stability, and bioaccessibility of these novel nanoliposomes. By altering the PC/HPC mass ratio from 1:0 to 0:1, an increase in the encapsulation efficiency (EE), loading capacity (LC), polydispersity index (PDI), and bioaccessibility of OPs-loaded nanoliposomes was observed. Additionally, there was a decrease in thiobarbituric acid reactive substances (TBARS), peroxide value (POV), non-volatile aldehyde, and ketone. The stability of salt decreased when using HPC alone (0:1). The performance of OPs-loaded nanoliposomes with a PC/HPC mass ratio of 1:3 was found to be satisfactory in terms of storage and pH stability. Fluorescence spectroscopy, Differential Scanning Calorimetry (DSC) and Fourier Transform Infrared spectroscopy (FTIR) revealed a tighter lipid aggregation, enhanced intermolecular van der Waals forces, and hydrogen bond formation in membranes of nanoliposomes containing HPC. The addition of HPC to the nanoliposomes delayed the release of peptides in simulated without affecting osteogenic activity. These results provide guidance for the development of oxidation-resistant nanoliposomes loaded with OPs products.

Hematoma-like dynamic hydrogelation through natural glycopeptide molecular recognition for infected bone fracture repair.

Infected bone fractures remain a major clinical challenge for orthopedic surgeons. From a tissue regeneration perspective, biomaterial scaffolds with antibacterial and osteoinductive activities are highly desired, while advanced materials capable of mimicking the pathological microenvironment during the healing process of infected tissues remain an area deserving more research. Hematoma, the gel-like blood coagulum, plays an essential role in bone fracture repair because of its ability to serve as a dynamic and temporary scaffold with cytokines for both pathogen elimination and tissue healing. In light of this, we designed a dynamic hydrogel with hematoma-like antimicrobial or reparative performance for infected bone fracture repair in this study. The proposed dynamic hydrogel network was based on the reversible recognition of a natural glycopeptide antibiotic vancomycin (Van) and its target dipeptide D-Ala-D-Ala (AA), which could serve as a hematoma-like scaffold for obliterating bacteria in the fracture region and promoting bone repair by introducing an endogenous osteogenic peptide (OGP). In vivo experiments demonstrated that the hydrogel could rapidly eradicate bacteria, improve bone regeneration and restore the local inflammatory microenvironment. Together, findings from this study imply that the use of hematoma-like dynamic hydrogel could lead to a biomimetic revolution in surgical strategies against susceptible bone fractures.

Define of Optimal Addition Period of Osteogenic Peptide to Accelerate the Osteogenic Differentiation of Human Pluripotent Stem Cells.

The addition of growth factiors is commonly applied to improve the osteogenic differentiation of stem cells. However, for human pluripotent stem cells (hPSCs), their complex differentiation processes result in the unknown effect at different stages. In this study, we focused on the widely used bone forming peptide-1 (BFP-1) and investigated the effect and mechanisms of its addition on the osteogenic induction of hPSCs as a function of the supplementation period. Monolayer-cultured hPSCs were cultured in osteogenic induction medium for 28days, and the effect of BFP-1 peptide addition at varying weeks was examined. After differentiation for varying days (0, 7, 14, 21 and 28), the differentiation efficiency was determined by RT-PCR, flow cytometry, immunofluorescence, and alizarin red staining assays. Moreover, the expression of marker genes related to germ layers and epithelial-mesenchymal transition (EMT) was investigated at day 7. Peptide treatment during the first week promoted the generation of mesoderm cells and mesenchymal-like cells from hiPSCs. Then, the upregulated expression of osteogenesis marker genes/proteins was detected in both hESCs and hiPSCs during subsequent inductions with BFP-1 peptide treatment. Fortunately, further experimental design confirmed that treating the BFP-1 peptide during 7-21days showed even better performance for hESCs but was ineffective for hiPSCs. The differentiation efficiency of cells could be improved by determining the optimal treatment period. Our study has great value in maximizing the differentiation of hPSCs by adding osteogenesis peptides based on the revealed mechanisms and promoting the application of hPSCs in bone tissue regeneration.

