Design of Integrin ?vβ6 or ?IIbβ3-specific Antagonists using Disintegrin Scaffolds

Abstract

Integrins are a family of heterodimeric receptors that are expressed on the surface of most cells where they mediate cell-cell and cell-extracellular matrix interactions Integrin ?vβ6 is epithelial-specific and strongly induced during wound healing inflammation and carcinogenesis Integrin ?IIbβ3 plays a critical role in platelet aggregation that is essential for hemostasis and thrombosis It was reported that proteins with ARGDLXXL and KGD motifs can selectively bind to integrins ?vβ6 and ?IIbβ3 respectively In this study we propose to use rhodostomin (Rho) and trimucrin (Tmu) snake venom disintegrins as scaffolds to develop integrins ?vβ6 or ?IIbβ3-specific antagonists In the study on the design of integrin ?vβ6-specific antagonists we found that the 48ARGDRP and 48ARGDKP mutants exhibited high affinity with the IC50 values of 24 0 and 36 4 nM In contrast the 48ARGD(D/E)P and 48ARGDLP mutants exhibited low affinity with the IC50 values of > 5000 and 357 3 nM These results suggest that the binding of integrin ?vβ6 prefers the C-terminal residue adjacent to the RGD motif with positively charged residues but not with negatively charged and hydrophobic residues In summary we found that the relative affinity of 48ARGDXP mutants to integrin ?vβ6 were 48ARGDRP > 48ARGDKP > 48ARGDWP > 48ARGDMP > 48ARGDAP > 48ARGDLP> 48ARGDDP We also incorporated the 48ARGDLAAL amino acid sequence of CagL in Helicobacter pylori the integrin ?vβ6-binding loop into Rho and it exhibited low affinity with the IC50 value of about 2000 nM These results suggested that the corporation of the RGD motif into different scaffolds may exhibit different conformations In addition 52RP derived mutants exhibited better activity than mutants derived from 52LA suggesting that binding of integrins ?vβ6 prefers 52RP in Rho From the docking results of 48ARGDRP binding to integrin ?vβ6 we can see the difference interactions that LAP binding to integrins ?vβ6 processes which may provide us a new perspective for future design in selectivity In the study on the design of integrin ?IIbβ3-specific antagonist we found that 41KKKRT-50AKGDRR 41MKKGT-50AKGDRR and 41IEEGT-50AKGDRP mutants had the IC50 values of 127 1 115 6 and 511 2 nM These results indicate that the binding of integrin ?IIbβ3 prefers positively charged residues in the linker region of Tmu The results of this study will serve as the basis for the design of integrin ?vβ6- or ?IIbβ3-specific drugs for the treatments of fibrosis and myocardial infarction

Date of Award	2018 Aug 27
Original language	English
Supervisor	Woei-Jer Chuang (Supervisor)