title: Investigation on cellular uptake and pharmacodynamics of DOCK2-inhibitory peptides conjugated with cell-penetrating peptides.
abstract: Protein-protein interaction between dedicator of cytokinesis 2 (DOCK2) and Ras-related C3 botulinum toxin substrate 1 (Rac1) is an attractive intracellular target for transplant rejection and inflammatory diseases. Recently, DOCK2-selective inhibitory peptides have been discovered, and conjugation with oligoarginine cell-penetrating peptide (CPP) improved inhibitory activity in a cell migration assay. Although a number of CPPs have been reported, oligoarginine was only one example introduced to the inhibitory peptides. In this study, we aimed to confirm the feasibility of CPP-conjugation approach for DOCK2-inhibitory peptides, and select preferable sequences as CPP moiety. First, we evaluated cell permeability of thirteen known CPPs and partial sequences of influenza A viral protein PB1-F2 using an internalization assay system based on luciferin-luciferase reaction, and then selected four CPPs with efficient cellular uptake. Among four conjugates of these CPPs and a DOCK2-inhibitory peptide, the inhibitory activity of a novel CPP, PB1-F2 fragment 5 (PF5), conjugate was comparable to oligoarginine conjugate and higher than that of the non-conjugated peptide. Finally, internalization assay revealed that oligoarginine and PF5 increased the cellular uptake of inhibitory peptides to the same extent. Hence, we demonstrated that CPP-conjugation approach is applicable to the development of novel anti-inflammatory drugs based on DOCK2 inhibition by investigating both cellular uptake and bioactivity.
authors.affiliation: Pharmaceutical Research Division, Takeda Pharmaceutical Company, Ltd., Fujisawa 251-8555, Japan.
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Adachi
authors.affiliation: Pharmaceutical Research Division, Takeda Pharmaceutical Company, Ltd., Fujisawa 251-8555, Japan.
authors.forename: Kotaro
authors.initials: K
authors.lastname: Sakamoto
authors.affiliation: Pharmaceutical Research Division, Takeda Pharmaceutical Company, Ltd., Fujisawa 251-8555, Japan.
authors.forename: Tadashi
authors.initials: T
authors.lastname: Umemoto
authors.affiliation: Pharmaceutical Research Division, Takeda Pharmaceutical Company, Ltd., Fujisawa 251-8555, Japan.
authors.forename: Yasunori
authors.initials: Y
authors.lastname: Fukuda
authors.affiliation: Pharmaceutical Research Division, Takeda Pharmaceutical Company, Ltd., Fujisawa 251-8555, Japan.
authors.forename: Akiyoshi
authors.initials: A
authors.lastname: Tani
authors.affiliation: Pharmaceutical Research Division, Takeda Pharmaceutical Company, Ltd., Fujisawa 251-8555, Japan. Electronic address: taiji.asami@takeda.com.
authors.forename: Taiji
authors.initials: T
authors.lastname: Asami
mesh_terms: D057846:Cell-Penetrating Peptides / Q000378:metabolism*; D020690:GTPase-Activating Proteins; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism*; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans
chemical_list: D057846:Cell-Penetrating Peptides; C402110:DOCK2 protein, human; D020690:GTPase-Activating Proteins; D020662:Guanine Nucleotide Exchange Factors
keywords: Cell-penetrating peptide; Cellular uptake; Dedicator of cytokinesis 2; Protein–protein interaction