Influence of surface engineering on 3D printed Ti lattice structure towards enhanced tissue integration: An in vitro and in vivo study

Reconstruction of segmental defects are popularly approached with surface engineered additively manufactured scaffolds owing to its enhanced post-surgery tissue integration properties. The present work is aimed at fabrication of Ti lattice structures using 3D printing, with a novel approach of silane chemistry-based surface modification of those Ti-surfaces with osteogenic peptides (OGP). The lattice structures with 0.6 mm strut-diameter having 0.5 mm inter-strut distance were chosen for fabrication using an extrusion-based 3D printing. Based on the evidence, it could be concluded that extrusion-based 3D printing is an optimal alternative as compared to those high cost incurring additive manufacturing processes. Therefore, OGP were grafted on the pristine Ti-surfaces using a silane chemistry based novel vapour deposition process. In vitro assessments of the surface modified scaffolds using human amniotic derived mesenchymal stem cells showed evidence of enhanced cell adhesion and viability. In vivo subcutaneous study in rat models of the surface modified Ti-scaffolds also showed enhanced tissue integration in terms of Collagen I deposition around the boundary of the tissue-integrated struts as compared to those of pristine scaffolds. The study has established that the novel surface modification technique is capable to engineer the Ti-surfaces towards enhanced tissue integration in vivo.

Protection of osteogenic peptides in nanoliposomes: Stability, sustained release, bioaccessibility and influence on bioactive properties

This study prepared osteogenic peptides (OPs) from cod meat by hydrolysis and subsequently encapsulated them in nanoliposomes (NLP) to enhance bioaccessibility. The characterization, stability, controlled release behavior and bioactivity of OPs-loaded nanoliposomes (OPs-NLP) were investigated as well. The highest loading capacity (27.32%) was achieved in NLP loaded with 6 mg/mL of OPs. The particle size, zeta potential, and encapsulation efficiency of OPs-NLP were 70.59 nm, −11.98 mV, and 75.24%, respectively. The interaction between OPs and empty NLP was through hydrogen bonding and hydrophobic. The OPs-NLP showed the greatest stability during storage at 4 °C. The in vitro release profile of OPs from OPs-NLP fitted a one-level kinetic model best. The osteogenic activity of OPs was unaffected by NLP encapsulation, and the bioaccessibility of OPs was notably improved. These findings suggest that OPs-NLP has the potential to be used in functional foods.

A Bone Targeting Nanoparticle Loaded OGP to Restore Bone Homeostasis for Osteoporosis Therapy.

Restoring bone homeostasis is the key to the treatment of osteoporosis. How to increase osteogenic ability or inhibit osteoclast activity has always been a topic of great concern. In recent years, short peptides with biological activity have received great attention in bone repair. However, the application of short peptides is still limited due to the lack of a stable and targeted delivery system. Poly(lactic-co-glycolic acid) (PLGA) nanoparticles modified by alendronate (AL) to transport osteogenic peptides (OGP) (AL-PLGA@P NPs) are designed. Benefiting from the high affinity of AL for hydroxyapatite, AL-PLGA@P NPs have the ability to target bone. In this delivery system, OGP that promotes osteogenesis synergizes with AL, which inhibits osteoclasts, to regulate bone homeostasis, which gives them more advantages in the treatment of osteoporosis. The data shows that nanoparticles can selectively deliver peptides to the bone surface without systemic toxicity. Moreover, nanoparticles can upregulate osteogenesis-related factors (ALP, Runx-2, and BMP2) and downregulate osteoclast-related factors (TRAP and CTSK) in vitro. With AL-PLGA@P NPs, bone microarchitecture and bone mass are improved in ovariectomized osteoporosis rats. Therefore, this study proposes a novel osteoporosis-based drug system that effectively improves bone density.

Functionalized Decellularized Bone Matrix Promotes Bone Regeneration by Releasing Osteogenic Peptides.

