Systematical Mutational Analysis of FRATtide against Osteoclast Differentiation by Alanine Scanning.

Osteoporosis, a global bone disease, results in decreased bone density, mass, and microarchitecture deterioration, increasing fracture risk. In previous research, FRATtide, a peptide derived from a glycogen synthase kinase-3 binding protein, effectively hindered osteoclast differentiation to yield therapeutically potent derivatives via single and double stapling. However, FRATtide's structure-activity relationship remains unclear. This study synthesized 25 FRATtide-derived peptides through systematic alanine scanning and evaluated their activities. Substitutions in Pro, Leu, Leu, Val, Leu, Ser Asn, Leu, Ile, Glu, Arg, Ser, and Arg showed reduced activity, while FRT13 and FRT20 with Gly and Arg substitutions, respectively, displayed enhanced activities. F-actin binding and bone resorption assays on FRT13 and FRT20 showed better inhibition of osteoclast differentiation and bone resorption compared with FRATtide. This study elucidated FRATtide's structure-activity relationship, thereby facilitating future structural optimization for osteoporosis treatment.