Biochemical pharmacology of total retro-inverso analogues of bradykinin and angiotensin II: Molecular recognition by G-protein-coupled receptors and angiotensin converting enzyme

Total retro-inverso (TRI) analogues of bradykinin (BK), the B₂a-selective kinin antagonist d-Arg⁰[Hyp₃,d-Phe⁷,Leu⁸]BK, angiotensin II (AT II) and the AT II antagonist Saralasin ([Sar¹,Val⁵,Ala⁸]AT II) were prepared by conventional solid-phase synthesis. Molecular recognition of TRI peptidomimetics by G-protein-coupled receptors was studied by competitive radioligand displacement experiments. TRI analogues of d-Arg⁰[Hyp³,d-Phe⁷,Leu⁸]BK specifically bound to the kidney medulla B₂a bradykinin receptor with affinities (K_d) ranging from 64 M to 4 M. Conversely, TRI analogues of BK, AT II and Saralasin did not bind to either the B_2a bradykinin receptor or the rat AT_1a AT II receptor, respectively. These studies indicate that the TRI strategy is more compatible with the synthesis of antagonists than agonists. Three TRI peptidomimetics of d-Arg⁰[Hyp³,d-Phe⁷,Leu⁸]BK were weak inhibitors of angiotensin converting enzyme. All other TRI peptidomimetics had no effect upon ACE activity. These data endorse the utility of the TRI strategy for the synthesis of protease-resistant antagonists of peptide hormones and neuropeptides.     

Intracellular translocation of the decapeptide carboxyl terminal of Gi3 alpha induces the dual phosphorylation of p42/p44 MAP kinases

The carboxyl terminal of heterotrimeric G protein alpha subunits binds both G protein-coupled receptors and mastoparan (MP), a tetradecapeptide allostere. Moreover, peptides corresponding to the carboxyl domains of G(i)3alpha and G(t) display intrinsic biological activities in cell-free systems. Thus, the purpose of this study was to develop a cell penetrant delivery system to further investigate the biological properties of a peptide mimetic of the G(i)3alpha carboxyl terminal (G(i)3alpha(346-355); H-KNNLKECGLY-NH2). Kinetic studies, using a CFDA-conjugated analogue of G(i)3alpha(346-355), confirmed the rapid and efficient intracellular translocation of TP10-G(i)3alpha(346-355) (t(0.5) = 3 min). Translocated G(i)3alpha(346-355), but not other bioactive cargoes derived from PKC and the CB1 cannabinoid receptor, promoted the dual phosphorylation of p42/p44 MAPK without adverse changes in cellular viability. The relative specificity of this novel biological activity was further confirmed by the observation that translocated G(i)3alpha(346-355) did not influence the exocytosis of beta-hexoseaminidase from RBL-2H3, a secretory event stimulated by other cell penetrant peptide cargoes and MP. We conclude that TP10-G(i)3alpha(346-355) is a valuable, non-toxic research tool with which to study and modulate signal transduction pathways mediated by heterotrimeric G proteins and MAPK.     

Effects of vasopressin-mastoparan chimeric peptides on insulin release and G-protein activity.

Two chimeric peptides, consisting of the linear vasopressin receptor V1 antagonist PhAc-D-Tyr(Me)-Phe-Gln-Asn-Arg-Pro-Arg-Tyr, in the N-terminus and mastoparan in the C-terminus connected directly (M375) or via 6-aminohexanoic acid (M391), have been synthesised. At 10 microM concentration, these novel peptides increased insulin secretion from isolated rat pancreatic islet cells 18-26-fold at 3.3 mM glucose and 3.5-5-fold at 16.7 mM glucose. PTX pretreatment of the islets decreased the peptide-induced insulin release. M375 and M391 bind to V1a vasopressin receptors with affinities lower than the unmodified vasopressin antagonist, but with K(D) values of 3.76 nM and 9.02 nM, respectively, both chimeras are high affinity ligands. The GTPase activity and GTPgammaS binding in the presence of these peptides has been characterised in Rin m5F cells. Comparison of the influence of the peptides M375 and M391 on GTPase activity in native and pertussis toxin-treated cells suggests a selective activation of G alpha(i)/G alpha(o) subunits, combined with a suppression of other GTPases, primarily G alpha(s). However, the GTPgammaS binding data show that the peptides retain some of the activating property even in PTX-treated cell membranes. In conclusion, the conjugation of mastoparan with the V1a receptor antagonists produce peptides with properties different from the parent peptides that could be used to elucidate the role of different G proteins in insulin release.     

Lipidic mitochondrial inclusions in experimentally-induced in vitro myopathy of mouse diaphragm.

1. Treatment of murine skeletal muscle with the Ca2(+)-channel agonist, Bay K 8644, induces myopathic changes including configurational changes of the mitochondria. 2. During the progression of the myopathy the mitochondria apparently engulf neighboring lipid droplets. 3. Mitochondrial changes are most marked when damage to the muscle is most severe, are prevented under conditions that should reduce Bay K 8644-induced Ca2(+)-influx, and are not modified by various maneuvers designed to prevent the generation of free radical species.     

