Cellular Delivery of Impermeable Effector Molecules in the Form of

NOVEMBER/DECEMBER 2001 Volume 12, Number 6 © Copyright 2001 by the American Chemical Society

REVIEWS Cellular Delivery of Impermeable Effector Molecules in the Form of Conjugates with Peptides Capable of Mediating Membrane Translocation Peter M. Fischer,* Eberhard Krausz, and David P. Lane Cyclacel Limited, Dundee Technopole, James Lindsay Place, Dundee DD1 5JJ, Scotland, UK. Received May 1, 2001

Most molecules that are not actively imported by living cells are impermeable to cell membranes, including practically all macromolecules and even many small molecules whose physicochemical properties prevent passive membrane diffusion. The use of peptide vectors capable of transporting such molecules into cells in the form of covalent conjugates has become an increasingly attractive solution to this problem. Not only has this technology permitted the study of modulating intracellular target proteins, but it has also gained importance as an alternative to conventional cellular transfection with oligonucleotides. Peptide vectors derived from viral, bacterial, insect, and mammalian proteins endowed with membrane translocation properties have now been proposed as delivery vectors. These are discussed comprehensively and critically in terms of relative utility, applications to compound classes and specific molecules, and relevant conjugation chemistry. Although in most cases the mechanisms of membrane translocation are still unclear, physicochemical studies have been carried out with a number of peptide delivery vectors. Unifying and distinguishing mechanistic features of the various vectors are discussed. Until a few years ago speculations that it might be possible to deliver peptides, proteins, oligonucleotides, and impermeable small molecules with the aid of cellular delivery peptides not only to target cells in vitro, but in vivo, was received with scepticism. However, the first studies showing pharmacological applications of conjugates between macromolecules and peptide delivery vectors are now being reported, and therapies based on such conjugates are beginning to appear feasible.

INTRODUCTION

Biological membranes are natural barriers central to compartmentalization in living systems. They prevent unchecked influx and efflux of solutes from cells and * To whom correspondence should be addressed. E-mail: [email protected]. Phone: +44 (0)1382 206062. Fax: +44 (0)1382 206067.

cellular organelles. All biological membranes contain a phospholipid bilayer as the basic structural unit. Phospholipids consist of hydrophobic tails and hydrophilic heads. Under physiological conditions they aggregate spontaneously into a bilayer, where the fatty acyl chains are sequestered within the hydrophobic interior and the polar heads face the aqueous surfaces. Disposed within the fluid but discrete bilayer structure are proteins and

10.1021/bc0155115 CCC: $20.00 © 2001 American Chemical Society Published on Web 10/24/2001

826 Bioconjugate Chem., Vol. 12, No. 6, 2001

protein complexes that act as specific pumps and channels to permit active transport of solutes. More primitive channels are found in bacterial membranes, nuclei, and mitochondria. These function essentially as molecular sieves with defined size exclusion limits. The ability to introduce membrane-impermeable molecules into mammalian cells has been an invaluable tool for the study of intracellular processes. Both physical and chemical methods have been developed to this end. Microinjection, electroporation, freeze-thaw techniques, etc., belong to the former, whereas the latter involve transient cell membrane permeabilization with the aid of solvents such as toluene or dimethyl sulfoxide, hypotonic buffers, and various detergents. The main problem with these methods is the fact that they are not readily reproducible, they are not quantitative, and they generally compromise cellular integrity. Furthermore, such methods are obviously only relevant for in vitro cell biology applications but not suitable for in vivo use. One of the main constraints in the design of drugs whose interaction targets are located within host cells is thus membrane permeability. Only compounds within a narrow range of molecular size, net charge, and polarity are able to diffuse effectively through the lipid bilayers of biological membranes and can thus reach their pharmacological target (1). The vast majority of molecules, including many natural products and biomolecules, i.e., compounds often possessing inherent biochemical selectivity and potency, are thus apparently precluded from therapeutic application. The conventional mode of cellular entry for hydrophilic macromolecules is endocytosis. This process involves absorption of the macromolecules to the plasma membrane or to membrane-associated receptors, followed by energy-dependent formation of vesicles. The endocytic machinery of the cell then tags and directs the internalized molecules to appropriate compartments for either destruction or recycling. In general, molecules that enter cells via the endocytic route are not released to the cytoplasm in unaltered form, if at all. Liposomes have been used widely in order to enhance the cellular delivery of biomolecules via the endocytic route. Liposomal delivery, as well as liposomes in general, have been reviewed very comprehensively elsewhere (2, 3). Efficient cellular uptake via endocytosis is generally observed but the efficiency is then limited downstream by insufficient endosomal escape, restricted diffusion, especially of highmolecular weight substances, in the gellike cytosol, dissociation of the complexes, or nuclear entry. Enhanced endosomal escape has been achieved through modification of liposomes, i.e., pH-sensitive liposomes (4, 5), or association with helper lipids such as DOPE, or complexation with endosome-disrupting or destabilizing additives, i.e., PEG (5, 6), cholesterol (7), or lytic peptides (8-10). The nuclear barrier may be crossed efficiently by attachment of nuclear localization signals (11). Although numerous encouraging strategies to overcome various limitations of liposomal delivery are being developed, there is still a need for further improvement and consideration of alternative delivery strategies. For this reason, delivery systems addressing their molecular cargo to cells through endocytosis are only partly or not at all suitable if the molecular targets are cytosolic or nuclear, as is usually the case. Many of the peptide vectors described below, however, can effect cell entry of their molecular conjugates through mechanisms best described as penetration (12). Although poorly understood, these mechanisms appear to be energy-independent and address conjugates to the cytosol and/or the nucleus. No

Fischer et al.

distinct membrane receptors seem to be involved and membrane translocation is nonselective in terms of cell type. A recent study compared the cellular uptake of a number of different delivery peptides by a combination of high-performance liquid chromatography and confocal laser-scanning microscopy (13). The presence of both endocytic and nonendocytic modes of uptake were observed. Contrary to current opinion, the authors propose a general ability of peptides to cross plasma membranes and suggest that effective delivery with certain peptides may be due to prevention of wash-out in certain cases, e.g., enhancement of binding to intracellular components. While there is probably insufficient evidence at present to support this hypothesis, it highlights the fact that the mechanisms of cell membrane translocation by delivery vector peptides are far from clear. An additional complication stems from the fact that each vector-effector conjugate will probably behave differently, depending on the nature of different effector molecules. Infective organisms such as viruses and bacteria possess proteins endowed with properties to penetrate living cells indiscriminately (14). For example, the HIV TAT protein, the HBV VP22 protein, pore-forming toxins such as tetanus toxin (15), anthrax toxin (16), cytolysins (17), diphtheria toxin (18), etc., can gain entry into cells in essentially receptor-independent fashion. Peptides derived from some of these proteins, particularly those of viral origin, have been developed as delivery vectors and these are discussed below. Secreted proteins, some membrane proteins, and proteins of membrane-enveloped organelles of eukaryotic cells are partially or fully transported across membranes and contain signal sequences which direct them to the correct subcellular compartment. Most signal sequences are N-terminal extensions of the functional proteins and as a rule are removed proteolytically after translocation. Surprisingly, no sequence homology has been found for sequences directing proteins to the same subcellular compartment, and only general features such as charge distribution and hydrophobicity seem to be common. The secreted proteins of prokaryotes and proteins destined for the endoplasmic reticulum of eukaryotes generally contain a signal sequence consisting of a few charged residues, followed by an uninterrupted stretch of 7-16 apolar residues. Mitochondrial signal sequences, and those of many bacterial proteins consist of 12-30 apolar, polar, and charged residues, arranged in a pattern potentially allowing formation of secondary structures with polar/charged and apolar faces (helical amphiphilicity) or ends (segmental amphiphilicity). Mammalian and insect host defense peptides, including e.g., melittin, protegrins, defensins, tachyplesins, cecropins, etc., as well as pore-forming proteins and peptides in general (19), also fall within this class of substances. Numerous studies on such peptides have shown that they are capable of forming amphiphilic structures and that this propensity correlates with membrane activity (20). Both classes of signal peptides have led to the development of vector systems, and these are discussed more fully below. Amphiphilic peptides have gained some importance in cellular delivery of oligonucleotides, particularly as an endosomal entrapment escape device. Yet other endogenous mammalian proteins possess the innate ability to cross cell membranes due to their function in intercellular signaling. At least in some cases such proteins do not appear to rely on membrane receptors or transporter systems. The best-documented case falling within this category concerns homeoproteins. Because much pioneering work on peptide-based cellular

Reviews

Bioconjugate Chem., Vol. 12, No. 6, 2001 827

Table 1. Summary of the Best-Characterized Delivery Peptide Vectors sequence

name (origin of sequence)

literature reference

RQIKIWFQNRRMKWKK kkwkmrrnqfwvkvqr RRWRRWWRRWWRRWRR RRMKWKK GRKKRRQRRRPPQ YGRKKRRQRRR rrrrrrr GALFLGWLGAAGSTMGA GALFLGFLGAAGSTMGAWSQPKSKRKV MGLGLHLLVLAAALQGA MGLGLHLLVLAAALQGAWSQPKKKRKV PLSSIFSRIGDP GWTLNSAGYLLGKINLKALAALAKKIL RGGRLSYSRRRFSTSTGR AAVALLPAVLLALLAP AAVLLPVLLAAP VTVLALGALAGVGVG VAYISRGGVSTYYSDTVKGRFTRQKYNKRA KLALKLALKALKAALKLA WEAKLAKALAKALAKHLAKALAKALKACEA

pAntp(43-58); Penetratin retro-inverso pAntp(43-58) W/R Penetratin pAntp(52-58) HIV TAT HIV TAT r7 gp41 fusion sequence MPG (gp41fusion sequence - SV40 NLS) Caiman crocodylus Ig(v) light chain Caiman crocodylus Ig(v) light chain - SV40 NLS PreS2-TLM Transportan SynB1 MPS (kaposi FGF signal sequence) MPS (kaposi FGF signal sequence) MPS (human integrin β3 signal sequence) P3 Model amphiphilic peptide KALA

67 69 70 73 124 136 138 168 196 168 168 145 225 52 166 178 234 222 215 214

delivery vectors was originated with this system, a separate section is devoted to this topic. APPLICATIONS OF VECTOR CONJUGATES

Polypeptides. Only the very shortest of peptides, such as di- and tripeptides, can enter cells nonspecifically, either using general peptide transporter systems (21), or by diffusion (22). Although very short peptides may be rendered permeable by derivatization with membraneactive lipids such as myristic acid (23, 24), native polypeptides cannot traverse membrane barriers, unless they participate in specific cellular or subcellular processes, in which case appropriate and highly specific receptor and transporter systems exist. While some of the methods developed in the context of gene transfer are also applicable to cellular delivery of polypeptides, e.g., transient chemical cell permeabilisation (25) or liposomal delivery (4, 26, 27), it is the recent development of peptidic cell delivery vectors that has made possible the routine intracellular administration of polypeptides. Traditionally, delivery vector-derivatized polypeptides have been used as tools in order to elucidate signaling pathways and generally to probe protein-protein interactions in cellular systems. Perhaps the most surprising finding with vectorized polypeptides has been their ability to internalize into cells and there to exert their biological function almost irrespective of molecular mass. Thus, short effector peptides containing only a few amino acid residues can be transported across membranes, as well as entire proteins. For example, the 20-kDa p16INK4a tumor suppressor and the 120-kD β-galactosidase proteins have been shown to penetrate into cells using the pAntp and TAT peptide vectors (refer to Table 1), respectively (28, 29). Oligonuceotides. Apart from receptor-mediated approaches (30, 31) and viral transfection methods, which have gained therapeutic importance for gene therapy but are not discussed here, carrier-mediated oligonucleotide and gene delivery falls into two categories. The first depends on the reversible association of condensing molecules with oligonucleotides. The carriers that have been used most frequently are composed of polymers, particularly polylysines (32) and polyethyleneimine (33), lipids (34-36), or cationic peptides that bind to anionic sites on the oligonucleotides (34). In all these cases, noncovalent association between the carriers and the oligonucleotides leads to compaction and condensation of the oligonucleotides into small particles that gain entry

into cells by nonspecific fluid-phase pinocytosis (37). Some synthetic peptide-based carriers falling within this category have been used successfully, e.g., AlkCWK18 (38), and others (39, 40). Because pinocytotic uptake usually leads to endosomal trapping of the oligonucleotide complexes, helper molecules such as chloroquine (facilitator of endocytic exit through limitation of DNA degradation by lysosomal enzymes (41, 42)) and glycerol (enhanced uptake of particles and labilization of vesicular membranes (43)) are being used, and peptides capable of rupturing endosomal membranes at the low pH of this compartment have been developed. These are discussed under the amphiphilic peptide vectors below. The second category of carrier-mediated oligonucleotide delivery concerns vectors capable of mediating direct cytosolic delivery of oligonucleotides across the plasma membrane. Several peptide vectors in this category have been described and are referred to specifically below. Early examples using antisense oligonucleotides and pAntp vector conjugates showed not only successful cellular delivery of such constructs, but also clear antisense effects: inhibition of neurite outgrowth due to suppression of amyloid precursor protein neosynthesis (44) and cell death due to down-regulation of Cu/Zn superoxide (45), respectively. Nanoparticles. Intracellular magnetic labeling of cells with ferro/supermagnetic substances can be achieved, e.g., by fluid phase endocytosis, receptor-mediated endocytosis, or phagocytosis. The cellular uptake of dextrancoated superparamagnetic iron oxide particles was shown to be over 100-fold more efficient when the particles were derivatized with an HIV TAT-delivery vector peptide (46). In this approach, a monodispersed superparamagnetic iron oxide colloid was cross-linked and aminated. The pendant amino groups were first derivatized with the heterobifunctional cross-linker N-succinimidyl 3-(2-pyridyldithio)propionate, followed by disulfide exchange using a cysteinylated TAT(48-57) vector peptide. The resulting peptidyl conjugate particles had an average size of 41 nm and contained ca. 6.7 TAT peptides. Internalization of these particles into lymphocytes was very efficient, resulting in up to 107 particles/cell, permitting NMR imaging techniques. This technology has recently been applied to the in vivo tracking and recovery of progenitor cells (47). Organic Molecules. Not only biomolecules, but also many small-molecule natural products, as well as numerous synthetic drug candidates, are membrane-


impermeable due to unfavorable physicochemical properties. Here too, administration in vectorized form may be useful. Furthermore, effective therapeutic application of many organic molecules is prevented because of their poor solubility, absorption, or distribution. It is conceivable that some of these difficulties could be overcome with drug-vector peptide conjugates. One such problem is the delivery of neuropharmaceuticals across the blood-brain barrier. Various strategies, including peptide-vector approaches, have been proposed for the brain delivery of drugs (48). Thus it was shown that the anticancer agent doxorubicin was able to cross the blood-brain barrier in a brain perfusion model (49), as well as after parenteral administration to mice, when covalently linked to Pegelin (50, 51) or pAntp vector peptides (52). VECTORS

