Siegmund Reissmann

Relationships between Cargo, Cell Penetrating Peptides and Cell Type for Uptake of Non-Covalent Complexes into Live Cells

Modulating signaling pathways for research and therapy requires either suppression or expression of selected genes or internalization of proteins such as enzymes, antibodies, nucleotide binding proteins or substrates including nucleoside phosphates and enzyme inhibitors. Peptides, proteins and nucleotides are transported by fusing or conjugating them to cell penetrating peptides or by formation of non-covalent complexes. The latter is often preferred because of easy handling, uptake efficiency and auto-release of cargo into the live cell. In our studies complexes are formed with labeled or readily detectable cargoes for qualitative and quantitative estimation of their internalization. Properties and behavior of adhesion and suspension vertebrate cells as well as the protozoa Leishmania tarentolae are investigated with respect to proteolytic activity, uptake efficiency, intracellular localization and cytotoxicity. Our results show that peptide stability to membrane-bound, secreted or intracellular proteases varies between different CPPs and that the suitability of individual CPPs for a particular cargo in complex formation by non-covalent interactions requires detailed studies. Cells vary in their sensitivity to increasing concentrations of CPPs. Thus, most cells can be efficiently transduced with peptides, proteins and nucleotides with intracellular concentrations in the low micromole range. For each cargo, cell type and CPP the optimal conditions must be determined separately.

Cell penetration: scope and limitations by the application of cell-penetrating peptides

The penetration of polar or badly soluble compounds through a cell membrane into live cells requires mechanical support or chemical helpers. Cell‐penetrating peptides (CPPs) are very promising chemical helpers. Because of their low cytotoxicity and final degradation to amino acids, they are particularly favored in in vivo studies and for clinical applications. Clearly, the future of CPP research is bright; however, the required optimization studies for each drug require considerable individualized attention. Thus, CPPs are not the philosophers stone. As of today, a large number of such transporter peptides with very different sequences have been identified. These have different uptake mechanisms and can transport different cargos. Intracellular concentrations of cargos can reach a low micromole range and are able to influence intracellular reactions. Internalized ribonucleic acids such as small interfering RNA (siRNA) and mimics of RNA such as peptide nucleic acids, morpholino nucleic acids, and triesters of oligonucleotides can influence transcription and translation. Despite the highly efficient internalization of antibodies, enzymes, and other protein factors, as well as siRNA and RNA mimics, the uptake and stabile insertion of DNA into the genome of the host cells remain substantially challenging.

Structure and Alignment of the Membrane-Associated Peptaibols Ampullosporin A and Alamethicin by Oriented 15N and 31P Solid-State NMR Spectroscopy

Ampullosporin A and alamethicin are two members of the peptaibol family of antimicrobial peptides. These compounds are produced by fungi and are characterized by a high content of hydrophobic amino acids, and in particular the alpha-tetrasubstituted amino acid residue ?-aminoisobutyric acid. Here ampullosporin A and alamethicin were uniformly labeled with (15)N, purified and reconstituted into oriented phophatidylcholine lipid bilayers and investigated by proton-decoupled (15)N and (31)P solid-state NMR spectroscopy. Whereas alamethicin (20 amino acid residues) adopts transmembrane alignments in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) or 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) membranes the much shorter ampullosporin A (15 residues) exhibits comparable configurations only in thin membranes. In contrast the latter compound is oriented parallel to the membrane surface in 1,2-dimyristoleoyl-sn-glycero-3-phosphocholine and POPC bilayers indicating that hydrophobic mismatch has a decisive effect on the membrane topology of these peptides. Two-dimensional (15)N chemical shift -(1)H-(15)N dipolar coupling solid-state NMR correlation spectroscopy suggests that in their transmembrane configuration both peptides adopt mixed alpha-/3(10)-helical structures which can be explained by the restraints imposed by the membranes and the bulky alpha-aminoisobutyric acid residues. The (15)N solid-state NMR spectra also provide detailed information on the helical tilt angles. The results are discussed with regard to the antimicrobial activities of the peptides.

A high-affinity bradykinin receptor in membranes from rat myometrium is coupled to pertussis toxin-sensitive G-proteins of the Gi family.

