Letters A Novel Cell-Penetrating Peptide Sequence Derived by Structural Minimization of a Snake Toxin Exhibits Preferential Nucleolar Localization Gandhi Ra ´dis-Baptista, †,‡,# Beatriz G. de la Torre, † and David Andreu* ,† Department of Experimental and Health Sciences, Pompeu Fabra UniVersity, Dr Aiguader, 80, E-08003 Barcelona, Spain, and Department of Biochemistry, Center for Biological Sciences, Federal UniVersity of Pernambuco, Cidade UniVersita ´ria, 50732-970 Recife-PE, Brazil ReceiVed July 28, 2008 Abstract: Structural simplification of a 42-residue venom peptide by N-to-C-terminal splicing led to two sequences [YKQCHKKG- GXKKGSG, where X ) nil (1) or 6-aminohexanoyl (2)], both efficiently uptaken by HeLa cells and, most interestingly, specifically localized at the nucleolus. Retro-2 was uptaken less efficiently, but a single (His f Ile) replacement recovered the translocation ability. None of the peptides were cytotoxic up to 100 µM. Enantio-1 did not translocate, suggesting that peptide uptake was receptor-mediated. Cell penetrating peptides (CPPs a ) are short cationic amphi- pathic sequences with high affinity for lipid membranes and cell-surface proteoglycans. 1,2 CPPs can translocate membrane systems, localize in different cell compartments, and often mediate the intracellular delivery of assorted cargos, including drugs, imaging agents, biopolymers (nucleic acids, polypep- tides), liposomes, and nanoparticles 3-5 and have for this reason raised considerable interest as therapeutic tools. The first recognized CPP sequences were the transactivator of transcrip- tion from HIV-1, named Tat protein, 6 and the antennapedia (Antp) homeodomain from Drosophila. 7 Later, short fragments of Tat [e.g., Tat(47-57) or Tat(48-60)] 8 and a 16-amino acid segment of Antp [Antp(43-58), named “penetratin”] 9 were demonstrated to be sufficient to translocate through the plasma membranes. Since then, an increasing number of natural and synthetic CPPs have been identified and utilized to translocate and deliver into the cell cytoplasm and nucleus distinct types of cargos both in vitro and in vivo. 10-13 Transduction peptides, containing or not containing homing nuclear domains, have also been identified in other proteins like VP22 from Herpes simplex virus type I, 14 fibroblast growth factors 1 and 2, 15 or the opioid neuropeptide dynorphin. 16 Some eukaryotic antimicrobial pep- tides also display cell-penetrating behavior, somehow expected from the structural similarities (cationic character, amphipathic nature) that underlie their ability to interact with lipid bilayer components, and eventually penetrate cells and target specific organelles. 17 Examples along these lines include the amphibian, histone 2A-derived peptide buforin II 18 or salivary histatin 5, which translocates into the cytoplasm of Leishmania and accumulates into the mitochondrion. 19 Hybrid buforin 2-ma- gainin 2 sequences have been shown to translocate efficiently across the bacterial membrane and deliver into the cytoplasm the antimicrobial R-helical portion of magainin 2. 20 Animal venoms are a very rich source of bioactive polypep- tides that in several cases have been turned into actual or candidate drugs. Snakes, 21 scorpions, 22 spiders, 23 or sea snails, 24 to name just a few, produce and secrete a valuable diversity of toxins capable of interacting and interfering with distinct molecular targets in the cell. Hitherto, a very limited number of animal toxins able to translocate the cytoplasmic membrane and localize into distinct cell compartments are known. Two examples are maurocalcine, 25 a scorpion toxin of 33 amino acids and three disulfide bonds that localizes in the cytoplasm and release calcium ions, and crotamine, 26 a rattlesnake toxin of 42 amino acids and also three disulfide bonds that selectively translocates into actively proliferating cells, both in vivo and in vitro, during the G1/S phase of the cell cycle and localizes to the nucleus. For both peptides, the uptake mechanism appears to involve initial binding to heparan sulfate proteoglycans, followed by endocytosis for crotamine 27 and macropinocytosis for maurocalcine. 28 In a solution NMR study, crotamine was shown to adopt an R fold 29,30 (Figure 1), similar to that of the human antimicrobial peptide -defensin 2 or to scorpion toxins targeting sodium channels. Structure-guided deconstruction of complex, highly folded bioactive peptides has been advantageously used to identify the structural traits essential for their biological activity, 31,32 i.e., their pharmacophore, and in turn to design minimalist, thera- peutically useful versions of such peptides. 33 Although identi- fication of which particular section of a protein will translocate still relies more on experimental than on predictive approaches, 34 it seemed worthwhile to perform a structural dissection of * To whom correspondence should be addressed. Phone: +34-933160868. Fax: +34-933160901. E-mail: david.andreu@upf.edu. † Pompeu Fabra University. ‡ Federal University of Pernambuco. # Present address: Institute for Marine SciencessLabomar, Federal University of Ceará, Avenida Abolição, 3207, Fortaleza-CE, 60165-081, Brazil. a Abbreviations: Ahx, 6-aminohexanoic acid; CPP, cell-penetrating peptide; DIC, differential interference contrast; FITC, fluorescein isothio- cyanate; Fmoc, 9-fluorenylmethyloxycarbonyl; HPLC, high performance liquid chromatography; MALDI-TOF MS, matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Figure 1. 3D structure of crotamine (PDB entry no. 1h5o) 29,30 showing proximity between the N- (R-helix, residues 1-9, blue) and C-terminal (random, residues 28-32) regions (both highlighted in blue), which is the basis for the design of peptides 1 and 2 and retro versions 3 and 4. J. Med. Chem. 2008, 51, 7041–7044 7041 10.1021/jm8009475 CCC: $40.75  2008 American Chemical Society Published on Web 11/05/2008