Some of the more prominent vector peptides are summarized in Table 1. Although various different membrane translocation mechanisms are implicated, depending on the vectors, certain low-order structural similarities between the peptides can be discerned. Apart from the highly lipophilic signal peptide-derived vectors, amphiphilicity and the preponderance of positively charged amino acid residues (Arg and Lys) are noted. Recent systematic approaches to investigate the latter observation demonstrated that natural Arg-rich peptides, including RNA-binding and DNA-binding peptides derived from all types of organisms (viruses and yeasts to human), as well as synthetic polyarginines, can translocate across membranes and are able to deliver proteins (53). Furthermore, selected Lys- and Arg-rich peptides derived from a phage-displayed library were capable of delivering β-galactosidase in vitro and in vivo (54). Many of the vectors also possess oligonucleotide-binding properties, as indeed do most proteins with nuclear localization signals (55, 56) and in many cases DNA-binding and tranlocation capacities appear to be interdependent. Few studies have been reported where different vectors have been applied to the same delivery problem, and it is thus difficult to assess accurately the relative advantages and disadvantages of the various vector systems. In one study, three vector systems (Penetratin, MPS, and model amphiphilic peptide; refer to Table 1) were compared for the delivery of SH3 domain blocker peptides (57). Here Penetratin was found to be the superior vector, with the other two peptides either reducing biochemical affinity of the blocker peptide in vectorized form and the vector displaying cellular toxicity, respectively. Homeoprotein Vectors. The homeobox encodes the 60-amino acid sequence of the homeodomain in the products of many developmental genes. These products are often regulatory proteins, which affect the expression of other developmental genes through binding to cognate DNA sequences. The fact that a synthetic peptide (pAntp) encompassing the entire homeodomain of the Drosophila homeoprotein antennapedia was sufficient to bind to specific DNA had already been demonstrated (58), when it was discovered that this peptide was also capable of penetrating nerve cells in culture and to accumulate in their nuclei through an energy-independent mechanism (59). Initially, R-2,8-polysialic acid (PSA) chains specific to the neural cell adhesion molecule were proposed as the neuronal cell surface receptors (60). However, the ability of pAntp to penetrate efficiently into a large number of cells devoid of PSA suggested the existence of a more general cell internalization mechanism (61). Even lymphocytes, which are notoriously difficult to transfect, take up the pAntp peptide efficiently (62). After delivery

Fischer et al.

into cells, pAntp conjugates are generally localized to the cytosol and the nucleus. However, mitochondrial delivery is also possible with the aid of chimaeric pAntp mitochondrial signal sequence vectors (63). Homeodomains are composed of three R-helices, with a β-turn between helices 2 and 3 (64) and are highly conserved among homeoproteins and across species (65). The region of the Antp homeodomain responsible for internalization of the protein by cells was mapped to the third helix (66), and this finding led to the demonstration that a 16-residue peptide corresponding to that helix is necessary and sufficient for the translocation of biological membranes (67). Subsequent structure-activity studies based on that peptide showed that cell internalization was receptor-independent and correlated in some way with a proposed bioactive amphipathic structure (68). Thus membrane translocation was equally efficient at 4 °C and 37 °C for the native sequence, as well as retroand inverso-peptide isomers. The same was independently shown to be true for retro-inverso analogues of the same sequence (69). An analogue derived from pAntp(4358), and consisting entirely of Arg and Trp residues (7072), has also been studied, and the exclusive presence of cationic and hydrophobic groups in this peptide support the notion that amphipathicity is involved in the mechanism of action. The minimal sequence necessary and sufficient for functional membrane translocation of pAntp peptides, previously thought to correspond to pAntp(4358), has recently been mapped to the C-terminal heptamer encompassing residues 52-58 (73). A pAntpderived vector, designed from known structure-activity relationships and incorporating peptidomimetic elements was also reported recently (74). The fact that several homeoproteins and homeodomains investigated to date appear to internalize into cells suggests that additional peptides, mechanistically related to pAntp(43-58), may yet be discovered (12, 65, 75, 76). Although pAntp(43-58) adopts amphipathic helical structure in various solvents and in the presence of SDS micelles (77), helicity does not appear to be a prerequisite for activity, since Pro-containing substitution analogues presumably incapable of helical structures nevertheless traversed membranes (78). In fact it has been shown recently that the pAntp(43-58)’s secondary structure in membrane-mimicking environments is dominated by β-structures (79, 80). The peptide exhibits high surface activity in the presence of phospholipids, and translocation properties appear to be linked with the disorder induced by the peptide in charged lipid layers. The cellular uptake mechanism appears to involve only peptide-membrane lipid interactions (81), and the translocation does not apparently involve pore formation (82, 83). However, although models have been proposed (72, 84), the exact mechanism responsible for membrane translocation by pAntp peptides remains largely elusive. The question of how certain proteins without a signal sequence can be secreted has been raised in connection with homeoproteins (84). A detailed study (85, 86) with the homeoprotein Engrailed, which, when expressed in cells, is secreted into the culture medium, revealed a short sequence (termed ∆1) that appears to be necessary for nuclear export and secretion. The sequence in question is positioned N-terminally of the cellular import sequence (helix 3) and partly overlaps with it. This situation is suggestive of an import-export model that may apply to other homeoproteins, and messenger proteins in general. Furthermore, these findings may lead to the discovery of vector peptides capable not only of cellular import, but also cellular trafficking.

Reviews

The use of pAntp vectors in order to probe proteinprotein interactions with short peptide sequences in the context of whole-cell function has been very extensive, while protein transduction has been analyzed to a much lower extent (28, 76). Thus peptides involved in regulation of cell proliferation (28, 57, 87-102), cell migration (103, 104), cellular immune processes (105), and peptidic active-site antagonists and other modulators of cellular processes (106-110), have all been delivered to cells in vitro, and, in some cases, in vivo, and effector mechanismbased functional read-outs have generally been obtained. Applications involving successful cellular delivery of antisense oligonucleotides (44, 111, 112) and antisense peptide nucleic acids (PNAs) (113, 114), leading to antisense target-related effects, have also been reported, again including in vivo applications. HIV TAT Vectors. The human immunodeficiency virus (HIV) encodes several regulatory proteins not present in other retroviruses. One of these is the 86residue TAT protein, which trans-activates certain viral genes and is essential to viral replication. Independent studies using bacterially expressed (115) and chemically synthesized (116) TAT protein, respectively, showed that this protein could enter cells when added to the medium of cells in culture. Furthermore, the protein located to the nucleus and was capable of trans-activating viral promoters. The domain responsible for translocation was shown to encompass the basic domain of the TAT protein. A peptide extending from residues 37-72 enabled cellular delivery of protein cargoes such as β-galactosidase, horseradish peroxidase, RNAse A, a Pseudomonas exotoxin A fragement not only in vitro, but also in vivo (117, 118). Similarly, a vector based on the sequence 37-62 was successfully used to deliver an antibody Fab fragment to cells (119, 120). A peptide containing the TAT sequence including residues 38-60 was shown to adopt an R-helical conformation under certain conditions; in particular, the sequence 38-45 appeared capable of forming an helix with amphipathic characteristics, whereas the basic sequence 49-57 was unstructured (121). The latter sequence was demonstrated to contain a nuclear localization sequence (NLS) (122). Furthermore, using a β-galactosidase-TAT cellular expression vector, it was demonstrated that the TAT-NLS was able to confer nuclear entry and binding to nuclear components through a novel import pathway (123). A detailed study (124) involving peptides derived from the TAT(37-60) sequence showed that the amphipathic helix was in fact not required for cellular peptide uptake. In contrast, the whole basic domain from the TAT sequence, which appears unstructured in solution (125), was necessary for cell internalization. The minimal sequence necessary and sufficient for membrane translocation was TAT(48-57). Although intact HIV-1 TAT protein contains an RGD cell adhesion site (126) and enters cells by energy-dependent absorptive endocytosis (127), uptake of TAT peptide constructs does not apparently involve classical receptor-, transporter-, endosomeor adorptive-endocytosis-mediated processes. However, unlike the inverted micelle model of membrane transduction favored for pAntp, a mechanism involving direct penetration of the lipid bilayer caused by the localized positive charge of the vector, in which the momentum of the molecule drives the covalently attached cargo into the cytoplasm, has been proposed (128). On the other hand, a similar mechanism is indicated by studies with equine TAT protein which suggest that the structural elements responsible for the cell membrane transduction


properties of TAT and homeoproteins could be related by evolution (129). Short TAT peptide vectors have been used widely in order to introduce various functional peptides and oligonucleotides into cells (112, 130-132). Furthermore, TAT peptide vectors have been used particularly successfully for cellular transduction with functional proteins. An interesting example is the design of a transducing, modified, apoptosis-promoting caspase-3 protein, in which endogenous proteolytic cleavage sites were substituted with HIV protease-specific ones (133). The application of this TAT peptide-caspase construct to HIV-infected cells led to apoptosis, while remaining without effect on uninfected cells. Although entire proteins can be delivered into living cells using TAT peptide vectors (118, 128, 134-136), membrane transduction appears to be a stringent process that involves partial or complete unfolding of the protein cargo (137). For example, although delivery of correctly folded TAT-GFP fusion protein into cells was efficient, it was accompanied by significant loss of GFP fluorescence emission (128). Very recently, a detailed structure-activity relationship study (138) based on the HIV TAT(49-57) sequence RKKRRQRRR (139) revealed that while further truncation of the 9mer sequence led to abolition of membranetranslocation properties, the sequence could be retro-, enantio-, and retroenantio-modified with beneficial effects. Furthermore, of the nine residues only Gln54 was particularly sensitive to Ala substitution. Replacement of the two Lys and the Gln residues with Arg, i.e., formation of an Arg9 homopolymer, afforded a peptide with improved cell internalization properties. This could be further enhanced by using D-Arg residues exclusively (r9). Of the D-Arg homopolymers the 9mer had optimal properties but here truncation down to the 7mer (r7) was tolerated in terms of internalization properties. Corresponding Lys, Orn, and His oligomers were less effective at entering cells (140). Comparison of r9 with HIV TAT(47-59) and pAntp(43-58) showed that the former entered cells significantly faster than the native TAT sequence and the pAntp sequence. Peptoid analogues of the HIV TAT sequence (141, 142), i.e., peptidomimetics containing N- rather than CR-substituted R-amino acid monomers, were also studied. Here similar findings as with the Arg homopolymers were made. Additionally, cellular uptake was proportional to the length of the alkyl chain in the N-alkylguanidine monomer side chains. In this study the energetics of cellular uptake of the peptides and peptoids were assessed not only in terms of temperature-dependence, but also by performing penetration assays in the presence or absence of sodium azide. While temperature did not apparently influence membrane translocation (consistent with results obtained elsewhere with many different vectors), azide, an inhibitor of oxidative phosphorylation (143), was found to inhibit cell internalization. An application of the r7 vector, conjugated through a pH-sensitive linker to cyclosporin A, has been reported (144). Cyclosporin A is ineffective for topical applications due to its poor skin-penetration properties. In contrast, it was found that the r7 conjugate was transported efficiently into mouse and human skin, reached dermal T lymphocytes, and inhibited cutaneous inflammation. HBV PreS2 Vector. A permeable peptide, referred to as PreS2-TLM (translocation motif), was recently reported (145). The presence of a masked fusion peptide in the PreS2 surface antigen of hepatitis-B virus had previously been postulated (146), when it was observed that protelolytic digestion of viral particles enabled


infection of otherwise nonpermissive cells (147). The PreS2-TLM corresponds to an amphipathic R-helix between residues 41 and 52 of the PreS2 protein. The peptide appears to be able to penetrate a variety of cells, including plant cells, by an energy-independent mechanism. Once inside a cell, it distributes evenly over the cytosol. Recombinant PreS2 protein, when infused into the portal vein of anesthetised rats, was detected in various liver cells, including cell layers distant from capillaries (145). Interestingly, in comparison to the TATpeptide, Penetratin, and other peptides rich in positively charged residues, PreS2-TLM contains only one negatively and one positively charged amino acid. Therefore, this peptide may follow a different translocation mechanism. HSV VP22 Vector. A 38-kDa structural protein, VP22, from herpes simplex virus-1 has the remarkable property of intercellular transport (148). When expressed in a subpopulation of cells, the protein spreads to every cell in a monolayer, where it concentrates in the nucleus and binds chromatin. VP22 is also a target for phosphorylation in the nucleus after viral infection, presumably as a result of casein kinase II activity (149). Apparently, intercellular trafficking occurs via a nonclassical Golgiindependent mechanism that may involve the actin cytoskeleton. The utility of VP22 as a transport vector was demonstrated using fusion proteins with a 12-residue epitope tag or the 27-kDa jellyfish green fluorescent protein (GFP). In the former case, transport properties were found using either endogenous synthesis, or by application to the medium and uptake. The GFP fusion protein was expressed in COS-1 cells and was observed to move to surrounding untransfected cells in the same way as wt-VP22. Although intercellular transport of GFP-VP22 constructs was discussed controversially (150), recent reports have confirmed intercellular transport, not only to proliferating cells, but also to terminally differentiated cells (151-154). Furthermore, transport was quantitated (155) and in vivo delivery demonstrated (156). VP22-mediated protein transport has also been used in order to deliver thymidine kinase (TK) to neuroblastomas in live mice, rendering the tumors sensitive to the TK-activated prodrug gangciclovir and causing tumor regression (157). Functional application relevant to gene therapy was shown with a chimaeric protein consisting of VP22 linked to the tumor suppressor protein p53 (158). Not only was spreading between cells seen using this protein, but induction of apoptosis in p53negative human osteosarcoma cells, resulting in a widespread cytotoxic effect. A mutant VP22 protein lacking the C-terminal 34 residues was found to be synthesized in transfected cells at similar levels as wt-VP22; however, it was not detected in the nuclei of surrounding cells. This indicates that the sequence responsible for imparting membrane permeability to the protein is located in the C-terminus and that, as has been found in many similar cases, a short permeable peptide may be derived from the sequence of this protein. As far as can be ascertained, this has not as yet been achieved. Identification of such peptides would be desirable as immunogenicity is likely to be a problem with full-length VP22 chimaeras (134). Viral VP22 is known to be a T-cell antigen (159) and VP22-reactive T-cells may play a role in the control of recurrent HSV infection. Nevertheless, VP22-mediated delivery of transgene products is now considered a promising strategy for successful gene therapy, overcoming current problems associated with limited delivery (160). Apolar Signal Peptide Vectors. Although hydrophobic signal sequences lack primary sequence identity,

Fischer et al.

