title: Cell-penetrating peptide-conjugated XIAP-inhibitory cyclic hexapeptides enter into Jurkat cells and inhibit cell proliferation.
abstract: X-Linked inhibitor of apoptosis protein (XIAP) is a member of the inhibitor of apoptosis protein family that is overexpressed in human cancers. There is great interest in the development of XIAP inhibitors, which are predicted to promote apoptosis in cancer cells and thus have therapeutic potential. A cyclic hexapeptide (CH), CPFKQC, which is one of the consensus motifs that can bind to the baculovirus IAP repeat 2 domain of XIAP, has been identified using phage-displayed combinatorial chemistry techniques [Tamm I, Trepel M, Cardo-Vila M, Sun Y, Welsh K, Cabezas E, Swatterthwait A, Arap W, Reed JC & Pasqualini R (2003) Peptides targeting caspase inhibitors. J Biol Chem278, 14401-14405]. In this study, we designed and synthesized covalently linked conjugates of CHs, cyclo[Cys-Pro-Xaa-Lys-Gln-Glu(-CO-)-NH2] (Xaa = various amino acids; cyclization via a peptide bond between the N-terminal amino group of Cys1 and the side-chain carboxylic acid of Glu6), and a cell-penetrating peptide (CPP), Ac-Cys-Trp-(Arg)8-Lys-NH2. CH-CPP conjugates (CHCPPs) with aromatic and hydrophobic Xaa residues, such as Phe (CHCPP 1), 2,6-dimethyl-phenylalanine (CHCPP 2) and 3-(1-naphthyl)-alanine/3-(2-naphthyl)-alanine (CHCPPs 3 and 4), potently inhibited the proliferation of Jurkat cells in a dose-dependent manner, whereas analogues with nonaromatic or less hydrophobic amino acids at the Xaa residue were less potent or caused no inhibition. A morphological study of nuclei after treatment with CHCPPs 1-4 revealed that nuclear fragmentation occurred, suggesting that these conjugates induce apoptosis. A kinetic study of the uptake of fluorescein-labelled CHCPP 2 into the cells showed that the conjugates were translocated within a few minutes. The cellular uptake of fluorescein isothiocyanate-labelled CHCPP 1 and CPP was greatly reduced in high-K+ buffers, suggesting that CPP and its conjugate are translocated by a mechanism associated with cell membrane potential. Competitive binding studies performed using fluorescence correlation spectroscopy demonstrated that CHCPP 1 binds to the baculovirus IAP repeat 2 domain of XIAP via the CH (Xaa = Phe) moiety. CHCPPs 1 and 2 showed the most potent inhibitory activity of the CHCPPs and embelin, a nonpeptide inhibitor of XIAP, suggesting that they are good templates for the design of a new class of anticancer drug.
authors.affiliation: Department of Biochemistry, Tohoku Pharmaceutical University, Sendai, Japan. ysasaki@tohoku-pharm.ac.jp
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Sasaki
authors.forename: Motoko
authors.initials: M
authors.lastname: Minamizawa
authors.forename: Akihiro
authors.initials: A
authors.lastname: Ambo
authors.forename: Shigeki
authors.initials: S
authors.lastname: Sugawara
authors.forename: Yukiko
authors.initials: Y
authors.lastname: Ogawa
authors.forename: Kazuo
authors.initials: K
authors.lastname: Nitta
mesh_terms: D017304:Annexin A5 / Q000378:metabolism; D017209:Apoptosis / Q000187:drug effects; D016227:Benzoquinones / Q000494:pharmacology; D001692:Biological Transport; D002352:Carrier Proteins / Q000737:chemistry; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D049109:Cell Proliferation / Q000187:drug effects*; D002470:Cell Survival / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D006801:Humans; D019169:Jurkat Cells; D008564:Membrane Potentials / Q000187:drug effects; D008856:Microscopy, Fluorescence; D015394:Molecular Structure; D009842:Oligopeptides / Q000737:chemistry / Q000493:pharmacokinetics / Q000494:pharmacology*; D010456:Peptides, Cyclic / Q000737:chemistry / Q000493:pharmacokinetics / Q000494:pharmacology*; D011188:Potassium / Q000494:pharmacology; D011419:Propidium / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D013050:Spectrometry, Fluorescence; D051636:X-Linked Inhibitor of Apoptosis Protein / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism
chemical_list: D017304:Annexin A5; D016227:Benzoquinones; D002352:Carrier Proteins; D009842:Oligopeptides; D010456:Peptides, Cyclic; D051636:X-Linked Inhibitor of Apoptosis Protein; C496866:XIAP protein, human; D011419:Propidium; D011188:Potassium; C010945:embelin