Construction of a small peptide library related to inhibitor OM99-2 and its structure-activity relationship to β-secretase

Aim:To develop probes for detecting the binding specificity between β-secretaseand substrate,and provide reliable biological activity data for further researchingencircling substrate-based inhibitors.Methods:To prepare the inhibitors,thehydroxyethylene(HE)segment including P1and P1′ was synthesized after multi-step reactions;the combination of all segments was then completed through solidphase synthesis.Recombinant human β-secretase ectodomain(amino acid resi-dues 1-460)was expressed as a secreted protein with a C-terminal His tag in insectcells using baculovirus infection,and all compounds were evaluated in thisβ-secretase enzyme assay.In order to understand the interaction in detail,the theoretical methods,namely molecular dynamics(MD)simulation and mo-lecular mechanics-generalized-born surface area(MM-GBSA)analysis,were per-formed on the complex of β-secretase and OM99-2 to obtain the geometrical andenergetical information.Results:We designed and constructed a positional scan-ning combinatorial library including 16 compounds;all members of the librarywere synthesized based on HE dipeptide isostere.Structure-activity relationshipstudies at the P4-P1and P1′-P4′ positions led to the discoveries of P and P’sidesbinding specificity and potent inhibitors 14,18,and 22.The binding free energyon the whole system and every residue were compared to the biological assayresult.Conclusion:The removal of P4′ yielded inhibitor 22(A3*B2)with highpotency;further truncation of P3′ gave inhibitor 18(A3*B1)with equal activity,implying that the right side of the inhibitors play a less important role and could beeasily simplified,while change on the P side may cause substantial results.