the critical characteristic which correlates with membrane activity appears to be residue-average hydrophobicity in the h region (161), and neither the positively charged N-terminal segment nor the C-terminal signal peptidase cleavage site are necessary for membrane translocation (162). The h region, comprising 7-16 nonconserved residues, is thus the dominant structure determining membrane-translocating signal sequence function (163, 164) and has been identified in numerous signal peptides (165). Synthetic peptides corresponding to the h region of signal sequences from Kaposi FGF (166), integrin β3 (167), and Caiman crocodylus Ig(v) light chain (168) have been developed as delivery vectors. Kaposi FGF and β3-integrin derived delivery peptides are also known as MPS (membrane permeable sequences) in the literature. As with other peptide vectors, cell permeability is not limited to particular cell types, and membrane translocation appears to be independent of ATP, since cells depleted of their metabolic ATP pool were nevertheless able to internalize vector peptide (166). However, the mechanism of membrane translocation here is clearly different to the cases discussed above, since transport of apolar signal peptide vectors is temperature-dependent (166). Presumably membrane translocation in this case is dependent in some way on membrane fluidity. Again the precise mechanism remains unclear, although it is clear that import is not dependent on caveloae (164). Conformational analysis of a delivery vector constructed by fusing the signal sequence of caiman crocodylus Ig(v) light chain (169) and the NLS of the SV40 large T antigen (170) showed that the peptide adopted a disordered structure in water, R-helical structure in the presence of trifluoroethanol or detergent micelles, but mainly antiparallel β-sheet form in the presence of lipids, regardless of the nature of the phosphate headgroup, and embedded in the lipid cores (171). Unlike other apolar signal peptide vectors, this particular peptide was observed to internalize efficiently into cells at low temperature. The apolar signal peptide vectors have been applied extensively and successfully in dissecting intracellular signaling pathways (172, 173), particularly those concerning intracellular integrin signaling (166, 174) and blocking of signaling to the nucleus through specific NLSs (167). Recent studies with the latter approach also suggest potential in vivo utility of apolar signal peptide vectors (175-177). As with the HIV TAT peptide, large macromolecular cargo can be imported into cells. Thus a 12mer apolar signal peptide vector was fused to the 41kDa protein glutathione S-transferase, and the resulting construct was imported into cells efficiently (178). Although several examples of functional molecular cargoes being imported into living cells using these vectors are documented (164, 179), a question remains concerning their universal applicability. This question concerns intracellular targeting and localization, since hydrophobic signal peptides generally direct the polypeptides they form part of to the endoplasmic reticulum (180). At least in one case (181), where antisense oligonucleotides were fused with these vectors, efficient membrane translocation was observed, but no appreciable antisense effect was obtained. Examination of the cellular distribution revealed endosomal trapping of the conjugates. Interestingly, this was the case regardless of whether a NLS was appended to the vector sequence. In a different approach (182), the K-FGF vector was conjugated with a polycationic linker, which was then complexed electrostatically with an oligonucleotide. In this case cytosolic and nuclear accumulation of the

Reviews

construct after application to cell cultures was shown, although no functional read-out was reported. Fusion Peptide from HIV gp41. Because of their role in cell fusion, the hydrophobic amino-terminal regions of transmembrane proteins of various enveloped viruses, including the orthomyxoviruses, paramyxoviruses, and retroviruses, are known as fusion peptides (183). Viral fusion reactions fall into two classes, low pHdependent (see below) and pH-independent. Viruses with low pH-dependent activity fuse with membranes of acidic endosomes, whereas those with pH-independent activity fuse with both plasma membranes and endosomes. Thus, cells expressing the fusion proteins of simian virus 5, respiratory syncytial virus or HIV fuse at neutral pH. A fusion peptide consisting of 28 contiguous hydrophobic amino acid residues was shown to be present in the HIV gp41 glycoprotein (184), and its function in syncytium formation was demonstrated (185). Certain mutations in the conserved N-terminal fusion domain of gp41 (186, 187), as well as antibodies specific for this domain, inhibited HIV infection, syncytium formation, and HIV cytopathy (188, 189). Furthermore, the ability of synthetic gp41 fusion peptides to perturb and lyse artificial lipid bilayers has been demonstrated and the membranotropic ability shown to be dependent on peptide length, sequence, and temperature (190-192). Using human erythrocyte membranes it was shown that at subhaemolytic doses N-terminal gp41 fusion peptides bind to erythrocytic lipids, whereas at doses of g5 µM haemolysis takes place (193). The potential utility as a cellular delivery vector of a gp41-derived peptide (residues 1-17), also containing the NLS of SV40 large T-antigen (170, 194), was suggested (195). Until recently it has been assumed that peptide vectors had to be tethered covalently to their cargo in order to effect cellular delivery. This concept has now been challenged. The fact that cellular transfection with oligonucleotides can be mediated by cationic liposomes and other cationic amphiphilic species is well established (37). Here, packaging and compaction resulting from electrostatic and hydrophobic interactions with anionic oligonucleotides is important. Similar effects have recently been shown with the above gp41/SV40T vector peptide, when noncovalently complexed with oligonucleotides (196). Some of the claimed advantages over traditional transfection methods of this system are as follows: delivery does not follow the endosomal pathway, it is not cytotoxic, and it is not affected by serum proteins. More surprisingly, noncovalent association of the vector with a hydrophobic peptide has recently been shown to permit cellular delivery (197). High-affinity interaction between the peptide and the vector, presumably involving the hydrophobic N-terminus of the latter, resulting in vector-peptide complexes (ca. 20:1 ratio), were demonstrated. It remains to be seen how general this promising approach is in terms of polypeptide delivery. Amphiphilic Peptide Vectors. Lipid-enveloped viruses infect their host cells by fusing with either the plasma membrane or the endosome membrane after endocytosis of the virion. The former process is mediated by hydrophobic fusion sequences (see above), which are normally expressed on the surface of the fusion proteins, whereas the fusion sequence of, e.g., influenza virus haemagglutinin (HA), is exposed only at the low pH of endosomes, at pH 5-6 (198, 199). The low pH results in a change of conformation of HA leading to the exposure of the amino terminus, which consists of an amphiphilic helix ending in a short hydrophobic sequence of about 10 residues. Short synthetic peptides containing the first


17-23 residues of the HA2 subunit of HA also display pH-dependent lytic properties, with little activity at neutral pH but with >100-fold increase in transfection efficiency at pH 5. The lytic properties of these anionic peptides are only revealed as the Asp and Glu side-chain carboxyl groups are protonated, allowing the peptides to adopt amphiphilic helical structures (200, 201). Oligonucleotide-peptide conjugates containing such HA peptides have been used successfully in order to overcome the frequent phenomenon of endosomal trapping after transfection (39, 202, 203). While certain anionic peptides cause membrane fusion at acidic pH, corresponding cationic peptides may have the same effect at alkaline pH. Thus amphiphilic anionic and cationic peptides formally derived from the influenza virus HA fusion segment, have been proposed as vehicles for oligonucleotide transfection (8, 204, 205). A study examining a large number of influenza fusion sequences, as well as many other different lytic peptides (19), led to the design of the amphipathic endosomolytic vector peptide JTS-1 (GLFEALLELLESLWELLEA), whose application for gene delivery, in conjunction with the synthetic poly-Lys (40, 206, 207) substitute YKAK8WK, has been advocated (208). Many other naturally occurring and designed amphiphilic lytic peptides, including, e.g., ones derived from the wasp venom component mastoparan (209), from the C-terminus of the HIV-1 gp41 protein (210), and from synthetic ion channel peptides (119), are known and have been studied as delivery vectors (134). In many of these constructs containing endosomolytic peptides, DNA-binding and compacting sequences, and NLSs have also been incorporated (11, 211, 212). A particularly interesting system is represented by the so-called GALA (213) and KALA peptides. The latter peptide (WEAKLAKALAKALAKHLAKALAKALKACEA (214)) undergoes a pH-dependent random coil to amphiphilic R-helical conformational change as the pH increases from 5.0 to 7.5. KALA is efficient at causing membrane leakage from neutral and negatively charged liposomes and causes complete leakage of entrapped contents over the pH range of 4.0-8.5. Furthermore, KALA binds to oligonucleotides and plasmid DNA and assists their nuclear delivery when complexes are prepared at a 10/1 +/- charge ratio. In fact such complexes mediated transfection of a variety of cell lines. It seems likely that here direct plasma membrane translocation to the cytosol, as well as endocytic uptake and release, may be involved. For a similar model R-helical amphiphilic peptide (KLALKLALKALKAALKLA, (215, 216)), nonendocytic uptake mechanisms were in fact demonstrated. Further examples of designed cationic amphiphilic R-helical delivery peptides are known (217). Not only do peptide-based oligonucleotide delivery systems now offer a viable alternative to traditional in vitro lipofection methods (33, 34, 36, 218), but synthetic peptide-based gene delivery systems consisting of Lysrich oligonucleotide binding regions and pH-sensitive endosomolytic motifs have been developed for in vivo gene delivery and expression (38). In general it should be remembered, however, that amphiphilic peptides are generally cytolytic and cannot therefore be expected to provide delivery vectors permitting delivery of effector molecules at high therapeutic indeces. Anti-DNA Antibody-Derived Peptide Vectors. The fact that certain anti-DNA Abs are capable of entering living cells has been known for some time (219-221). Studying naturally occurring polyreactive anti-DNA mAbs derived from the spleen cells of a nonimmunized lupus mouse, Avrameas et al. (222) found that many


hybridomas cloned secreted cell-penetrating IgG mAbs. These Abs, or their Fab′ and (Fab′)2 fragments, were capable of penetrating various different cell types through an energy-dependent mechanism, entering the nucleus, and accumulating there. This process did not apparently alter either cell viability or metabolism and internalization was mediated in some way by mAb binding to cell surface structures. Interestingly, only polyreactive antiDNA mAbs could penetrate cells. They appear to represent clones of natural polyreactive autoantibodies that escape normal control and which are expanded in lupus disease. Penetrating mAbs or their fragments were effective vectors for the delivery of haptens, oligonucleotides, and polypeptides (389 > Mr > 40000). Sequencing of three mAbs revealed high homology in the VH CDR2. A synthetic peptide corresponding to one of these CDR2 sequences did not bind antigen or translocate membranes. A peptide corresponding to the VH CDR3, however, had some penetration properties. When both sequences were linked in the form of a peptide, full DNA-binding, polyreactivity, and efficient cell penetration properties were observed. This peptide vector (P3) was capable of carrying molecular cargo into cells in the same way as the parent mAb. It has been speculated that by combining the CDR2 and CDR3 of different penetrating anti-DNA mAbs or their mimotopes (223), peptide vectors with various properties may be designed (222). Incubation of cells with complexes between penetrating mAb and plasmid DNA encoding a tumor antigen resulted in expression of the antigen in no more than 0.1% of cells. It is unclear whether this is due to misrouting of the internalized plasmids or hindrance to internalization arising from structural alterations imposed on the mAbs through plasmid binding. The latter is more probable, as site-directed mutagenesis studies of penetrating anti-DNA mAbs have shown that the same residues are involved in DNA binding and mediation of cell entry (221). Vector P3 was also ineffective in oligonucleotide delivery. Extension of the peptide with 19 Lys residues to achieve DNA condensation (38), however, afforded an efficient gene delivery vehicle. For example, efficient tranfection of cells with plasmids carrying the genes of the green fluorescent protein or luciferase was reported (224). Transportans. Transportan is a 27-residue peptide vector (225). Its sequence is composed of 12 residues derived from the neuropeptide galanin, connected, via a Lys residue, with 14 residues corresponding to the wasp venom peptide mastoparan. The former represents the smallest active galanin-receptor ligand with agonist properties. Mastoparan has the ability to penetrate membranes, where it creates short-lived pores by translocating into the inner leaflet; it inhibits Golgi transport and facilitates the opening of mitochondrial permeability pores. Transportan appears to enter cells in an energyindependent manner unrelated to endocytosis. Like other peptide vectors, it does not seem to be selective for particular cell types. It first localizes to the outer membrane, followed by redistribution into the nuclear membrane and uptake into the nuclei and concentration in the nucleoli. Because of its derivation from bioactive peptides, it can be expected that this vector may have undesirable biological effects. Thus it lowers GTPase activity in Bowes cell membranes with an apparent IC50 of 50 µM, although apparently not exhibiting cytotoxic effects at concentrations below 20 µM. An improved version, termed transportan 2, containing an inactive mastoparan analogue, has been proposed to overcome these problems (226). The utility of transportans as

Fischer et al.

delivery vectors has not as yet been examined extensively. Nevertheless, comparative studies with the Antp vector (227) and using cellular and in vivo delivery of PNAs suggest promise (114). A truncation analysis of transportan has also been reported recently (228). PHARMACOLOGY IN LIVING ANIMALS

The utility of peptidic delivery vectors in the context of cell biology has been demonstrated extensively. However, the ultimate aim of the technology is to provide novel therapeutic modalities for macromolecules. Although the first report on peptide vector-mediated systemic delivery of polypeptides into whole animals dates back to 1994 (118), comparatively few in vivo studies with vector conjugates have been reported since then and more detailed pharmacological studies are required. In the case of the 11mer TAT vector it was demonstrated that a fusion protein with the 120-kD β-galactosidase protein could be delivered in vivo (29). After intraperitoneal injection of this construct into mice, specific galactosidase activity could be developed in all tissues, including the brain. In fact several in vivo protein transduction applications have been reported using this vector; the subject was reviewed recently (135). The pAntp delivery peptide system, as well as several other vectors discussed above, have also been used for in vivo applications (105, 106, 114, 229). The main concern regarding therapeutic utitlity concerns the rapid and highly efficient uptake of vector conjugates by tissues. For example, it was shown that intraperitoneal administration of 0.6 nmol of a HIV TAT peptide-derived vector to mice resulted in complete transduction into all cells present in whole blood after only 30 min exposure, with no additional increases at 60 min postadministration (230). Pharmacokinetically this situation suggests that targeting to tissues in vivo will be difficult and that high doses may be required. Little appears to be known about the inherent toxicity of peptidic delivery vectors; however, cationic peptides such as polylysines are known to be toxic. Thus the potential for rate-limiting toxicity, if high conjugate doses are required, clearly exists. A further area requiring examination concerns the peptidic nature of the delivery vectors in relation to immunogenicity. Since the majority of applications will presumably require repeated administration of conjugates, avoiding the induction of an immune response to peptidic conjugates will be important. One of the advantages with the peptide vectors is the fact that they appear to be able to cross the blood-brain barrier (49, 114). Furthermore, topical applications have recently been shown to hold promise (106, 144). VECTOR CONJUGATION METHODS

Polypeptide-Vector Conjugates. In the majority of vector peptide applications, the effector parts of the conjugates are peptides themselves. In these cases the two sequences are usually synthesized contiguously. This approach is practically feasible for conjugates up to about 50 amino acid residues in length. For longer constructs, genetic engineering methods or chemoselective conjugation can be used. The latter approach is also sometimes preferred for shorter constructs when a modular approach is desired. Apart from ligation techniques, protein conjugation methods are not discussed here as there are excellent treatises on this subject (231). Methods that have found application in the preparation of permeable polypeptide conjugates are summarized in Scheme 1, and some are discussed in more detail below in the context