In rat myometrial membranes, two 3H-Bradykinin binding sites with KD values of 16 pM and 1.0 nM were identified. Employed at pM concentrations, bradykinin stimulated high affinity GTPases. This effect was abolished by the bradykinin antagonist, [D-Arg(Hyp3-Thi5,8, D-Phe7)]bradykinin (10 microM), and by treatment of membranes with pertussis toxin. Myometrial membranes contained two pertussis toxin substrates of 40 and 41 kDa, which corresponded immunologically to alpha-subunits of Gi-type G-proteins. The faster migrating substrate was tentatively identified as Gi2 alpha-subunit. The electrophoretic mobility of the slower migrating Gi alpha-subunit was very similar to that of the Gi3 alpha-subunit. Go alpha-subunits were not detected. Thus, in uterine smooth muscle, G-proteins of the Gi-family (Gi2, Gi3) couple high-affinity bradykinin receptors to their effector enzymes.

Transduction of Peptides and Proteins Into Live Cells by Cell Penetrating Peptides

Internalization of peptides and proteins into live cells is an essential prerequisite for studies on intracellular signal pathways, for treatment of certain microbial diseases and for signal transduction therapy, especially for cancer treatment. Cell penetrating peptides (CPPs) facilitate the transport of cargo‐proteins through the cell membrane into live cells. CPPs which allow formation of non‐covalent complexes with the cargo are used primarily in this study due to the relatively easy handling procedure. Efficiency of the protein uptake is estimated qualitatively by fluorescence microscopy and quantitatively by SDS–PAGE. Using the CPP cocktail JBS‐Proteoducin, the intracellular concentrations of a secondary antibody and bovine serum albumin can reach the micromolar range. Internalization of antibodies allows mediation of intracellular pathways including knock down of signal transduction. The high specificity and affinity of antibodies makes them potentially more powerful than siRNA. Thus, CPPs represent a significant new possibility to study signal transduction processes in competition or in comparison to the commonly used other techniques. To estimate the highest attainable intracellular concentrations of cargo proteins, the CPPs are tested for cytotoxicity. Cell viability and membrane integrity relative to concentration of CPPs are investigated. Viability as estimated by the reductive activity of mitochondria (MTT‐test) is more sensitive to higher concentrations of CPPs versus membrane integrity, as measured by the release of dead cell protease. Distinct differences in uptake efficiency and cytotoxic effects are found using six different CPPs and six different adhesion and suspension cell lines. J. Cell. Biochem. 112: 3824–3833, 2011.

Development of conformationally restricted analogues of bradykinin and somatostatin using constrained amino acids and different types of cyclization.

The structure-based design of peptide drugs requires the knowledge of the bioactive conformation. Studies on this receptor-bound 3D structure require linear or cyclic analogues with strongly reduced flexibility, but high biological activity, since only analogues with retained potency have preserved the bioactive conformation. Constrained amino acids containing double bonds or bulky substituents at the N(alpha)-, C(alpha)- and C(beta)-atom as well as at the aromatic ring atom were successfully applied to obtain potent and stable analogues of bradykinin and somatostatin, which due to their restricted conformation were suitable objects for conformational studies. Besides the generation of constrained cyclic analogues with improved biological and pharmacological properties, cyclic peptides were used as convenient models for the study of turn formations. Cyclization of the linear peptide bradykinin was performed by linking the N-terminus and the C-terminus, and in both bradykinin and somatostatin by cyclization using the amino acid side chains and by backbone cyclization. The later requires the introduction of N(alpha)-functionalised amino acids for ring closure which can be performed either through incorporation of N(alpha)-functionalised amino acids or dipeptide building units. Conformational analysis of a cyclic bradykinin analogue by means of NMR-studies together with molecular dynamics simulation led to a quasicyclic 3D structure with two turns and together with other 3D structures provided a pharmacophore model of bradykinin antagonists.

Potentiation of bradykinin actions by analogues of the bradykinin potentiating nonapeptide BPP9α

Abstract Synthetic analogues of the bradykinin potentiating nonapeptide BPP9α indicate significantly different structural requirements for potentiation of the bradykinin (BK)-induced smooth muscle contraction (GPI) and the inhibition of isolated somatic angiotensin I-converting enzyme (ACE). The results disprove the ACE inhibition as the only single mechanism and also the direct interaction of potentiating peptides with the bradykinin receptors in transfected COS-7 cells as molecular mechanism of potentiation. Our results indicate a stimulation of inositol phosphates (IP n ) formation independently from the B2 receptor. Furthermore, the results with La3+ support the role of extracellular Ca2+ and its influx through corresponding channels. The missing effect of calyculin on the GPI disproves the role of phosphatases in the potentiating action. These experimental studies should not only contribute to a better understanding of the potentiating mechanisms but also incorporate a shift in the research towards the immune system, in particular towards the immunocompetent polymorphonuclear leukocytes. The chemotaxis of these cells can be potentiated most likely by exclusive inhibition of the enzymatic degradation of bradykinin. Thus the obtained results give evidence that the potentiation of the bradykinin action can occur by different mechanisms, depending on the system and on the applied potentiating factor.

