• A rhegnylogic strategy for the synthesis of signal transduction modulatory, cell penetrating peptides

      Jones, Sarah; Ostlund, Pernilla; Langel, Ulo; Zorko, Matjaz; Nicholl, Iain D.; Howl, John D. (Wiley InterScience, 2006)
      INTRODUCTION: Many cell-penetrating peptides (CPP) have been utilised as biologically inert vectors. A majority of these studies employ sychnologically organised constructs in which a bioactive cargo (message) is chemically conjugated to the CPP (address). Previously, we have adopted a sychnologic strategy to modulate intracellular signal transduction. Using chimeric constructs composed of the CPP transportan 10, conjugated to partial sequences that correspond to functional domains of signal transduction proteins, we have selectively modulated a variety of cellular activities including secretion and activation of p42/p44 mitogen-activated protein kinases [1, 2]. However, a QSAR-based algorithm can now be used to predict CPP that reside within the primary sequences of proteins [3]. We have adapted this strategy to identify CPP within signal transducing proteins including functional domains that govern protein-protein interactions. Data presented herein indicate that it is now feasible to identify rhegnylogic sequences, containing vectoral-independent discontinuously organised pharmacophores, that are cell penetrant modulators of signal transduction pathways.
    • Applications of cell-penetrating peptides as signal transduction modulators.

      Jones, Sarah; Howl, John D. (Washington, D.C.: CRC Press, 2006)
      THIS BOOK: Since the first Handbook of Cell-Penetrating Peptides was prepared in 2001, the wealth of new information on the use of these peptides as transport systems has in fact served to confound the field. The constant internal change in the field of cell-penetrating peptides (CPPs) is due to recent research uncovering apparent ambiguities in cellular uptake. There is still neither a common terminology nor a uniform explanation for the penetrative mechanism of cell-penetrating peptides. In this second edition of the Handbook of Cell-Penetrating Peptides, the authors summarize the current state of the field including recent reevaluations of earlier studies of CPP mechanisms. Beginning with an overview of the classes of peptides and their individual uptake mechanisms, from the earlier lipid models to the more recent endocytotic pathways, the book demonstrates the diversity and the opportunity for these biologically active proteins to serve as future drug leads. The text then covers the use of CPPs in gene modulation, addressing the application of antisense and decoy oligonucleotides, as well as the new avenue of research targeting specific tumors and other tissues-questions that had barely been asked when the first edition was published. (CRC Press)
    • Chimeric peptides as tumour-selective delivery systems.

      Jones, Sarah; Howl, John D. (Society for Neuro-oncology and Duke University Press, 2005)
      The cell-type-specific targeting of cytotoxic agents and other functional moieties can be achieved by using peptidyl address motifs that selectively bind protein targets expressed at high density at the cell membrane. Indeed, numerous studies have confirmed the utility of ligands for G protein–coupled receptors as components of heterofunctional peptide chimeras that are selective biological probes. Our current efforts are directed toward the further development of chimeric peptidyl constructs that employ sequences derived from GPCR ligands or cell penetrant motifs to affect the selective delivery of cytotoxins and signal transduction modulators to tumor cells. We have designed and synthesized a range of hybrid constructs consisting of cytotoxins (peptide and non-peptide) covalently linked to an address peptide derived from the C-terminal of gastrin (G7; H-AYGWMDF-NH2). The G7 homing motif targets a novel binding site expressed by U373MG astrocytic tumor cells that is distinct from classical CCK1/CCK2 receptors. Moreover, biological responses following activation of this novel membrane-bound protein may offer additional therapeutic advantages. For example, G7 receptor activation is reported to inhibit the motility of malignant astrocytoma in vivo while avoiding the growth-promoting effects of gastrin (Pannequin et al., J. Pharmacol. Exp. Ther. 302, 274, 2002). We evaluated the cytotoxicity of our chimeric peptides by comparing changes in cellular viability using MTT conversion assays. Our data indicate that chimeric peptides dose-dependently and rapidly (<8 h) reduced the viability of U373MG cells. Moreover, as a chimeric amino-terminal extension, the G7 address motif enhanced the cytotoxicity of both mastoparan (H-INLKALAALAKKIL-NH2) and D(KLAKLAK)2 peptides reported to stimulate necrosis and/or apoptosis of eukarytoic cells. In conclusion, hybrid G7 chimeras enhance the efficacy of cytotoxic agents and may be valuable probes to investigate and manipulate additional aspects of astrocytoma cell biology. This work was supported by The Wellcome Trust.
    • Identification and biological applications of rhegnylogically-organized cell penetrating peptides.