Reviews


Scheme 1. Chemoselective Conjugation Methods

of oligonucleotide-peptide conjugation. Of particular importance are peptide heterodimers formed through disulfide bond formation (Scheme 1a). Specific and efficient methods for the synthesis of such heterodimers by titration of thiol-activated peptides with thiol-containing peptides have been developed (232). In principle the same considerations as for polypeptide constructs apply for peptide nucleic acid (PNA)-peptide hybrids. These can be obtained by synthesis in tandem (233) or through conjugation (114). Ligation of an aldehyde group in one reactant with a 1,2-aminothiol function in the second reactant is sitespecific and rapid, resulting in a conjugate containing a stable thiazolidine ring (Scheme 1d). This method has been used to prepare functionally active permeable peptides, and it was found that the thiazolidine-linked conjugates were equipotent with those containing a conventional amide linkage (234). The required C-terminal peptide aldehydes were elaborated by periodate oxidation of a 1,2-diaminopropanediol or 1,2-amino alcohol group. Oligonucleotide-Peptide Conjugates. The fact that thiol groups can readily be introduced during solidphase oligonucleotide synthesis at either the 3′-end by using appropriate thiol linkers (235, 236), or 5′-terminally (237-240), has often been exploited in order to obtain conjugates. Thus thiolated oligonucleotides have been conjugated with cysteinyl vector peptides through disulfide bond formation (44, 112, 114, 168, 181, 241). To favor formation of the heterodimeric conjugate in such reactions, disulfide-exchange between the free thiol in one raction partner with a pyridylsulfenyl-activated form in the other is usually employed (242, 243) (Scheme 1a). Maleimide-thiol chemistry (244) provides the required selectivity in order to obtain unambiguous oligonucleotide-peptide hybrids from unprotected precursors in most cases (Scheme 1c). Coupling of thiolated oligonucleotides with maleimido-peptides has thus been used successfully for the synthesis of thioether-conjugated hybrids (245, 246). Specifically, thiolated oligodeoxy-

nucleotides were shown to react selectively and efficiently with maleimide-derivatized 18mer TAT and 22mer Cterminal gp41 vector peptides (247). Alternatively, 5′amino-modified oligonucleotides can be maleimidefunctionalized and then conjugated with Cys-containing peptides (248, 249) or with thiol-peptides obtained from SPPS using a disulfide bond-based linker (250). The latter method was applied to prepare oligonucleotide conjugates with a fusogenic peptide (203). Similar thioether conjugation can be achieved using bromoacetylinstead of maleimide-functionalized peptides (251) (Scheme 1b). Nonselective covalent attachment of peptides to DNA can also be achieved, e.g., by using a method based on the DNA alkylating moiety cyclopropylpyrroloindole (CPI). A bromoacetyl-CPI derivative is first alkylated with a cysteinyl peptide, and the resulting complex is then conjugated to DNA through N-alkylation of purine bases. Unsatisfactory reactivity during macromolecular condensations in solution frequently derives from limited and incompatible solubility of macromolecular substrates. This situation has been shown to be ameliorated to a large extent by employing solid-phase segment condensation methods (252). This approach is applicable to the synthesis of peptide-oligonucleotide hybrids: for example, protected solid phase-bound oligonucleotides can be derivatized 5′-terminally with an aminoalkyl group, followed by amidation with suitably side-chain protected NR-Fmoc-blocked peptide acids. The protected immobilized hybrids are then liberated from the support through appropriate deprotection and cleavage (253). A linkerfree version using a 3′-terminal aminothymidine building block, followed by stepwise Fmoc-based SPPS, has also been reported (254). Although various approaches toward total stepwise solid-phase synthesis of oligonucleotidepeptide hybrids have been taken (255-258), this attractive method of synthesis cannot as yet be regarded as mature enough for general application. Native ligation techniques (259), originally devised for the splicing of unprotected peptide segments in the


chemical synthesis of proteins, have also been adapted to the conjugation of peptides with oligonucleotides (Scheme 1e). In one such approach (260), oligodeoxynucleotides are equipped with a S-tert-butylsulfenylcysteine phosphoester at the 3′ end during solid-phase synthesis. After cleavage and deprotection, the product is condensed with a peptidyl benzyl thioester. The latter is also obtained through solid-phase assembly using an activated thiosuccinate to introduce the thioester moiety. The ligation reaction is catalyzed by the water-soluble reducing agent tris(2-carboxyethyl)phosphine, and an auxiliary thiol nucleophile is employed in order to facilitate thiol exchange. The reaction proceeds through nucleophilic attack of the oligonucleotide Cys thiol group on the peptide thioester. The resulting thioester ligation product undergoes rapid intramolecular rearrangement to the amide, due to the favorable geometry of the Cys amino and thioester carbonyl groups. A similar approach was also reported (261), in which the oligonucleotide is equipped 5′-terminally with a guanosine-5′-phosphorothioate. A phenyl thioester group was then introduced via S-alkylation of the terminal phosphorothioate moiety with phenyl R-bromothioacetate. The 5′-thioester oligonucleotide product was then transthioesterified with a stoichiometric amount of a peptide containing N-terminal Cys. As in the preceding example, facile intramolecular attack of the free peptidyl R-amino group on the newly formed thioester bond led to rearrangement to the stable amide-bonded conjugate. In effect, the ligation mechanism provides entropy activation for the eventual amide bond formation (262) and is therefore particularly attractive for condensation of macromolecular species. Ligations can be carried out in aqueous or mixed aqueous/ organic media, even in the presence of powerful solubilizing agents such as urea or guanidinium hydrochloride. Furthermore, due to the near-complete chemoselectivity of native ligation, fully deprotected peptide and oligonucleotide components can be employed. Template-directed ligation of peptides to oligonucleotides has also been reported (263): An activated peptide thioester was reacted with a 5′-thiolated oligodeoxynucleotide1, resulting in a thioester linked peptideoligodeoxynucleotide1. This was then brought into close proximity with a 3′-amino-modified oligodeoxynucleotide2 by hybridization to adjacent sites on a complementary oligodeoxynucleotide template. The nucleophilic amine then attacked the thioester carbonyl group of the peptide-oligodeoxynucleotide1, forming an amide bond between the oligodeoxynucleotide2 and the peptide, and releasing the 5′-thiol-oligodeoxynucleotide1. This method relies on a template to promote the ligation reaction and to confer specificity for a particular combination of oligonucleotides and peptide. Because here ligation is carried out in an addressable format, different combinations of oligonucleotides and peptides should be able to be joined simultaneously. The full scope of this approach does not appear to have been explored to date. Selective conjugation of biosynthetic oligonucleotides with peptides is more difficult. In the case of RNA, periodate oxidation of the 2′,3′-cis diol group of the 5′terminal ribose and coupling of, for example, peptide hydrazides or thiosemicarbazides to the dialdehyde product would appear to offer a possibility (264, 265). It has been shown that modified nucleotides can be incorporated enzymatically into both RNA and DNA (266, 267). It should therefore be possible similarly to introduce peptides, suitably modified with TTP or UTP moieties. A synthetic route for the preparation of peptides carrying

Fischer et al.

such a group, linked through an acylphosphoramidate linkage, appears to be available (268). CONCLUSIONS

While the in vitro and in vivo cellular delivery of membrane-impermeable therapeutic agents, including inherently selective macromolecules such as proteins and oligonucleotides, now seems feasible, the next question to address will be how such agents might be delivered in a tissue- and cell type-selective manner. Here too, significant advances have been made (134, 269, 270), again using peptide-based approaches. In particular, targeted nonviral systems for gene transfer have shown promise (271-273). In fact it may soon prove possible to combine cellular delivery and targeting functions using truly modular and discriminating delivery vector systems. Despite these prospects, more detailed answers to many mechanistic questions are still required. Thus it remains unclear in most cases if and to what extent target cells are damaged by the delivery vectors. Very little is as yet known about the ability of delivery vector conjugates to penetrate into tissues, e.g., tumors. Also, intrinsic cell selectivity has not been addressed in sufficient detail. Generally absorption, distribution, metabolism, immunogenicity, and cellular fate of delivery peptide vector conjugates will need to be studied in depth before the current promise of these agents can be realized for therapeutic applications. LITERATURE CITED (1) Lipinski, C. A., Lombardo, F., Dominy, B. W., and Feeney, P. J. (1997) Experimental and computational approaches to estimate solubility and permeability in drug discovery and development setting. Adv. Drug Delivery Rev. 23, 3-25. (2) Allen, T. M. (1998) Liposomal drug formulations: rationale for development and what we can expect for the future. Drugs 56, 747-756. (3) Drummond, D. C., Meyer, O., Hong, K., Kirpotin, D. B., and Papahadjopoulos, D. (1999) Optimizing liposomes for delivery of chemotherapeutic agents to solid tumors. Pharmacol. Rev. 51, 691-743. (4) Tachibana, R., Harashima, H., Shono, M., Azumano, M., Niwa, M., Futaki, S., and Kiwada, H. (1998) Intracellular regulation of macromolecules using pH-sensitive liposomes and nuclear localization signal: qualitative and quantitative evaluation of intracellular trafficking. Biochem. Biophys. Res. Commun. 251, 538-544. (5) Lutwyche, P., Cordeiro, C., Wiseman, D. J., St-Louis, M., Uh, M., Hope, M. J., Webb, M. S., and Finlay, B. B. (1998) Intracellular delivery and antibacterial activity of gentamicin encapsulated in pH-sensitive liposomes. Antimicrob. Agents Chemother. 42, 2511-2520. (6) Maurer, N., Mori, A., Palmer, L., Monck, M. A., Mok, K. W. C., Mui, B., Akhong, Q. F., and Cullis, P. R. (1999) Lipidbased systems for the intracellular delivery of genetic drugs. Mol. Membr. Biol. 16, 129-140. (7) El Ouahabi, A., Thiry, M., Pector, V., Fuks, R., Ruysschaert, J. M., and Vandenbranden, M. (1997) The role of endosome destabilizing activity in the gene transfer process mediated by cationic lipids. FEBS Lett. 414, 187-192. (8) Kamata, H., Yagisawa, H., Takahashi, S., and Hirata, H. (1994) Amphiphilic peptide enhance the efficiency of liposomemediated DNA transfection. Nucleic Acids Res. 22, 536-537. (9) Kichler, A., Mechtler, K., Behr, J.-P., and Wagner, E. (1997) Influence of membrane-active peptides on lipospermine/DNA complex mediated gene transfer. Bioconjugate Chem. 8, 213221. (10) Plank, C., Zatloukal, K., Cotten, M., Mechtler, K., and Wagner, E. (1992) Gene transfer into hepatocytes using asialoglycoprotein receptor mediated endocytosis of DNA

Reviews complexed with an artificial tetra-antennary galactose ligand. Bioconjugate Chem. 3, 533-539. (11) Zanta, M. A., Belguise-Valladier, P., and Behr, J. P. (1999) Gene delivery: a single nuclear localization signal peptide is sufficient to carry DNA to the cell nucleus. Proc. Natl. Acad. Sci. U.S.A. 96, 91-96. (12) Lindgren, M., Ha¨llbrink, M., Prochiantz, A., and Langel, U ¨ . (2000) Cell-penetrating peptides. Trends Pharmacol. Sci. 21, 99-103. (13) Scheller, A., Wiesner, B., Melzig, M., Bienert, M., and Oehlke, J. (2000) Evidence for an amphipathicity independent cellular uptake of amphipathic cell-penetrating peptides. Eur. J. Biochem. 267, 6043-6049. (14) Pugsley, A. P. (1996) Bacterial toxins deliver the goods. Proc. Natl. Acad. Sci. U.S.A. 93, 8155-8156. (15) Boquet, P., and Duflot, E. (1982) Tetanus toxin fragment forms channels in lipid vesicles at low pH. Proc. Natl. Acad. Sci. U.S.A. 79, 7614-7618. (16) Ballard, J. D., Collier, R. J., and Starnbach, M. N. (1996) Anthrax toxin-mediated delivery of a cytotoxic T-cell epitope in vivo. Proc. Natl. Acad. Sci. U.S.A. 93, 12531-12534. (17) Rossjohn, J., Feil, S. C., McKinstry, W. J., Tweten, R. K., and Parker, M. W. (1997) Structure of a cholesterol-binding, thiol-activated cytolysin and a model of its membrane form. Cell 89, 685-692. (18) Stenmark, H., Moskaug, J. O., Madshus, I. H., Sandvig, K., and Olsnes, S. (1991) Peptides fused to the amino-terminal end of diphtheria toxin are translocated to the cytosol. J. Cell. Biol. 113, 1025-1032. (19) Ojcius, D. M., and Young, J. D. E. (1991) Cytolytic poreforming proteins and peptides: is there a common structural motif? Trends Biochem. Sci. 16, 225-229. (20) Fujii, G., Horvath, S., Woodward, S., Eiserling, F., and Eisenberg, D. (1992) A molecular model for membrane fusion based on solution studies of an amphiphilic peptide from HIV gp41. Protein Sci. 1, 1454-1464. (21) Brandsch, M., Brandsch, C., Prasad, P. D., Ganapathy, V., Hopfer, U., and Leibach, F. H. (1995) Identification of a renal cell line that constitutively expresses the kidney-specific highaffinity H+/peptide cotransporter. FASEB J. 9, 1489-1496. (22) Stevenson, C. L., Augustijns, P. F., and Hendren, R. W. (1999) Use of Caco-2 cells and LC/MS/MS to screen a peptide combinatorial library for permeable structures. Int. J. Pharmaceut. 177, 103-115. (23) Souroujon, M. C., and Mochly-Rosen, D. (1998) Peptide modulators of protein-protein interactions in intracellular signaling. Nat. Biotechnol. 16, 919-924. (24) O’Brian, C. A., Ward, N. E., Liskamp, R. M., de Bont, D. B., Earnest, L. E., van Boom, J. H., and Fan, D. (1991) A novel N-myristylated synthetic octapeptide inhibits protein kinase C activity and partially reverses murine fibrosarcoma cell resistance to adriamycin. Invest. New Drugs 9, 169-179. (25) Johnson, J. A., Gray, M. O., Karliner, J. S., Chen, C.-H., and Mochly-Rosen, D. (1996) An improved permeabilization protocol for the introduction of peptides into cardiac mycocytes. Circ. Res. 79, 1086-1099. (26) Selzman, C. H., Shames, B. D., Reznikov, L. L., Miller, S. A., Meng, X., Barton, H. A., Werman, A., Harken, A. H., Dinarello, C. A., and Banerjee, A. (1999) Liposomal delivery of purified inhibitory-κBR inhibits tumor necrosis factor-Rinduced human vascular smooth muscle proliferation. Circ. Res. 84, 867-875. (27) Guan, H. H., Budzynski, W., Koganty, R. R., Krantz, M. J., Reddish, M. A., Rogers, J. A., Longenecker, B. M., and Samuel, J. (1998) Liposomal formulations of synthetic MUC1 peptides: effects of encapsulation versus surface display of peptides on immune responses. Bioconjugate Chem. 9, 451458. (28) Kato, D., Miyazawa, K., Ruas, M., Starborg, M., Wada, I., Oka, T., Sakai, T., Peters, G., and Hara, E. (1998) Features of replicative senescence induced by direct addition of antennapedia-p16INK4A fusion protein human diploid fibroblasts. FEBS Lett. 427, 203-208. (29) Schwarze, S. R., Ho, A., Vocero-Akbani, A., and Dowdy, S. F. (1999) In vivo protein transduction: delivery of a biologically active protein into the mouse. Science 285, 1569-1572.

