Repertoire of Structure–Activity-Based Novel Modified Peptides Elicits Enhanced Osteogenic Potential

Biologically active peptides in milk proteins can be used as effective dietary supplements for management of bone-associated issues including osteoporosis. A bioactive peptide derived from milk, viz. VLPVPQK/PepC, has been validated previously from our lab for its osteoanabolic action. In this study, we report 14 novel variants of PepC, designed in silico, based on the structure-activity relationship, aiming to enhance its osteogenic effect that holds tremendous therapeutic utility for bone-related injuries. PepC was computationally modified at seven positions of its original sequence, resulting in 14 modified synthetic peptides for functional predictions and in vitro assessment by comparative analysis of modified peptides by PepC for improved ability in osteogenic functional assays (proliferation potential, antioxidant ability, gene and protein expression, cytotoxic effect, bone mineralization) using calvarial osteoblasts. For most peptides with the highest Peptide7 response relative to PepC (p < 0.05), enhanced osteoanabolic response was observed. Further observations on Peptide7 have therefore been investigated in depth (qPCR, immunoblotting, LCMS/MS, and PCA analysis). Peptide7 displayed a rise in the expression of osteogenes (Osterix, Opg, Bmp2, and Runx2, p < 0.05) and protein (Runx2 and Bmp2, p < 0.05). Besides, LCMS/MS findings suggest Peptide7 escapes intestinal peptidases degradation. Experimental evidence supports an improved osteological reaction to newly modified peptides and hence exploitation in the preparation of functional foods or supplements.