      Howl, John D.; Jones, Sarah (Australian Peptide Association, 2007)
      Introduction: Many different cell penetrating peptides (CPPs) have been utilized as vectors to affect the highly efficient intracellular delivery of bioactive moieties. A majority of such studies employ sychnologically-organized tandem combinations of a cargo (message) and a CPP (address). To date, bioactive cargoes have included peptides, proteins and a range of oligonucleotides attached either by direct chemical conjugation or as a component of a larger macromolecular complex. Moreover, a majority of CPPs, including the commonly used sequences Tat and penetratin, are designed to be both biologically and toxicologically inert. More recently, a QSAR-based algorithm has been developed to predict cryptic polycationic CPP motifs within the primary sequences of proteins. As described here, this novel technology has enabled the study of rhegnylogic CPPs in which multiple pharmacophores for cellular penetration and desirable biological activities are discontinuously organized within the primary sequence of single peptide. This organization differs from the more commonly utilized sychnologic strategy which joins functionally discrete and continous address and messages together in a tandem construct.
    • Mitoparan and target-selective chimeric analogues: membrane translocation and intracellular redistribution induces mitochondrial apoptosis.

      Jones, Sarah; Martel, Cecile; Belzacq-Casagrande, Anne-Sophie; Brenner, Catherine; Howl, John D. (Amsterdam: Elsevier, 2008)
      Mastoparan, and structurally-related amphipathic peptides, may induce cell death by augmentation of necrotic and/or apoptotic pathways. To more precisely delineate cytotoxic mechanisms, we determined that [Lys(5,8)Aib(10)]mastoparan (mitoparan) specifically induces apoptosis of U373MG and ECV304 cells, as demonstrated by endonuclease and caspase-3 activation and phosphatidylserine translocation. Live cell imaging confirmed that, following translocation of the plasma membrane, mitoparan specifically co-localizes with mitochondria. Complementary studies indicated that mitoparan induces swelling and permeabilization of isolated mitochondria, through cooperation with a protein of the permeability transition pore complex VDAC, leading to the release of the apoptogenic factor, cytochrome c. N-terminal acylation of mitoparan facilitated the synthesis of chimeric peptides that incorporated target-specific address motifs including an integrin-specific RGD sequence and a Fas ligand mimetic. Significantly, these sychnologically-organised peptides demonstrated further enhanced cytotoxic potencies. We conclude that the cell penetrant, mitochondriotoxic and apoptogenic properties of mitoparan, and its chimeric analogues, offer new insights to the study and therapeutic induction of apoptosis.
    • Mitoparans: mitochondriotoxic cell penetrating peptides and novel inducers of apoptosis.

      Jones, Sarah; Martel, Cecile; Belzacq-Casagrande, Anne-Sophie; Brenner, Catherine; Howl, John D. (Australian Peptide Association, 2007)
      Introduction: The amphipathic helical peptide mastoparan (MP; H-INLKALAALAKKIL-NH2) inserts into biological membranes to modulate the activity of heterotrimeric G proteins and other targets. Moreover, whilst cell free models of apoptosis demonstrate MP to facilitate mitochondrial permeability transition and release of apoptogenic cytochrome c, MP-induced death of intact cells has been attributed to its non-specific membrane destabilising properties (necrotic mechanisms). However, MP and related peptides are known to activate other signalling systems, including p42/p44 MAP kinases and could therefore, also modulate cell fate and specific apoptotic events. The ability of MP to facilitate mitochondrial permeability in cell free systems has lead to proposals that MP could be of utility in tumour therapeutics provided that it conferred features of cellular penetration and mitochondrial localization. We have recently reported that our highly potent amphipathic MP analogue mitoparan (mitP; [Lys5,8Aib10]MP; Aib = -aminoisobutyric acid) specifically promotes apoptosis of human cancer cells, as was confirmed by in situ TUNEL staining and activation of caspase-3. Moreover, we have also demonstrated that mitP penetrates plasma membranes and redistributes to co-localize with mitochondria. Complementary studies, using isolated mitochondria, further demonstrated that mitP, through co-operation with a protein of the permeability transition pore complex voltage-dependent anion channel (VDAC), induced swelling and permeabilization of mitochondria, leading to the release of the apoptogenic factor cytochrome c. An expanding field of peptide and cell penetrating peptide (CPP) research has focussed on the selective targeting of tumours by engineering constructs that incorporate cell-specific or tissue–specific address motifs. Peptidyl address motifs could enhance the selectivity of drug delivery whilst the improved cellular uptake offered by CPP enhances bioavailability. Thus and as a potential therapeutic strategy, we extended our findings to design target-specific mitP analogues. The integrin-specific address motif RGD and a Fas ligand mimetic WEWT were incorporated by N-terminal acylation of mitP to produce novel tandem-linked chimeric peptides.