Bioconjugate Chem., Vol. 12, No. 6, 2001 835 (30) Perales, J. C., Ferkol, T., Molas, M., and Hanson, R. W. (1994) An evaluation of receptor-mediated gene transfer using synthetic DNA-ligand complexes. Eur. J. Biochem. 226, 255266. (31) Leamon, C. P., and Low, P. S. (1991) Delivery of macromolecules into living cells: a method that exploits folate receptor endocytosis. Proc. Natl. Acad. Sci. U.S.A. 88, 55725576. (32) Wolfert, M. A., and Seymour, L. W. (1996) Atomic force microscopic analysis of the influence of the molecular weight of poly-L-lysine on the size of polyelectrolyte complexes formed with DNA. Gene Ther. 3, 269-273. (33) Boussif, O., Lezoualc’h, F., Zanta, M. A., Mergny, M. D., Scherman, D., Demeneix, B., and Behr, J.-P. (1995) A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine. Proc. Natl. Acad. Sci. U.S.A. 92, 7297-7301. (34) Behr, J.-P., Demeneix, B., Loeffler, J.-P., and Perez-Mutul, J. (1989) Efficient gene transfer into mammalian primary endocrine cells with lipopolyamine-coated DNA. Proc. Natl. Acad. Sci. U.S.A. 86, 6982-6986. (35) Felgner, J. H., Kumar, R., Sridhar, C. N., Wheeler, C. J., Tsai, Y. J., Border, R., Ramsey, P., Martin, M., and Felgner, P. L. (1994) Enhanced gene delivery and mechanism studies with a novel series of cationic lipid formulations. J. Biol. Chem. 269, 2550-2561. (36) Felgner, P. L., Gadek, T. R., Holm, M., Roman, R., Chan, H. W., Wenz, M., Northrop, J. P., Ringold, G. M., and Danielsen, M. (1987) Lipofection: a highly efficient, lipidmediated DNA-transfection procedure. Proc. Natl. Acad. Sci. U.S.A. 84, 7413-7417. (37) Behr, J.-P. (1994) Gene transfer with synthetic cationic amphiphiles: prospects for gene therapy. Bioconjugate Chem. 5, 382-389. (38) Wadhwa, M. S., Collard, W. T., Adami, R. C., McKenzie, D. L., and Rice, K. G. (1997) Peptide-mediated gene delivery: influence of peptide structure on gene expression. Bioconjugate Chem. 8, 81-88. (39) Plank, C., Oberhauser, B., Mechtler, K., Koch, C., and Wagner, E. (1994) The influence of endosome-disruptive peptides on gene transfer using synthetic virus-like gene transfer systems. J. Biol. Chem. 269, 12918-12924. (40) Plank, C., Tang, M. X., Wolfe, A. R., and Szoka, F. C. (1999) Branched cationic peptides for gene delivery: role of type and number of cationic residues in formation and in vitro activity of DNA polyplexes. Hum. Gene Ther. 10, 319-332. (41) Wagner, E., Zenke, M., Cotten, M., Beug, H., and Birnstiel, M. L. (1990) Transferrin-polycation conjugates as carriers for DNA uptake into cells. Proc. Natl. Acad. Sci. U.S.A. 87, 34103414. (42) Zauner, W., Kichler, A., Schmidt, W., Mechtler, K., and Wagner, E. (1997) Glycerol and polylysine synergize in their ability to rupture vesicular membranes: a mechanism for increased transferrin-polylysine-mediated gene transfer. Exp. Cell Res. 232, 137-145. (43) Zauner, W., Kichler, A., Schmidt, W., Sinski, A., and Wagner, E. (1996) Glycerol enhancement of ligand-polylysine/ DNA transfection. BioTechniques 20, 905-913. (44) Allinquant, B., Hantraye, P., Mailleux, P., Moya, K., Buillot, C., and Prochiantz, A. (1995) Downregulation of amyloid precursor protein inhibits neurite outgrowth in vitro. J. Cell. Biol. 128, 919-927. (45) Troy, C. M., Derossi, D., Prochiantz, A., Greene, L. A., and Shelanski, M. L. (1996) Downregulation of Cu/Zn superoxide dismutase leads to cell death via the nitric oxide-peroxynitrite pathway. J. Neurosci. 16, 253-261. (46) Josephson, L., Tung, C.-H., Moore, A., and Weissleder, R. (1999) High-efficiency intracellular magnetic labeling with novel superparamagnetic-Tat peptide conjugates. Bioconjugate Chem. 10, 186-191. (47) Lewin, M., Carlesso, N., Tung, C. H., Tang, X. W., Cory, D., Scadden, D. T., and Weissleder, R. (2000) Tat peptidederivatized magnetic nanoparticles allow in vivo tracking and recovery of progenitor cells. Nat. Biotechnol. 18, 410-414. (48) Temsamani, J., Scherrmann, J. M., Rees, A. R., and Kaczorek, M. (2000) Brain drug delivery technologies: novel

836 Bioconjugate Chem., Vol. 12, No. 6, 2001 approaches for transporting therapeutics. Pharm. Sci. Technol. Today 3, 155-162. (49) Rousselle, C., Smirnova, M., Clair, P., Lefauconnier, J.M., Chavanieu, A., Calas, B., Scherrmann, J.-M., and Temsamani, J. (2001) Enhanced delivery of doxorubicin into the brain via a peptide-vector- mediated strategy: saturation kinetics and specificity. J. Pharmacol. Exp. Ther. 296, 124131. (50) Harwig, S. S. L., Swiderek, K. M., Lee, T. D., and Lehrer, R. I. (1995) Determination of disulfide bridges in protegrin PG-2, an anti-microbial peptide from porcine leukocytes. J. Pept. Sci. 1, 207-215. (51) Mangoni, M. E., Aumelas, A., Charnet, P., Roumestand, C., Chiche, L., Despaux, E., Grassy, G., Calas, B., and Chavanieu, A. (1996) Change in membrane permeability induced by protegrin 1: implication of disulfide bridges for pore formation. FEBS Lett. 383, 93-98. (52) Rousselle, C., Clair, P., Lefauconnier, J.-M., Kaczorek, M., Scherrmann, J.-M., and Temsamani. J. (2000) New advances in the transport of doxorubicin through the blood-brain barrier by a peptide vector-mediated strategy. Mol. Pharmacol. 57, 679-686. (53) Futaki, S., Suzuki, T., Ohashi, W., Yagami, T., Tanaka, S., Ueda, K., and Sugiura, Y. (2001) Arginine-rich peptides: an abundant source of membrane-permeable peptides having potential as carriers for intracellular protein delivery. J. Biol. Chem. 276, 5836-5840. (54) Mi, Z., Mai, J., Lu, X., and Robbins, P. D. (2000) Characterization of a class of cationic peptides able to facilitate efficient protein transduction in vitro and in vivo. Mol. Ther. 2, 339-347. (55) Richardson, W. D., Roberts, B. L., and Smith, A. E. (1986) Nuclear location signals in polyoma virus large-T. Cell 44, 77-85. (56) Wychowski, C., Benichou, D., and Girard, M. (1986) A domain of SV40 capsid polypeptide VP1 that specifies migration into the cell nucleus. EMBO J. 5, 2569-2576. (57) Kardinal, C., Konkol, B., Schulz, A., Posern, G., Lin, H., Adermann, K., Eulitz, M., Estrov, Z., Talpaz, M., Arlinghaus, R. B., and Feller, S. M. (2000) Cell-penetrating SH3 domain blocker peptides inhibit proliferation of primary blast cells from CML patients. FASEB J. 14, 1529-1538. (58) Mihara, H., and Kaiser, E. T. (1988) A chemically synthesized Antennapedia homeo domain binds to a specific DNA sequence. Science 242, 925-927. (59) Joliot, A., Pernelle, C., Deagostini-Bazin, H., and Prochiantz, A. (1991) Antennapedia homeobox peptide regulates neural morphogenesis. Proc. Natl. Acad. Sci. U.S.A. 88, 1864-1868. (60) Joliot, A. H., Triller, A., Volovitch, M., Pernelle, C., and Prochiantz, A. (1991) R-2,8-Polysialic acid is the neuronal surface receptor of antennapedia homeobox peptide. New Biol. 3, 1121-1134. (61) Perez, F., Joliot, A., Bloch-Gallego, E., Zahraoui, A., Triller, A., and Prochiantz, A. (1992) Antennapedia homeobox as a signal for the cellular internalization and nuclear addressing of a small exogenous peptide. J. Cell Sci. 102, 717-722. (62) Fenton, M., Bone, N., and Sinclair, A. J. (1998) The efficient and rapid import of a peptide into primary B and T lymphocytes and a lymphoblastoid cell line. J. Immunol. Methods 212, 41-48. (63) Niidome, T., Okuda, T., Hatakeyama, T., and Aoyagi, H. (2000) in Peptide Science 1999 (Fujii, N., Ed.) pp 239-240, The Japanese Peptide Society. (64) Gehring, W. J., Muller, M., Affolter, M., Percival-Smith, A., Billeter, M., Qian, Y. Q., Otting, G., and Wuthrich, K. (1990) The structure of the homeodomain and its functional implications. Trends Genet. 6, 323-329. (65) Prochiantz, A. (1999) Homeodomain-derived peptides. In and out of cells. Ann. New York Acad. Sci. 886, 172-179. (66) Le Roux, I., Duharcourt, S., Volovitch, M., Prochiantz, A., and Ronchi, E. (1995) Promoter-specific regulation of gene expression by an exogenously added homedomain that promotes neurite growth. FEBS Lett. 368, 311-314. (67) Derossi, D., Joliot, A. H., Chassaing, G., and Prochiantz, A. (1994) The third helix of the Antennapedia homeodomain

Fischer et al. translocates through biological membranes. J. Biol. Chem. 269, 10444-10450. (68) Derossi, D., Calvet, S., Trembleau, A., Brunissen, A., Chassaing, G., and Prochiantz, A. (1996) Cell internalization of the third helix of the Antennapedia homeodomain is receptor-independent. J. Biol. Chem. 271, 18188-18193. (69) Brugidou, J., Legrand, C., Mery, J., and Rabie, A. (1995) The retro-inverso form of a homeobox-derived short peptide is rapidly internalised by cultured neurones: a new basis for an efficient intracellular delivery system. Biochem. Biophys. Res. Commun. 214, 685-693. (70) Williams, E. J., Dunican, D. J., Green, P. J., Howell, F. V., Derossi, D., Walsh, F. S., and Doherty, P. (1997) Selective inhibition of growth factor-stimulated mitogenesis by a cellpermeable Grb2-binding peptide. J. Biol. Chem. 272, 2234922354. (71) Nur-E-Kamal, M. S. A., Kamal, J. M., Qureshi, M. M., and Maruta, H. (1999) The CDC42-specific inhibitor derived from ACK-1 blocks v-Ha-Ras-induced transformation. Oncogene 18, 7787-7793. (72) Derossi, D., Chassaing, G., and Prochiantz, A. (1998) Trojan peptides: the penetratin system for intracellular delivery. Trends Cell Biol. 8, 84-87. (73) Fischer, P. M., Zhelev, N. Z., Wang, S., Melville, J. E., Fåhraeus, R., and Lane, D. P. (1999) Structure-activity relationship of truncated and substituted analogues of the intracellular delivery vector Penetratin. J. Pept. Res. 55, 163172. (74) Garcia-Echeverria, C., Jiang, L., Ramsey, T. M., Sharma, S. K., and Chen, Y. N. P. (2001) A new Antennapedia-derived vector for intracellular delivery of exogenous compounds. Bioorg. Med. Chem. Lett. 11, 1363-1366. (75) Chatelin, L., Volovitch, M., Joliot, A. H., Perez, F., and Prochiantz, A. (1996) Transcription factor hoxa-5 is taken up by cells in culture and conveyed to their nuclei. Mech. Dev. 55, 111-117. (76) Han, K., Jeon, M.-J., Kim, K.-A., Park, J., and Choi, S. Y. (2000) Efficient intracellular delivery of GFP by homeodomains of Drosophila Fushi-tarazu and Engrailed proteins. Mol. Cells 10, 728-732. (77) Lindberg, M., and Graslund, A. (2001) The position of the cell penetrating peptide penetratin in SDS micelles determined by NMR. FEBS Lett. 497, 39-44. (78) Berlose, J. P., Convert, O., Derossi, D., Brunissen, A., and Chassaing, G. (1996) Conformational and associative behaviours of the third helix of antennapedia homeodomain in membrane-mimetic environments. Eur. J. Biochem. 242, 372-386. (79) Bellet-Amalric, E., Blaudez, D., Desbat, B., Graner, F., Gauthier, F., and Renault, A. (2000) Interaction of the third helix of Antennapedia homeodomain and a phospholipid monolayer, studied by elliptometry and PM-IRRAS at the air-water interface. Biochim. Biophys. Acta 1467, 131-143. (80) Magzoub, M., Kilk, K., Eriksson, L. E. G., Langel, U., and Graslund, A. (2001) Interaction and structure induction of cell-penetrating peptides in the presence of phospholipid vesicles. Biochim. Biophys. Acta 1512, 77-89. (81) Drin, G., Mazel, M., Clair, P., Mathieu, D., Kaczorek, M., and Temsamani, J. (2001) Physicochemical requirements for cellular uptake of pAntp peptide. Role of lipid-binding affinity. Eur. J. Biochem. 268, 1304-1314. (82) Thoren, P. E. G., Persson, D., Karlsson, M., and Norden, B. (2000) The Antennapedia peptide penetratin translocates across lipid bilayers - the first direct observation. FEBS Lett. 482, 265-268. (83) Drin, G., Demene, H., Temsamani, J., and Brasseur, R. (2001) Translocation of the pAntp peptide and Its amphipathic analogue AP-2AL. Biochemistry 40, 1824-1834. (84) Prochiantz, A. (2000) Messenger proteins: homeoproteins, TAT and others. Curr. Opin. Cell Biol. 12, 400-406. (85) Maizel, A., Bensaude, O., Prochiantz, A., and Joliot, A. (1999) A short region of its homeodomain is necessary for Engrailed nuclear export and secretion. Development 126, 3183-3190. (86) Joliot, A., Maizel, A., Rosenberg, D., Trembleau, A., Dupas, S., Volovitch, M., and Prochiantz, A. (1998) Identification of