Enantiomer-specific bioactivities of peptidomimetic analogues of mastoparan and mitoparan: characterization of inverso mastoparan as a highly efficient cell penetrating peptide

Retro-inverso transformation has commonly been employed as a strategy both for the synthesis of proteolytically stable peptide analogues and for the detailed investigation of structure activity relationships. Herein, we adopted a similar strategy to probe the structure activity relationships of two biologically active tetradecapeptides. Analogues of the α-helical mastoparan, and the highly potent apoptogenic analogue mitoparan, were synthesized using d-amino acids assembled in both endogenous (inverso) and reverse (retro-inverso) orientations. For a more comprehensive comparison, our studies also included the retro l-enantiomer of both peptides. Contrary to expectation, comparative investigations of cytotoxicity, mast cell degranulation, and cellular penetration demonstrated that, while retro-inverso transformation abrogated the associated biological activities of these helical peptides, inverso homologues retained their bioactivities. Moreover, inverso mastoparan demonstrated the highest translocation efficacy of all analogues with much improved uptake kinetics compared to other cell penetrating peptides (CPPs) including the commonly employed inert vectors penetratin and tat. Data presented herein thus propound the utility of inverso mastoparan as a highly efficient peptide vector. Furthermore, correlation analysis of plasma membrane translocation and intracellular uptake efficacy further supports a two-compartment model of CPP import whereby the intracellular accumulation of polycationic peptides is dependent upon both the efficiency of transport into the cell and their subsequent accretion at distinct subcellular loci.     

Bioportides: Bioactive cell-penetrating peptides that modulate cellular dynamics

The study and exploitation of cell-penetrating peptides (CPPs) now extends into a third exciting decade. Pharmacokinetic modulators, including the more common sequences Tat, penetratin and transportan-10, markedly enhance the intracellular delivery of small drugs, peptides, oligonucleotides and proteins. We introduced the term bioportide to distinguish cell penetrant peptides with intrinsic bioactivities from more typically inert CPP vectors. Our first examples included rhegnylogically organised bioportides, monomeric peptides presenting pharmacophores for both cellular internalization and bioactivity discontinuously distributed within the primary sequence. However, it is conceptually expedient to employ the same terminology to encompass s ychnologic bioportides that comprise an inert CPP vector conjugated to an otherwise impermeable bioactive peptide. In such cases the CPP provides an obvious address function whilst the bioactive cargo, often a protein mimetic sequence, is the message. Additional targeting sequences, usually added as chimeric extensions, can also be accommodated within the design of CPPs and bioportides to enable cell- and tissue-selective targeting. Thus, the identification and exploitation of bioportides provides further scope to employ CPPs as research tools, diagnostics and therapeutics spanning a range of pathologies.     

Mitoparan and target-selective chimeric analogues: membrane translocation and intracellular redistribution induces mitochondrial apoptosis

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.     

Biochemical pharmacology of total retro-inverso analogues of bradykinin and angiotensin II: Molecular recognition by G-protein-coupled receptors and angiotensin converting enzyme

Summary Total retro-inverso (TRI) analogues of bradykinin (BK), the B_2a -selective kinin antagonistd-Arg⁰[Hyp³,d-Phe⁷,Leu⁸]BK, angiotensin II (AT II) and the AT II antagonist Saralasin ([Sar¹, Val⁵, Ala⁸]AT II) were prepared by conventional solid-phase synthesis. Molecular recognition of TRI peptidomimetics by G-protein-coupled receptors was studied by competitive radioligand displacement experiments. TRI analogues ofd-Arg⁰[Hyp³,d-Phe⁷,Leu⁸]BK specifically bound to the kidney medulla B_2a bradykinin receptor with affinities (K _d ) ranging from 64 μM to 4 μM. Conversely, TRI analogues of BK, AT II and Saralasin did not bind to either the B_2a bradykinin receptor or the rat AT_1a AT II receptor, respectively. These studies indicate that the TRI strategy is more compatible with the synthesis of antagonists than ‘agonists’. Three TRI peptidomimetics ofd-Arg⁰[Hyp³,d-Phe⁷,Leu⁸]BK were weak inhibitors of angiotensin converting enzyme. All other TRI peptidomimetics had no effect upon ACE activity. These data endorse the utility of the TRI strategy for the synthesis of protease-resistant antagonists of peptide hormones and neuropeptides.     

Proteomimetic Cell Penetrating Peptides

QSAR analysis of the primary sequence of human cytochrome c indicated that numerous putative cell penetrating peptide sequences are located within major amino- and carboxyl-terminal helical domains. Two such sequences, Cyt c ⁷⁷⁻¹⁰¹ and the shorter homologue Cyt c ⁸⁶⁻¹⁰¹, readily translocate the plasma membrane of U373MG astrocytoma cells but differentially accumulate in extranuclear or nuclear compartments. Such sequences could be employed for the selective intracellular targeting of cytotoxins and other bioactive cargoes. Moreover, these peptides also induce apoptosis, indicating that they mimic the role of cytochrome c as a key regulator of programmed cell death. We propose that these domains may constitute an integral transduction domain within cytochrome c that enables the protein to directly translocate plasma membranes. These, and other studies, indicate that cell penetrating peptides (CPP) are valuable tools both for the purposes of intracellular delivery and for the direct manipulation of therapeutically relevant proteins and cellular processes. We propose that the term bioportide should be introduced to distinguish biologically active, proteomimetic CPP from inert sequences that include the majority of common vectors.