The decellularized bone matrix (DCB) provides a promising bone substitute for the treatment of bone defects because of its similar biochemical, biophysical, and mechanical properties to normal bone tissue. However, the decellularized procedure also greatly reduced its osteogenic induction activity. In this study, peptides derived from the knuckle epitope of bone morphogenetic protein-2 were incorporated into the thermo-sensitive hydrogel poloxamer 407, and the peptide-loaded hydrogel was then filled into the pores of DCB to construct a functionalized scaffold with enhanced osteogenesis. In vitro studies have shown that the functionalized DCB scaffold possessed appropriate mechanical properties and biocompatibility and exhibited a sustained release profile of osteogenic peptide. These performances critically facilitated cell proliferation and cell spreading of bone marrow mesenchymal stem cells and upregulated the expression of osteogenic-related genes by activating the Smad/Runx2 signaling pathway, thereby promoting osteogenic differentiation and extracellular matrix mineralization. Further in vivo studies demonstrated that the functionalized DCB scaffold accelerated the repair of critical radial defects in rabbits without inducing excessive graft-related inflammatory responses. These results suggest a clinically meaningful strategy for the treatment of large segmental bone defects, and the prepared osteogenic peptide modified composite DCB scaffold has great application potential for bone regeneration.

Protease-degradable hydrogels with multifunctional biomimetic peptides for bone tissue engineering.

Mimicking bone extracellular matrix (ECM) is paramount to develop novel biomaterials for bone tissue engineering. In this regard, the combination of integrin-binding ligands together with osteogenic peptides represents a powerful approach to recapitulate the healing microenvironment of bone. In the present work, we designed polyethylene glycol (PEG)-based hydrogels functionalized with cell instructive multifunctional biomimetic peptides (either with cyclic RGD-DWIVA or cyclic RGD-cyclic DWIVA) and cross-linked with matrix metalloproteinases (MMPs)-degradable sequences to enable dynamic enzymatic biodegradation and cell spreading and differentiation. The analysis of the intrinsic properties of the hydrogel revealed relevant mechanical properties, porosity, swelling and degradability to engineer hydrogels for bone tissue engineering. Moreover, the engineered hydrogels were able to promote human mesenchymal stem cells (MSCs) spreading and significantly improve their osteogenic differentiation. Thus, these novel hydrogels could be a promising candidate for applications in bone tissue engineering, such as acellular systems to be implanted and regenerate bone or in stem cells therapy.

Biofunctionalization of polyetheretherketone implant material by bone-forming peptide-2 immobilization

Polyetheretherketone (PEEK) has favorable biomechanical properties to be used as an implant material. Unfortunately, it is hydrophobic and does not promote cellular adhesion, which could result in poor integration with bone tissue. Bio-functionalization of PEEK surface with osteogenic peptides derived from bone extracellular matrix proteins is an excit-ing approach to encourage bone formation around the implant. In the current study, bone-forming peptide-2 was immo-bilized on PEEK surface using two different methods, using dopamine and a diglycidyl ether as conjugate compounds, respectively. Peptide quantification test revealed that the two strategies resulted in the most amount of peptides were attached with 0.5 mM concentration and no further peptides were grafted with a higher peptide concentration. Both methods showed good peptide stability after agitation in aqueous solution. Peptide grafting was confirmed with ATR-FTIR. Surface characterizations with AFM and wettability tests resulted in a significant increase in surface roughness and surface area ratio for the peptide-grafted PEEK compared to unmodified PEEK, which led to a signifi-cant enhancement in the wettability of the modified PEEK surface

Myosin heavy chain-derived peptide of Gadus morhua promotes proliferation and differentiation in osteoblasts and bone formation and maintains bone homeostasis in ovariectomized mice.