Reviews a signal sequence necessary for the unconventional secretion of Engrailed homeoprotein. Curr. Biol. 8, 856-863. (87) Mutoh, M., Lung, F. D., Long, Y. Q., Roller, P. P., Sikorski, R. S., and O’Connor, P. M. (1999) A p21(Waf1/Cip1)carboxylterminal peptide exhibited cyclin-dependent kinase-inhibitory activity and cytotoxicity when introduced into human cells. Cancer Res. 59, 3480-34888. (88) Chen, Y.-N. P., Sharma, S. K., Ramsey, T. M., Jiang, L., Martin, M. S., Baker, K., Adams, P. D., Bair, K. W., and Kaelin, W. G. (1999) Selective killing of transformed cells by cyclin/cyclin-dependent kinase 2 antagonists. Proc. Natl. Acad. Sci. U.S.A. 96, 4325-4329. (89) Bonfanti, M., Taverna, S., Salmona, M., D’Incalci, M., and Broggini, M. (1997) p21WAF1-derived peptides linked to an internalization peptide inhibit human cancer cell growth. Cancer Res. 57, 1442-1446. (90) Ball, K. L., Lain, S., Fåhraeus, R., Smythe, C., and Lane, D. P. (1996) Cell-cycle arrest and inhibition of Cdk4 activity by small peptides based on the carboxy-terminal domain of p21WAF1. Curr. Biol. 7, 71-80. (91) Lin, X., Nelson, P., and Gelman, I. H. (2000) SSeCKS, a major protein kinase C substrate with tumor suppressor activity, regulates G1/S progression by controlling the expression and cellular compartmentalization of cyclin D. Mol. Cell. Biol. 20, 7259-7272. (92) Holinger, E. P., Chittenden, T., and Lutz, R. J. (1999) Bak BH3 peptides antagonize Bcl-xL function and induce apoptosis through cytochrome c-independent activation of caspases. J. Biol. Chem. 274, 13298-13304. (93) Riedel, H., Yousaf, N., Zhao, Y., Dai, H., Deng, Y., and Wang, J. (2000) PSM, a mediator of PDGF-BB-, IGF-I-, and insulin-stimulated mitogenesis. Oncogene 19, 39-50. (94) Hall, H., Williams, E. J., Moore, S. E., Walsh, F. S., Prochiantz, A., and Doherty, P. (1996) Inhibition of FGFstimulated phosphatidylinositol hydrolysis and neutrite outgrowth by a cell-membrane premeable phosphopeptide. Curr. Biol. 6, 580-587. (95) Wang, J., Dai, H., Yousaf, N., Moussaif, M., Deng, Y., Boufelliga, A., Swamy, O. R., Leone, M. E., and Riedel, H. (1999) Grb10, a positive, stimulatory signaling adapter in platelet-derived growth factor BB-, insulin-like growth factor I-, and insulin-mediated mitogenesis. Mol. Cell. Biol. 19, 6217-6228. (96) Bandara, L. R., Girling, R., and La Thangue, N. B. (1997) Apoptosis induced in mammalian cells by small peptides that funcionally antagonize the Rb-regulated E2F transcription factor. Nat. Biotechnol. 15, 896-901. (97) Cussac, D., Vidal, M., Leprince, C., Liu, W.-Q., Cornille, F., Tiraboschi, G., Roques, B. P., and Garbay, C. (1999) A Sos-derived peptidimer blocks the Ras signaling pathway by binding both Grb2 SH3 domains and displays antiproliferative activity. FASEB J. 13, 31-38. (98) Fåhraeus, R., Lain, S., Ball, K. L., and Lane, D. P. (1998) Characterization of the cyclin-dependent kinase inhibitory domain of the INK4 family as a model for a synthetic tumour suppressor molecule. Oncogene 16, 587-596. (99) Fåhraeus, R., Paramio, J. M., Ball, K. L., Lain, S., and Lane, D. P. (1996) Inhibition of pRb phosphorylation and cellcycle progression by a 20-residue peptide derived from p16CDKN2/INK4A. Curr. Biol. 6, 84-91. (100) Kim, A. L., Raffo, A. J., Brandt-Rauf, P. W., Pincus, M. R., Monaco, R., Abarzui, P., and Fine, R. L. (1999) Conformational and molecular basis for induction of apoptosis by a p53 C-terminal peptide in human cancer cells. J. Biol. Chem. 274, 34924-34931. (101) Herbert, T. P., Fåhraeus, R., Prescott, A., Lane, D. P., and Proud, C. G. (2000) Rapid induction of apoptosis mediated by peptides that bind initiation factor eIF4E. Curr. Biol. 10, 793-796. (102) Giorello, L., Clerico, L., Pescarolo, M. P., Vikhanskaya, F., Salmona, M., Colella, G., Bruno, S., Mancuso, T., Bagnasco, L., Russo, P., and Parodi, S. (1998) Inhibition of cancer cell growth and c-Myc transcriptional activity by a c-Myc helix 1-type peptide fused to an internalization sequence. Cancer Res. 58, 3654-3659.

Bioconjugate Chem., Vol. 12, No. 6, 2001 837 (103) Fåhraeus, R., and Lane, D. P. (1999) The p16INK4a tumour suppressor protein inhibits Rvβ3 integrin-mediated cell spreading on vitronectin by blocking PKC-dependent localization of Rvβ3 to focal contacts. EMBO J. 18, 2106-2118. (104) Peck, D., and Isacke, C. M. (1998) Hyaluronan-dependent cell migration can be blocked by a CD44 cytoplasmic domain peptide containing a phosphoserine at position 325. J. Cell Sci. 111, 1595-1601. (105) Schutze-Redelmeier, M. P., Gournier, H., Garcia-Pons, F., Moussa, M., Joliot, A. H., Volovitch, M., Prochiantz, A., and Lemonnier, F. A. (1996) Introduction of exogenous antigens into the MHC class I processing and presentation pathway by Drosophila antennapedia homeodomain primes cytotoxic T cells in vivo. J. Immunol. 157, 650-655. (106) May, M. J., D’Acquisto, F., Madge, L. A., Glo¨ckner, J., Pober, J. S., and Ghosh, S. (2000) Selective inhibition of NFκB activation by a peptide that blocks the interaction of NEMO with the IκB kinase complex. Science 289, 1550-1554. (107) Liu, G. S., Cohen, M. V., Mochly-Rosen, D., and Downey, J. M. (1999) Protein kinase C- is responsible for the protection of preconditioning in rabbit cardiomyocytes. J. Mol. Cell. Cardiol. 31, 1937-1948. (108) Hildt, E., and Oess, S. (1999) Identification of Grb2 as a novel binding partner of tumor necrosis factor (TNF) receptor I. J. Exp. Med. 189, 1707-1714. (109) Troy, C. M., Stefanis, L., Prochiantz, A., Greene, L. A., and Shelanski, M. L. (1996) The contrasting roles of ICE family proteases and interleukin-1-β on apoptosis induced by trophic factor withdrawal and by copper/zinc superoxide dismutase down-regulation. Proc. Natl. Acad. Sci. U.S.A. 93, 5635-5640. (110) Theódore, L., Derossi, D., Chassaing, G., Llirbat, B., Kubes, M., Jordan, P., Chneiweiss, P., Godement, P., and Prochiantz, A. (1995) Intraneuronal delivery of protein kinase C pseudosubstrate leads to growth cone collapse. J. Neurosci. 15, 7158-7167. (111) Aldrian-Herrada, G., Desarmeńien, M. G., Orcel, H., Boissin-Agasse, L., Me´ry, J., Brugidou, J., and Rabie´, A. (1998) A peptide nucleic acid (PNA) is more rapidly internalized in cultured neurons when coupled to a retro-inverso delivery peptide. The antisense activity depresses the target mRNA and protein in magnocellular oxytocin neurons. Nucleic Acids Res. 26, 4910-4916. (112) Astriab-Fisher, A., Sergueev, D. S., Fisher, M., Ramsay Shaw, B., and Juliano, R. L. (2000) Antisense inhibition of P-glycoprotein expression using peptide-oligonucleotide conjugates. Biochem. Pharmacol. 60, 83-90. (113) Villa, R., Folini, M., Lualdi, S., Veronese, S., Daidone, M. G., and Zaffaroni, N. (2000) Inhibition of telomerase activity by a cell-penetrating peptide nucleic acid construct in human melanoma cells. FEBS Lett. 473, 241-248. (114) Pooga, M., Soomets, U., Hallbrink, M., Valkna, A., Saar, K., Rezaei, K., Kahl, U., Hao, J.-X., Xu, X.-J., WiesenfeldHallin, Z., Hokfelt, T., Bartfai, T., and Langel. U. (1998) Cell penetrating PNA constructs regulate galanin receptor levels and modify pain transmission in vivo. Nat. Biotechnol. 16, 857-861. (115) Frankel, A. D., and Pabo, C. O. (1988) Cellular uptake of the tat protein from human immunodeficiency virus. Cell 55, 1189-1193. (116) Green, M., and Loewenstein, P. M. (1988) Autonomous functional domains of chemically synthesized human immunodeficiency virus Tat trans-activator protein. Cell 55, 11791188. (117) Vives, E., Charneau, P., Van Rietschoten, J., Rochat, H., and Bahraoui, E. (1994) Effects of the tat basic domain on human immunodeficiency virus type 1 transactivation, using chemically synthesized tat protein and tat peptides. J. Gen. Virol. 68, 3343-3353. (118) Fawell, S., Seery, J., Daikh, Y., Moore, C., Chen, L. L., Pepinsky, B., and Barsoum, J. (1994) Tat-mediated delivery of heterologous proteins into cells. Proc. Natl. Acad. Sci. U.S.A. 91, 664-668. (119) Anderson, D. C., Manger, R., Schroeder, J., Woodle, D., Barry, M., Morgan, A. C., and Fritzberg, A. R. (1993) Enhanced in vitro tumour cell retention and internalization

838 Bioconjugate Chem., Vol. 12, No. 6, 2001 of antibody derivatized with synthetic peptides. Bioconjugate Chem. 4, 10-18. (120) Anderson, D. C., Nichols, E., Manger, R., Woodle, D., Barry, M., and Fritzberg, A. R. (1993) tumor cell retention of antibody Fab fragments is enhanced by attached HIV tat protein-derived peptide. Biochem. Biophys. Res. Commun. 194, 876-884. (121) Loret, E. P., Vives, E., Ho, P. S., Rochat, H., Van Rietschoten, J., and Johnson, W. C. (1991) Activating region of HIV-1 tat protein: vacuum UV circular dichroism and energy minimization. Biochemistry 30, 6013-6023. (122) Ruben, S., Perkins, A., Purcell, R., Joung, K., Sia, R., Burghoff, R., Haseltine, W. A., and Rosen, C. A. (1989) Structural and functional characterization of human immunodeficiency virus tat protein. J. Virol. 63, 1-8. (123) Efthymiadis, A., Briggs, L. J., and Jans, D. A. (1998) The HIV-1 Tat nuclear localization sequence confers novel nuclear import properties. J. Biol. Chem. 273, 1623-1628. (124) Vive`s, E., Brodin, P., and Lebleu, B. (1997) A truncated HIV-1 Tat protein basic domain rapidly translocates through the plasma membrane and accumulates in the cell nucleus. J. Biol. Chem. 272, 16010-16017. (125) Calnan, B. J., Biancalana, S., Hudson, D., and Frankel, A. D. (1991) Analysis of arginine-rich peptides from the HIV Tat protein reveals unusual features of RNA-protein recognition. Genes Dev. 5, 201-210. (126) Brake, D. A., Debouck, C., and Biesecker, G. (1990) Identification of an Arg-Gly-Asp (RGD) cell adhesion site in human immunodeficiency virus type 1 transactivation protein, tat. J. Cell. Biol. 111, 1275-1281. (127) Mann, D. A., and Frankel, A. D. (1991) Endocytosis and targeting of exogenous HIV-1 Tat protein. EMBO J. 10, 1733-1739. (128) Schwarze, S. R., Hruska, K. A., and Dowdy, S. F. (2000) Protein transduction: unrestricted delivery into all cells? Trends Cell Biol. 10, 290-295. (129) Ro¨sch, P., and Willbold, D. (1996) Is EIAV Tat protein a homeodomain? Science 272, 1672. (130) Lissy, N. A., Van Dyk, L. F., Becker-Hapak, M., VoceroAkbani, A., Mendler, J. H., and Dowdy, S. F. (1998) TCR antigen-induced cell death occurs from a late G1 phase cell cycle check point. Immunity 8, 57-65. (131) Ezhevsky, S. A., Nagahara, H., Vocero-Akbani, A. M., Gius, D. R., Wei, M. C., and Dowdy, S. F. (1997) Hypophosphorylation of the retinoblastoma protein (pRb) by cyclin D: Cdk4/6 complexes resul; ts in active pRb. Proc. Natl. Acad. Sci. U.S.A. 94, 10699-10704. (132) Gius, D. R., Ezhevsky, S. A., Becker-Hapak, M., Nagahara, H., Wei, M. C., and Dowdy, S. F. (1999) Transduced p16INK4a peptides inhibit hypophosphorylation of the retinoblastoma protein and cell cycle progression prior to activation of Cdk2 complexes in late G1. Cancer Res. 59, 2577-2580. (133) Vocero-Akbani, A. M., van der Heyden, N., Lissy, N. A., Ratner, L., and Dowdy, S. F. (1999) Killing HIV-infected cells by tranduction with an HIV protease-activated caspase-3 protein. Nat. Med. 5, 29-33. (134) Schwartz, J. J., and Zhang, S. (2000) Peptide-mediated cellular delivery. Curr. Opin. Mol. Ther. 2, 162-167. (135) Schwarze, S. R., and Dowdy, S. F. (2000) In vivo protein transduction: intracellular delivery of biologically active proteins, compounds and DNA. Trends Pharmacol. Sci. 21, 45-48. (136) Nagahara, H., Vocero-Akbani, A., Snyder, E. L., Ho, A., Latham, D. G., Lissy, N. A., Becker-Hapak, M., Ezhevsky, S. A., and Dowdy. S. F. (1998) Transduction of full-length TAT fusion proteins into mammalian cells: TAT-p27Kip1 induces cell migration. Nat. Med. 4, 1449-1452. (137) Bonifaci, N., Sitia, R., and Rubartelli, A. (1995) Nuclear translocation of an exogenous fusion protein containing HIV Tat requires unfolding. AIDS 9, 995-1000. (138) Wender, P. A., Mitchell, D. J., Pattabiraman, K., Pelkey, E. T., Steinman, L., and Rothbard, J. B. (2000) The design, synthesis, and evaluation of molecules that enable or enhance cellular uptake: peptoid molecular transporters. Proc. Natl. Acad. Sci. U.S.A. 97, 13003-13008.

Fischer et al. (139) Vives, E., Granier, C., Prevot, P., and Lebleu, B. (1997) Structure-activity relationship study of the plasma membrane translocating potential of a short peptide from HIV-1 Tat protein. Lett. Pept. Sci. 4, 429-436. (140) Mitchell, D. J., Kim, D. T., Steinman, L., Fathman, C. G., and Rothbard, J. B. (2000) Polyarginine enters cells more efficiently than other polycationic homopolymers. J. Pept. Res. 56, 318-325. (141) Tamilarasu, N., Huq, I., and Rana, T. M. (1999) High affinity and specific binding of HIV-1 TAR RNA by a Tatderived oligourea. J. Am. Chem. Soc. 121, 1597-1598. (142) Hamy, F., Felder, E. R., Heizmann, G., Lazdins, J., Aboulela, F., Varani, G., Karn, J., and Klimkait, T. (1997) An inhibitor of the Tat/TAR RNA interaction that effectively suppresses HIV-1 replication. Proc. Natl. Acad. Sci. U.S.A. 94, 3548-3553. (143) Sandvig, K., and Olsnes, S. (1982) Entry of the toxic proteins abrin, modeccin, ricin, and diphtheria toxin into cells. II. Effect of pH, metabolic inhibitors, and ionophores and evidence for toxin penetration from endocytotic vesicles. J. Biol. Chem. 257, 7504-7513. (144) Rothbard, J. B., Garlington, S., Lin, Q., Kirschberg, T., Kreider, E., McGrane, L. P., Wender, P. A., and Khavari, P. A. (2000) Conjugation of arginine oligomers to cyclosporin A facilitates topical delivery and inhibition of inflammation. Nat. Med. 6, 1253-1257. (145) Oess, S., and Hildt, E. (2000) Novel cell permeable motif derived from the PreS2-domain of hepatitis-B virus surface antigens. Gene Ther. 7, 750-758. (146) Gerlich, W. H., Lu, X., and Heermann, K. H. (1993) Studies on the attachment and penetration of hepatitis B virus. J. Hepatol. 17, S10-S14. (147) Lu, X., Block, T. M., and Gerlich, W. H. (1996) Proteaseinduced infectivity of hepatitis B virus for a human hepatoblastoma cell line. J. Virol. 70, 2277-2285. (148) Elliott, G., and O’Hare. (1997) Intercellular trafficking and protein delivery by a herpesvirus structural protein. Cell 88, 223-233. (149) Elliott, G., O’Reilly, D., and O’Hare, P. (1996) Phosphorylation of the herpes simplex virus type 1 tegument protein VP22. Virolology 226, 140-145. (150) Fang, B., Xu, B., Koch, P., and Roth, J. A. (1998) Intercellular trafficking of VP22-GFP fusion proteins is not observed in cultured mammalian cells. Gene Ther. 5, 14201424. (151) Aints, A., Dilber, M. S., and Smith, C. I. (1999) Intercellular spread of GFP-VP22. J. Gene Med. 1, 275-279. (152) Brewis, N., Phelan, A., Webb, J., Drew, J., Elliott, G., and O’Hare, P. (2000) Evaluation of VP22 spread in tissue culture. J. Virol. 74, 1051-1056. (153) Derer, W., Easwaran, H. P., Knopf, C. W., Leonhardt, H., and Cardoso, M. C. (1999) Direct protein transfer to terminally differentiated muscle cells. J. Mol. Med. 77, 609-613. (154) Elliott, G., and O’Hare, P. (1999) Intercellular trafficking of VP22-GFP fusion proteins. Gene Ther. 6, 149-151. (155) Wybranietz, W. A., Prinz, F., Spiegel, M., Schenk, A., Bitzer, M., Gregor, M., and Lauer, U. M. (1999) Quantification of VP22-GFP spread by direct fluorescence in 15 commonly used cell lines. J. Gene Med. 1, 265-74. (156) Lai, Z., Han, I., Zirzow, G., Brady, R. O., and Reiser, J. (2000) Intercellular delivery of a herpes simplex virus VP22 fusion protein from cells infected with lentiviral vectors. Proc. Natl. Acad. Sci. U.S.A. 97, 11297-11302. (157) Dilber, M. S., Phelan, A., Aints, A., Mohamed, A. J., Elliott, G., Smith, C. I. E., and O’Hare, P. (1999) Intercellular delivery of thymidine kinase prodrug activating enzyme by the herpes simplex virus protein, VP22. Gene Ther. 6, 1221. (158) Phelan, A., Elliott, G., and O’Hare, P. (1998) Intercellular delivery of functional p53 by the herpesvirus protein VP22. Nat. Biotechnol. 16, 440-443. (159) Koelle, D. M., Frank, J. M., Johnson, M. L., and Kwok, W. W. (1998) Recognition of herpes simplex virus type 2 tegument proteins by CD4 T cells infiltrating human genital herpes lesions. J. Virol. 72, 7476-7483.