Structure activity relationships for bradykinin antagonists on the inhibition of cytokine release and the release of histamine.

Highly potent bradykinin antagonists were found to inhibit bradykinin-induced release of cytokines but to stimulate histamine release. Both actions show structural requirements completely different from those for bradykinin B1 and B2 receptors, indicating that the release of some cytokines from spleen mononuclear cells and of histamine from rat mast cells is not mediated by these receptors. Most potent bradykinin antagonists release histamine at lower concentrations than does bradykinin itself. Dimers of bradykinin antagonists are the most potent compounds for histamine release. In contrast to enhanced histamine release, potent inhibition of cytokine release enhances the applicability of these compounds as anti-inflammatory drugs. Many of the peptides designed for high B2-receptor antagonism were found to be compared by their concentrations far more potent for inhibition of cytokine release than for smooth muscle contraction. Thus, for some antagonists inhibition of cytokine release was detected at concentrations as low as 10(-15) M. The rational design of peptide and nonpeptide bradykinin antagonists for therapeutic use requires not only knowledge about the potency but also knowledge about the structure-activity relationships of such important side effects as cytokine and histamine release.

Structural requirements for B2-agonists with improved degradation stability.

Studies on bradykinin (BK) have been impeded by the fact that this peptide is rapidly degraded by various kininases. Modifications enacted to stabilize the BK sequence have usually resulted in a loss of agonistic activity. In this study, new structural modifications were investigated with the aim to identify degradation-resistant agonists on the bradykinin B2-receptor. The efficacy and degradation stability of several potentially agonistic derivatives were examined using a B2-receptor model (FURA-stained rat fibroblasts) and rat serum kininases. Modifications of the investigated BK analogues included amino-terminal (D-Arg) or carboxy-terminal (Ile-Tyr) prolongation, various substitutions at positions 2, 5, 7, 8 (tetrahydroisoquinoline-3-carboxylic acid, octahydroindole-2-carboxylic acid, hydroxy-proline, beta-2-thienylalanine, 2,3-dehydro-phenylalanine, erythro-beta-phenylserine, erythro-alpha-amino-beta-phenyl-butyric acid, N-methyl-phenylalanine), or intramolecular cyclization via lactam bridges. Kinin inactivation was investigated in rat serum, where the activities of angiotensin I-converting enzyme (ACE), carboxypeptidase N (CPN), aminopeptidase P (APP) and aminopeptidase M (APM) could be differentiated by selective inhibitors. Analogues derived from phyllokinin (BK-Ile-Tyr-SO4) and cyclic peptides had no receptor affinity. Useful modifications compatible with agonistic activity included D-Arg0 (protects against APP), D-N-methyl-Phe7 and dehydro-Phe5 (protect against ACE), and erythro-phenylserine or erythro-amino-phenyl-butyric acid at position 8 (protect against ACE and CPN). Finally, the kinin derivatives D-Arg0-[Hyp3, Thi5, epsilonSer(betaPh)8]-BK and D-Arg0-[Hyp3, Thi5, epsilonAbu(betaPh)8]-BK proved to be potent B2-agonists with extensive stability against rat serum kininases.

Structure determination of a pseudotripeptide zinc complex with the COSMOS-NMR force field and DFT methods

A His-X-His pseudotripeptide zinc complex (X is a N-alkyl glycine derivative) similar to the catalytic center of the carbonic anhydrase was computer designed and experimentally synthesized. Using 2D-NMR techniques, all proton, carbon chemical shifts and nuclear overhauser effect signals were assigned. The three-dimensional structure of the complex was determined with the COSMOS (computer simulation of molecular structures) force field by applying 13C bond polarization theory chemical shift pseudo forces and restrictions for NOE distances. From molecular dynamics, simulated annealing simulations and geometry optimizations, the three best force field structures were taken for a final investigation by density functional theory calculations.