Gadus morhua is an important commercial fish rich in nutrients required for daily metabolism. However, the regulation of G. morhua peptides (GMP) on osteoblast growth remains unclear. In order to clarify the regulatory effects of GMP on osteoblasts, the effects of GMP on the growth of MC3T3-E1 cells were investigated, and the osteogenic peptides were identified and screened. The results showed that GMP promoted the proliferation and differentiation of osteoblasts by regulating the BMP/WNT signaling pathway at concentrations of 1-100 μg mL-1. Molecular docking studies showed that a decapeptide, MNKKREAEFQ (P-GM-1), had a high affinity for integrins 3VI4 and 1L5G (-CDOCKER interaction energy: 161.30, 212.27 kcal mol-1). Additionally, the proliferation rate of MC3T3-E1 cells was increased by 27%, and ALP activity was significantly increased under P-GM-1 treatment (100 μg mL-1). Moreover, P-GM-1 promotes bone formation, maintains bone homeostasis, and prevents osteoporosis in ovariectomized mice by regulating the BMP/Smad signaling pathway. This study confirmed the potential of GMP in the regulation of bone mineral density and provided a certain theoretical basis for the development of anti-osteoporosis active factors from GMP.

Self-Adaptive Antibacterial Scaffold with Programmed Delivery of Osteogenic Peptide and Lysozyme for Infected Bone Defect Treatment

Bone defects caused by disease or trauma are often accompanied by infection, which severely disrupts the normal function of bone tissue at the defect site. Biomaterials that can simultaneously reduce inflammation and promote osteogenesis are effective tools for addressing this problem. In this study, we set up a programmed delivery platform based on a chitosan scaffold to enhance its osteogenic activity and prevent implant-related infections. In brief, the osteogenic peptide sequence (YGFGG) was modified onto the surface of cowpea chlorotic mottle virus (CCMV) to form CCMV-YGFGG nanoparticles. CCMV-YGFGG exhibited good biocompatibility and osteogenic ability in vitro. Then, CCMV-YGFGG and lysozyme were loaded on the chitosan scaffold, which exhibited a good antibacterial effect and promoted bone regeneration for infected bone defect treatment. As a delivery platform, the scaffold showed staged release of lysozyme and CCMV-YGFGG, which facilitates the regeneration of infected bone defects. Our study provides a novel and promising strategy for the treatment of infected bone defects.

Integrated in Silico–in Vitro Rational Design of Osteogenic Peptides derived from the Armpit Epitope of Human Bone Morphogenetic Proteins

Bone morphogenetic proteins (BMPs) are multi-functional growth factors that initiate, promote and maintain cartilage and bone morphogenesis, differentiation and regeneration in both the developing embryo and adult. The proteins have a conformational wrist epitope and a linear knuckle epitope responsible for, respectively, type-I and type-II receptor binding, as well as a hybrid armpit epitope targeted by natural BMP antagonists. In this study, the recognition and interaction between human BMPs and their pan-antagonist Crossveinless was investigated systematically at molecular level. It is revealed that the armpit epitope shares a roughly common region over different BMPs, which consists of a loop segment and a turn segment that are sequentially discontinuous but spatially vicinal on these BMP protein surfaces. Turn segment is the primary binding site that can be bound effectively by Crossveinless using a tightly packed mode. The segment was further extended at its two termini to cover a complete double-stranded sheet of BMPs, which was then split from the interfacial context of BMP–Crossveinless complexes to derive a series of osteogenic peptides; they exhibit moderate intrinsic disorder in free state, but can be constrained into a native-like conformation by stapling a disulfide bridge across two strands of the sheet. The disulfide bridge was rationally designed and optimized to avoid disrupting the native interaction of BMP sheet peptides with the active pocket of Crossveinless. Biophysical assays substantiated that the binding affinities of resulting cyclic peptides were improved by 2–6-fold relative to their linear counterpart upon the stapling, in which the cyclic peptide Bmp7-sb1 (S[CLYFDDNSNVILC]K) derived from the double-stranded sheet region of BMP7 armpit epitope was determined to have the highest affinity to Crossveinless in all tested samples. These rationally designed epitope-derived peptides can be used as osteogenic agents to activate the human BMP signaling by competitively targeting their natural antagonist.