Reviews (160) Murphy, A. L., and Murphy, S. J. (1999) Catch VP22: the hitch-hiker’s ride to gene therapy? Gene Ther. 6, 4-5. (161) Hoyt, D. W., and Gierasch, L. M. (1991) Hydrophobic content and lipid interactions of wild-type and mutant OmpA signal peptides correlate with their in vivo function. Biochemistry 30, 10155-10163. (162) Andrews, D. W., Young, J. C., Mirels, L. F., and Czarnota, G. J. (1992) The role of the N region in signal sequence and signal-anchor function. J. Biol. Chem. 267, 7761-7769. (163) Von Heijne, G. (1990) The signal peptide. J. Membr. Biol. 115, 195-201. (164) Hawiger, J. (1999) Noninvasive intracellular delivery of functional peptides and proteins. Curr. Opin. Chem. Biol. 3, 89-94. (165) Prabhakaran, M. (1990) The distribution of physical, chemical and conformational properties in signal and nascent peptides. Biochem. J. 269, 691-696. (166) Lin, Y.-Z., Yao, S. Y., Veach, R. A., Torgerson, T. R., and Hawiger, J. (1995) Inhibition of nuclear translocation of transcription factor NF-κB by a synthetic peptide containing a cell membrane-permeable motif and nuclear localization sequence. J. Biol. Chem. 270, 14255-14258. (167) Liu, X.-Y., Timmons, S., Lin, Y.-Z., and Hawiger, J. (1996) Identification of a functionally important sequence in the cytoplasmic tail of integrin β3 by using cell-permeable peptide analogues. Proc. Natl. Acad. Sci. U.S.A. 93, 11819-11824. (168) Chaloin, L., Vidal, P., Lory, P., Me´ry, J., Lautredou, N., Divita, G., and Heitz, F. (1998) Design of carrier peptideoligonucleotide conjugates with rapid membrane translocation and nuclear localization properties. Biochem. Biophys. Res. Commun. 243, 601-608. (169) Briggs, M. S., and Gierasch, L. M. (1986) Molecular mechanisms of protein secretion: the role of the signal sequence. Adv. Protein Chem. 38, 109-180. (170) Kalderon, D., Richardson, W. D., Markham, A. F., and Smith, A. E. (1984) Sequence requirements for nuclear location of simian virus 40 large-T antigen. Nature 311, 3338. (171) Chaloin, L., Vidal, P., Heitz, A., Van Mau, N., Me´ry, J., Divita, G., and Heitz, F. (1997) Conformations of primary amphipathic carrier peptides in membrane mimicking environments. Biochemistry 36, 11179-11187. (172) Rojas, M., Yao, S. Y., and Lin, Y.-Z. (1996) Controlling epidermal growth factor (EGF)-stimulated Ras activation in intact cells by a cell-permeable peptide mimicking phosphorylated EGF receptor. J. Biol. Chem. 271, 27456-27461. (173) Chang, M., Zhang, L., Tam, J. P., and Sanders-Bush, E. (2000) Dissecting G protein-coupled receptor signaling pathways with membrane-permeable blocking peptides. J. Biol. Chem. 275, 7021-7029. (174) Lin, Y.-Z., Yao, S. Y., and Hawiger, J. (1996) Role of the nuclear localization sequence in fibroblast growth factor-1stimulated mitogenic pathways. J. Biol. Chem. 271, 53055308. (175) Kubota, T., Kushikata, T., Fang, J., and Krueger, J. M. (2000) Nuclear factor-kappaB inhibitor peptide inhibits spontaneous and interleukin-1β-induced sleep. Am. J. Physiol. Regul. Integr. Comput. Physiol. 279, R404-R413. (176) Fujihara, S. M., Cleaveland, J. S., Grosmaire, L. S., Berry, K. K., Kennedy, K. A., Blake, J. J., Loy, J., Rankin, B. M., Ledbetter, J. A., and Nadler, S. G. (2000) A D-amino acid peptide inhibitor of NF-κB nuclear localization is efficacious in models of inflammatory disease. J. Immunol. 165, 10041012. (177) Li, A.-J., Tsuboyama, H., Komi, A., Ikekita, M., and Imamura, T. (1998) Strong suppression of feeding by a peptide containing both the nuclear localization sequence of fibroblast growth factor-1 and a cell membrane-permeable sequence. Neurosci. Lett. 255, 41-44. (178) Rojas, M., Donahue, J. P., Tan, Z., and Lin, Y.-Z. (1998) Genetic engineering of proteins with cell membrane permeability. Nat. Biotechnol. 16, 370-375. (179) Hawiger, J. (1997) Cellular import of functional peptides to block intracellular signaling. Curr. Opin. Immunol. 9, 189194.

Bioconjugate Chem., Vol. 12, No. 6, 2001 839 (180) Tamm, L. K. (1991) Membrane insertion and lateral mobility of synthetic amphiphilic signal peptides in lipid model membrane. Biochim. Biophys. Acta 1071, 123-148. (181) Antopolsky, M., Azhayeva, E., Tengvall, U., Auriola, S., Jaä¨skelaïnen, I., Ro¨nko¨, S., Honkakoski, P., Urtti, A., Lo¨nnberg, H., and Azhayev, A. (1999) Peptide-oligonucleotide phosphorothioate conjugates with membrane translocation and nuclear localization properties. Bioconjugate Chem. 10, 598-606. (182) Dokka, S., Toledo-Vasquez, D., Shi, X., Wang, L., and Rojanasakul, Y. (1997) Cellular delivery of oligonucleotides by synthetic import peptide carrier. Pharmaceut. Res. 14, 1759-1764. (183) White, J. M. (1990) Viral and cellular membrane fusion proteins. Annu. Rev. Physiol. 52, 675-697. (184) Gallaher, W. R. (1987) Detection of a fusion peptide sequence in the transmembrane protein of human immunodeficiency virus. Cell 50, 327-328. (185) Freed, E. O., Myers, D. J., and Risser, R. (1990) Characterization of the fusion domain of the human immunodeficiency virus type 1 envelope glycoprotein gp41. Proc. Natl. Acad. Sci. U.S.A. 87, 4650-4654. (186) Freed, E. O., and Myers, D. J. (1992) Identification and characterization of fusion and processing domains of the human immunodeficiency virus type 2 envelope glycoprotein. J. Virol. 66, 5472-5478. (187) Delahunty, M. D., Rhee, I., Freed, E. O., and Bonifacino, J. S. (1996) Mutational analysis of the fusion peptide of the human immunodeficiency virus type 1: identification of critical glycine residues. Virology 218, 94-102. (188) Bergeron, L., Sullivan, N., and Sodroski, J. (1992) Target cell-specific determinants of membrane fusion within the human immunodeficiency virus type 1 gp120 third variable region and gp41 amino terminus. J. Virol. 66, 2389-2397. (189) Chanh, T. C., Dreesman, G. R., Kanda, P., Linette, G. P., Sparrow, J. T., Ho, D. D., and Kennedy, R. C. (1986) Induction of anti-HIV neutralizing antibodies by synthetic peptides. EMBO J. 5, 3065-3071. (190) Slepushkin, V. A., Melikyan, G. B., Sidorova, M. S., Chumakov, V. M., Andreev, S. M., Manulyan, R. A., and Karamov, E. V. (1990) Interaction of human immunodeficiency virus (HIV-1) fusion peptides with artificial lipid membranes. Biochem. Biophys. Res. Commun. 172, 952-957. (191) Rafalski, M., Lear, J. D., and DeGrado, W. F. (1990) Phospholipid interactions of synthetic peptides representing the N-terminus of HIV gp41. Biochemistry 29, 7917-7922. (192) Martin, I., Schaal, H., Scheid, A., and Ruysschaert, J.M. (1996) Lipid membrane fusion induced by the human immunodeficiency virus type 1 gp41 N-terminal extremity is determined by its orientation in the lipid bilayer. J. Virol. 70, 298-304. (193) Gordon, L. M., Curtain, C. C., Zhong, Y. C., Kirkpatrick, A., Mobley, P. W., and Waring, A. J. (1992) The aminoterminal peptide of HIV-1 glycoprotein 41 interacts with human erythrocyte membranes: peptide conformation, orientation and aggregation. Biochim. Biophys. Acta 25, 257274. (194) Dingwall, C., and Laskey, R. (1992) The nuclear membrane. Science 258, 942-947. (195) Vidal, P., Chaloin, L., Mery, J., Lamb, N., Lautredou, N., Bennes, R., and Heitz, F. (1996) Solid-phase synthesis and cellular localization of a C- and/or N-terminal labeled peptide. J. Pept. Sci. 2, 125-133. (196) Morris, M. C., Vidal, P., Chaloin, L., Heitz, F., and Divita, G. (1997) A new peptide vector for efficient delivery of oligonucleotides into mammalian cells. Nucleic Acids Res. 25, 2730-2736. (197) Morris, M. C., Robert-Hebmann, V., Chaloin, L., Mery, J., Heitz, F., Devaux, C., Goody, R. S., and Divita, G. (1999) A new potent HIV-1 reverse transcriptase inhibitor. A synthetic peptide derived from the interface subunit domains. J. Biol. Chem. 274, 24941-24946. (198) Du¨zgu¨nes, N., and Shavnin, S. A. (1992) Membrane destabilization by N-terminal peptides of viral envelope proteins. J. Membr. Biol. 128, 71-80.

840 Bioconjugate Chem., Vol. 12, No. 6, 2001 (199) Bullough, P. A., Hughson, F. M., Skehel, J. J., and Wiley, D. C. (1994) Structure of influenza haemagglutinin at the pH of membrane fusion. Nature 371, 37-43. (200) Murata, M., Sugahara, Y., Takahashi, S., and Ohnishi, S. (1987) pH-Dependent membrane fusion activity of a synthetic twenty amino acid peptide with the same sequence as that of the hydrophobic segment of influenza virus hemagglutinin. J. Biochem. 102, 957-962. (201) Mahato, R. I., Smith, L. C., and Rolland, A. (1999) Pharmaceutical perspectives of nonviral gene therapy. Adv. Genet. 41, 95-156. (202) Pichon, C., Freulon, I., Midoux, P., Mayer, R., Monsigny, M., and Roche, A.-C. (1997) Cytosolic and nuclear delivery of oligonucleotides mediated by an amphiphilic anionic peptide. Antisense Nucleic Acid Drug Dev. 7, 335-343. (203) Bongartz, J.-P., Aubertin, A.-M., Milhaud, P. G., and Lebleu, B. (1994) Improved biological activity of antisense oligonucleotides conjugated to a fusogenic peptide. Nucleic Acids Res. 22, 4681-4688. (204) Murata, M., Takahashi, S., Kagiwada, S., Suzuki, A., and Ohnishi, S. (1992) pH-Dependent membrane fusion and vesiculation of phospholipid large unimellar vesicles induced by amphiphilic anionic and cationic peptides. Biochemistry 31, 1986-1992. (205) Midoux, P., Kichler, A., Boutin, V., Maurizot, J.-C., and Monsigny, M. (1998) Membrane permeabilization and efficient gene transfer by a peptide containing several histidines. Bioconjugate Chem. 9, 260-267. (206) Schwartz, B., Ivanov, M.-A., Pitard, B., Escriou, V., Rangara, R., Byk, G., Wils, P., Crouzet, J., and Scherman, D. (1999) Synthetic DNA-compacting peptides derived from human sequence enhance cationic lipid-mediated gene transfer in vitro and in vivo. Gene Ther. 6, 282-292. (207) Bordier, B., Perala-Heape, M., Degols, G., Lebleu, B., Litvak, S., Sarih-Cottin, L., and He´le`ne, C. (1995) Sequencespecific inhibition of human immunodeficiency virus (HIV) reverse transcription by antisense oligonucleotides: comparative study in cell-free assays and in HIV-infected cells. Proc. Natl. Acad. Sci. U.S.A. 92, 9383-9387. (208) Gottschalk, S., Sparrow, J. T., Hauer, J., Mims, M. P., Leland, F. E., Woo, S. L. C., and Smith, L. C. (1996) A novel DNA-peptide complex for efficient gene transfer and expression in mammalian cells. Gene Ther. 3, 448-457. (209) Niidome, T., Urakawa, M., Takaji, K., Matsuo, Y., Ohmori, N., Wada, A., Hirayama, T., and Aoyagi, H. (1999) Influence of lipophilic groups in cationic alpha-helical peptides on their abilities to bind with DNA and deliver genes into cells. J. Pept. Res. 54, 361-367. (210) Gawrisch, K., Han, K.-H., Yang, J.-S., Bergelson, L. D., and Ferretti, J. A. (1993) Interaction of peptide fragment 828-848 of the envelope glycoprotein of human immunodeficiency virus type 1 with lipid bilayers. Biochemistry 32, 3112-3118. (211) Pichon, C., Arar, K., Stewart, A. J., Duc Dodon, M., Gazzolo, L., Courtoy, P. J., Mayer, R., Monsigny, M., and Roche, A.-C. (1997) Intracellular routing and inhibitory activity of oligonucleopeptides containing a KDEL motif. Mol. Pharmacol. 51, 431-438. (212) Singh, D., Kiarash, R., Kawamura, K., LaCasse, E. C., and Garie´py, J. (1998) Penetration and intracellular routing of nucleus-directed peptide-based shuttles (loligomers) in eukaryotic cells. Biochemistry 37, 5798-5809. (213) Subbarao, N. K., Parente, R. A., Szoka, F. C., Jr., Nadasdi, L., and Pongracz, K. (1987) The pH-dependent bilayer destabilization by an amphipathic peptide. Biochemistry 26, 2964-2972. (214) Wyman, T. B., Nicol, F., Zelphati, O., Scaria, P. V., Plank, C., and Szoka, F. C. (1997) Design, synthesis, and characterization of a cationic peptide that binds to nucleic acids and permeabilizes bilayers. Biochemistry 36, 3008-3017. (215) Oehlke, J., Scheller, A., Wiesner, B., Krause, E., Beyermann, M., Klauschenz, E., Melzig, M., and Bienert, M. (1998) Cellular uptake of an R-helical amphipathic model peptide with the potential to deliver polar compounds into the cell interior nonendocytically. Biochim. Biophys. Acta 1414, 127139.

Fischer et al. (216) Scheller, A., Oehlke, J., Wiesner, B., Dathe, M., Krause, E., Beyermann, M., Melzig, M., and Bienert, M. (1999) Structural requirements for cellular uptake of R-helical amphipathic peptides. J. Pept. Sci. 5, 185-194. (217) Niidome, T., Takaji, K., Urakawa, M., Ohmori, N., Wada, A., Hirayama, T., and Aoyagi, H. (1999) Chain length of cationic alpha-helical peptide sufficient for gene delivery into cells. Bioconjugate Chem. 10, 773-780. (218) Lewis, J. G., Lin, K.-Y., Kothavale, A., Flanagan, W. M., Matteucci, M. D., DePrince, R. B., Mook, R. A., Hendren, R. W., and Wagner, R. W. (1996) A serum-resistant cytofectin for cellular delivery of antisense oligodeoxynucleotides and plasmid DNA. Proc. Natl. Acad. Sci. U.S.A. 93, 3176-3181. (219) Alarcon-Segovia, D. (1978) Antibody to nuclear ribonucleoprotein penetrates live human mononuclear cells through Fc receptors. Nature 271, 67-79. (220) Yanase, K., Smith, R. M., Cizman, B., Foster, M. H., Peachey, L. D., Jarett, L., and Madaio, M. P. (1994) A subgroup of murine monoclonal anti-deoxyribonucleic acid antibodies traverse the cytoplasm and enter the nucleus in a time-and temperature- dependent manner. Lab. Invest. 71, 52-60. (221) Zack, D. J., Stempniak, M., Wong, A. L., Taylor, C., and Weisbart, R. H. (1996) Mechanisms of cellular penetration and nuclear localization of an anti-double strand DNA autoantibody. J. Immunol. 157, 2082-2088. (222) Avrameas, A., Ternynck, T., Nato, F., Buttin, G., and Avrameas, S. (1998) Polyreactive anti-DNA monoclonal antibodies and a derived peptide as vectors for the intracytoplasmic and intranuclear translocation of macromolecules. Proc. Natl. Acad. Sci. U.S.A. 95, 5601-5606. (223) Sibille, P., Ternynck, T., Nato, F., Buttin, G., Strosberg, D., and Avrameas, A. (1997) Mimotopes of polyreactive antiDNA antibodies identified using phage-display peptide libraries. Eur. J. Immunol. 27, 1221-1228. (224) Avrameas, A., Ternynck, T., Gasmi, L., and Buttin, G. (1999) Efficient gene delivery by a peptide derived from a monoclonal anti-DNA antibody. Bioconjugate Chem. 10, 8793. (225) Pooga, M., Ha¨llbrink, M., Zorko, M., and Langel, U ¨ . (1998) Cell penetration by transportan. FASEB J. 12, 67-77. (226) Pooga, M., Lindgren, M., Hallbrink, M., Brakenhielm, E., and Langel, U. (1998) Galanin-based peptides, galparan and transportan, with receptor-dependent and independent activities. Ann. New York Acad. Sci. 21, 450-453. (227) Lindgren, M., Gallet, X., Soomets, U., Haellbrink, M., Brkenhielm, E., Pooga, M., Brasseur, R., and Langel, U. (2000) Translocation properties of novel cell penetrating transportan and penetratin analogues. Bioconjugate Chem. 11, 619-626. (228) Soomets, U., Lindgren, M., Gallet, X., Hallbrink, M., Elmquist, A., Balaspiri, L., Zorko, M., Pooga, M., Brasseur, R., and Langel, U. (2000) Deletion analogues of transportan. Biochim. Biophys. Acta 1467, 165-176. (229) Bolton, S. J., Jones, D. N. C., Darker, J. G., Eggleston, D. S., Hunter, A. J., and Walsh, F. S. (2000) Cellular uptake and spread of the cell-permeable peptide penetratin in adult rat brain. Eur. J. Neurosci. 12, 2847-2855. (230) Ho, A., Schwarze, S. R., Mermelstein, S. J., Waksman, G., and Dowdy, S. F. (2001) Synthetic protein transduction domains: enhanced transduction potential in vitro and in vivo. Cancer Res. 61, 474-477. (231) Wong, S. S. (1993) Chemistry of protein conjugation and cross-linking, CRC Press, Boca Raton. (232) Schulz, A., Adermann, K., Eulitz, M., Feller, S. M., and Kardinal, C. (2000) Preparation of disulfide-bonded polypeptide heterodimers by titration of thio-activated peptides with thiol-containing peptides. Tetrahedron 56, 3889-3891. (233) Simmons, C. G., Pitts, A. E., Mayfield, L. D., Shay, J. W., and Corey, D. R. (1997) Synthesis and membrane permeability of PNA-peptide conjugates. Bioorg. Med. Chem. Lett. 7, 3001-3006. (234) Zhang, L., Torgerson, T. R., Liu, X.-Y., Timmons, S., Colosia, A. D., Hawiger, J., and Tam, J. P. (1998) Preparation of functionally active cell-permeable peptides by single-step

Reviews ligation of two peptide modules. Proc. Natl. Acad. Sci. U.S.A. 95, 9184-9189. (235) Gupta, K. C., Sharma, P., Kumar, P., and Sathyanarayana, S. (1991) A general method for the synthesis of 3′-sulfhydryl and phosphate group containing oligonucleotides. Nucleic Acids Res. 19, 3019-3025. (236) Zuckermann, R., Corey, D., and Schultz, P. (1987) Efficient methods for attachment of thiol specific probes to the 3′-ends of synthetic oligodeoxyribonucleotides. Nucleic Acids Res. 15, 5305-5321. (237) Sproat, B. S., Beijer, B., Rider, P., and Neuner, P. (1988) Synthesis of modified building blocks containing amino or thiol moieties: application of modified oligodeoxynucleotides. Nucleosides Nucleotides 7, 651-653. (238) Connolly, B. A., and Rider, P. (1985) Chemical synthesis of oligonucleotides contining a free sulphydryl group and subsequent attachment of thiol specific probes. Nucleic Acids Res. 13, 4485-4502. (239) Gaur, R. K., Sharma, P., and Gupta, K. C. (1989) A simple method for the introduction of thiol group at 5′-termini of oligodeoxynucleotides. Nucleic Acids Res. 17, 4404. (240) Sproat, B. S., Beijer, B., Rider, P., and Neuner, P. (1987) The synthesis of protected 5′-mercapto-2′, 5′-dideoxynucleoside-3′-O-phosphoramidites; uses of 5′-mercapto-oligodeoxyreibonucleotides. Nucleic Acids Res. 15, 4837-4848. (241) Vive`s, E., and Lebleu, B. (1997) Selective coupling of a highly basic peptide to an oligonucleotide. Tetrahedron Lett. 38, 1183-1186. (242) Bernatowicz, M. S., Matsueda, R., and Matsueda, G. R. (1986) Preparation of Boc-[S-(3-nitro-2-pyridinesulfenyl)]cysteine and its use for unsymmetrical disulfide bond formation. Int. J. Pept. Protein Res. 28, 107-112. (243) Whitesides, G. M., Houk, J., and Patterson, M. A. K. (1983) Activation parameters for thiolate-disulfide interchange reactions in aqueous solution. J. Org. Chem. 48, 112115. (244) Liu, F.-T., Zinnecker, M., Hamaoka, T., and Katz, D. H. (1979) New procedures for preparation and isolation of conjugates of proteins and a synthetic copolymer of D-amino acids and immunochemical characterization of such conjugates. Biochemistry 18, 690-697. (245) Ede, N. J., Tregear, G. W., and Haralambidis, J. (1994) Routine preparation of thiol oligonucleotides: application to the synthesis of oligonucleotide-peptide hybrids. Bioconjugate Chem. 5, 373-378. (246) Eritja, R., Pons, A., Escarceller, M., Giralt, E., and Albericio, F. (1991) Synthesis of defined peptide-oligonucleotide hybrids contining a nuclear transport signal sequence. Tetrahedron 47, 4113-4120. (247) Soukchareun, S., Haralambidis, J., and Tregear, G. (1998) Use of NR-Fmoc-cysteine(S-thiobutyl) derivatized oligodeoxynucleotides for the preparation of oligodeoxynucleotidepeptide hybrid molecules. Bioconjugate Chem. 9, 466-475. (248) Tung, C.-H., Rudolph, M. J., and Stein, S. (1991) Preparation of oligonucleotide-peptide conjugates. Bioconjugate Chem. 2, 464-465. (249) Harrison, J. G., and Balasubramanian, S. (1998) Synthesis and hybridization analysis of a small library of peptideoligonucleotide conjugates. Nucleic Acids Res. 26, 31363145. (250) Me´ry, J., Granier, C., Juin, M., and Brugidou, J. (1993) Disulfide linkage to polyacrylic resin for automated Fmoc peptide synthesis. Immunochemical applications of peptide resins and mercaptoamide peptides. Int. J. Pept. Protein Res. 42, 44-52. (251) Arar, K., Aubertin, A.-M., Roche, A.-C., Monsigny, M., and Mayer, R. (1995) Synthesis and antiviral activity of peptideoligonucleotide conjugates prepared by using NR-(bromoacetyl)peptides. Bioconjugate Chem. 6, 573-577. (252) Benz, H. (1994) The role of solid-phase fragment condensation (SPFC) in peptide synthesis. Synthesis 337-358. (253) Peyrottes, S., Mestre, B., Burlina, F., and Gait, M. J. (1999) Studies towards the synthesis of peptide-oligonucleotide conjugates. Nucleosides Nucleotides 18, 14431448.

Bioconjugate Chem., Vol. 12, No. 6, 2001 841 (254) Tetzlaff, C. N., Schwope, I., Bleczinski, C. F., Steinberg, J. A., and Richert, C. (1998) A convenient synthesis of 5′amino-5′-deoxythymidine and preparation of peptide-DNA hybrids. Tetrahedron Lett. 39, 4215-4218. (255) Haralambidis, J., Duncan, L., and Tregear, G. W. (1987) The solid-phase synthesis of oligonucleotides containing a 3′peptide moiety. Tetrahedron Lett. 28, 5199-5202. (256) Bergmann, F., and Bannwarth, W. (1995) Solid phase synthesis of directly linked peptide-oligodeocynucleotide hybrids using standard synthesis protocols. Tetrahedron Lett. 36, 1839-1842. (257) Basu, S., and Wickstrom, E. (1995) Solid-phase synthesis of a D-peptide-phosphorothioate oligodeoxynucleotide conjugate from two arms of a poly(ethylene glycol)-polystyrene support. Tetrahedron Lett. 36, 4943-4946. (258) De Napoli, L., Messere, A., Montesarchio, D., Piccialli, G., Benedetti, E., Bucci, E., and Rossi, F. (1999) A new solidphase synthesis of oligonucleotides 3′-conjugates with peptides. Bioorg. Med. Chem. 7, 395-400. (259) Dawson, P. E., Muir, T. W., Clark-Lewis, I., and Kent, S. B. H. (1994) Synthesis of proteins by native chemical ligation. Science 266, 776-779. (260) Stetsenko, D. A., and Gait, M. J. (2000) Efficient conjugation of peptides to oligonucleotides by “native ligation”. J. Org. Chem. 65, 4900-4908. (261) McPherson, M., Wright, M. C., and Lohse, P. A. (1999) Synthesis of an RNA-peptide conjugate by orthogonal ligation. Synlett S1, 978-980. (262) Kemp, D. S., and Carey, R. I. (1993) Synthesis of a 39peptide and 25-peptide by thiol capture ligations: observation of a 40-fold rate acceleration of the intramolecular O, N-acyltransfer reaction between peptide fragments bearing only cysteine protective groups. J. Org. Chem. 58, 2216-2222. (263) Bruick, R. K., Dawson, P. E., Kent, S. B. H., Usman, N., and Joyce, G. F. (1996) Template-directed ligation of peptides to oligonucleotides. Chem. Biol. 3, 49-56. (264) Schreiber, J. P., Hsiung, N., and Cantor, C. R. (1979) Fluorescence studies of the accessibility of the 3′ ends of the ribosomal RNAs in Escherichia coli ribosomes and subunits. Nucleic Acids Res. 6, 181-193. (265) Sto¨ffler-Meilicke, M., Sto¨ffler, G., Odom, O. W., Zinn, A., Kramer, G., and Hardesty, B. (1981) Localization of 3′ ends of 5S and 23S rRNAs in reconstituted subunits of Escherichia coli ribosomes. Proc. Natl. Acad. Sci. U.S.A. 78, 5538-5542. (266) Murasugi, A., and Wallace, R. B. (1984) Biotin-labeled oligonucleotides: enzymatic synthesis and use of hybridization probes. DNA 3, 269-277. (267) Leary, J. J., Brigati, D. J., and Ward, D. C. (1983) Rapid and sensitive colorimetric method for visualizing biotinlabeled DNA probes hybridized to DNA or RNA immobilized on nitrocellulose: bio-blots. Proc. Natl. Acad. Sci. U.S.A. 80, 4045-4049. (268) Grandas, A., Robles, J., and Pedroso, E. (1995) Phosphitylation of primary carboxamides. Synthesis of peptide-oligonucleotide conjugates with acylphosphoramidate linkages. Nucleosides Nucleotides 14, 825-828. (269) Arap, W., Pasqualini, R., and Ruoslahti, E. (1998) Cancer treatment by targeted drug delivery to tumor vasculature in a mouse model. Science 279, 377-380. (270) Pasqualini, R., and Ruoslahti, E. (1996) Organ targeting in vivo using phage display peptide libraries. Nature 380, 364-366. (271) Shewring, L., Collins, L., Lightman, S. L., Hart, S., Gustafsson, K., and Fabre, J. W. (1997) A nonviral vector system for efficient gene transfer to corneal endothelial cells via membrane integrins. Transplantation 64, 763-769. (272) Bachmann, A. S., Surovoy, A., Jung, G., and Moelling, K. (1998) Integrin receptor-targeted transfer peptides for efficient delivery of antisense oligodeoxynucleotides. J. Mol. Med. 76, 126-132. (273) Michael, S. I., and Curiel, D. T. (1994) Strategies to achieve targeted gene delivery via the receptor-mediated endocytosis pathway. Gene Ther. 1, 223-232.

BC0155115

Cellular Delivery of Impermeable Effector Molecules in the Form of

Recommend Documents