Related Hexapeptide Research Articles

Structure−Activity Relationship Studies Optimizing the Antiproliferative Activity of Novel Cyclic Somatostatin Analogues Containing a Restrained Cyclic β-Amino Acid

The cyclic somatostatin analogue cyclo[Pro(1)-Phe(2)-D-Trp(3)-Lys(4)-Thr(5)-Phe(6)] (L-363,301) displays high biological activity in inhibiting the release of growth hormone, insulin, and glucagon. According to the sequence of L-363,301, we synthesized a number of cyclic hexa- and pentapeptides containing nonnatural alpha- and beta-amino acids. The N- fluorenylmethoxycarbonyl protected cyclic beta-amino acid [1S, 2S, 5R]-2-amino-3,5-dimethyl-2-cyclohex-3-enecarboxylic acid (cbetaAA), for the replacement of the Phe(6)-Pro(1) moiety of L-363,301, was synthesized in two steps by an enantioselective multicomponent reaction using (-)-8-phenylmenthol as a chiral auxiliary. The resulting peptide cyclo[cbetaAA(1)-Tyr(2)-D-Trp(3)-Nle(4)-Thr(Trt)(5)] (Trt = triphenylmethyl) shows high antiproliferative effects in an in vitro assay with A431 cancer cells. The same peptide without the Trt group does not reveal any biological activity, whereas L-363,301 and closely related hexapeptides show only minor activity. By comparison of the solution structure of cyclo[cbetaAA(1)-Tyr(2)-D-Trp(3)-Nle(4)-Thr(Trt)(5)] with the structure of l-363,301, a nearly perfect match of the betaII'-turn region with d-Trp in the i + 1 position was observed. The cyclic beta-amino acid cbetaAA is likely needed for the bioactive conformation of the peptide.

10-Helical conformations in oxetane β-amino acid hexamers

The first structural investigations were undertaken on a β-hexapeptide 7 in which the peptide backbone is constrained by cis-substituted oxetane rings. Detailed NMR studies in CDCl 3 and C 6D 6 together with molecular mechanics conformational analysis identify a well-defined left-handed helical structure stabilised by 10-membered hydrogen-bonded rings. Comparison with two related hexapeptides 8 and 9 suggests a similar structural preference for these systems.

Peptide inhibitors of DNA cleavage by tyrosine recombinases and topoisomerases

The study of biochemical pathways requires the isolation and characterization of each and every intermediate in the pathway. For the site-specific recombination reactions catalyzed by the bacteriophage λ tyrosine recombinase integrase (Int), this has been difficult because of the high level of efficiency of the reaction, the highly reversible nature of certain reaction steps, and the lack of requirements for high-energy cofactors or metals. By screening synthetic peptide combinatorial libraries, we have identified two related hexapeptides, KWWCRW and KWWWRW, that block the strand-cleavage activity of Int but not the assembly of higher-order intermediates. Although the peptides bind DNA, their inhibitory activity appears to be more specifically targeted to the Int-substrate complex, insofar as inhibition is resistant to high levels of non-specific competitor DNA and the peptides have higher levels of affinity for the Int-DNA substrate complex than for DNA alone. The peptides inhibit the four pathways of Int-mediated recombination with different potencies, suggesting that the interactions of the Int enzyme with its DNA substrates differs among pathways. The KWWCRW and KWWWRW peptides also inhibit vaccinia virus topoisomerase, a type IB enzyme, which is mechanistically and structurally related to Int. The peptides differentially affect the forward and reverse DNA transesterification steps of the vaccinia topoisomerase. They block formation of the covalent vaccinia topoisomerase-DNA intermediate, but have no apparent effect on DNA religation by preformed covalent complexes. The peptides also inhibit Escherichia coli topoisomerase I, a type IA enzyme. Finally, the peptides inhibit the bacteriophage T4 type II topoisomerase and several restriction enzymes with 2000-fold lower potency than they inhibit integrase in the bent-L pathway.

Effect of Conformational Constraints on the Topography of Complex Potential Energy Surfaces

The extent to which structural modifications affect the topography of complex molecular energy landscapes is largely unknown. Through quantitative mapping of the potential energy surfaces of two related hexapeptides, we find that a conformational constraint can completely alter the character of the molecular energy landscape. The linear peptide exhibits a single-funnel potential topography, while its cyclic analog exhibits a shallow energy landscape with competing basins and unevenly distributed roughness.

Neurotensin and Neuromedin N Are Differently Metabolized in Ventral Tegmental Area and Nucleus Accumbens

Whole homogenates and membrane-bound and cytosoluble fractions prepared from rat ventral tegmental area (VTA) and nucleus accumbens were examined for their content of peptidasic activities and for their ability to metabolize neurotensin and its natural related hexapeptide neuromedin N. No qualitative differences were observed between these two brain regions concerning the presence and the subcellular distribution of a series of activities able to hydrolyze various specific fluorimetric enzymatic substrates. However, aminopeptidase B, endopeptidase 24-15, and endopeptidase 24-11 were significantly lower in the VTA than in the nucleus accumbens membrane preparations, while proline endopeptidase was detected in significantly higher amount only in the cytosolic fraction prepared from nucleus accumbens. Both neurotensin and neuromedin N were metabolized more rapidly in the nucleus accumbens than in the VTA. Furthermore, the degradation rate of neuromedin N was considerably faster than that of neurotensin whatever the cerebral area examined. Studies carried out with highly specific peptidase inhibitors revealed that endopeptidase 24-15 mainly contributed to the catabolism of neurotensin in homogenates and membrane-bound preparations of nucleus accumbens and VTA, while aminopeptidase B appeared predominantly responsible for the rapid disappearance of neuromedin N in both cerebral tissues. The possibility that the different metabolic processes of the two peptide congeners could explain their distinct pharmacological profiles observed after their microinjection in the nucleus accumbens and in the VTA is discussed.

Opioid peptides. Synthesis and biological properties of dermorphin related hexapeptides

The Gly 4 and/or Tyr 5 residues in dermorphin hexapeptide (H-Tyr- d-Ala-Phe-Gly-Tyr-Pro-OH) were replaced by Nα-methyl- or d-amino acids in order to examine the effect on opioid activity. Two pseudopeptides (H-Tyr- d-Ala-Phe-Gly-ψ (NHCO)-Xaa-Pro-OH, Xaa — Tyr or Phe) in which the Gly 4Xaa bond is reversed, were also prepared. Metabolic stability, analgesia and selectivity of these compounds for different receptor populations have been investigated. Results suggest that the 12 new analogues showed a negligible affinity for the K binding site and some selectivity for μ- or δ receptors. In some cases the analgesic potencies seems to be related to enzymatic stability of the peptides.

Potentiation by thiorphan and bestatin of the naloxone-insensitive analgesic effects of neurotensin and neuromedin n

Neurotensin induced significant antinociceptive activity as measured in a variety of nociceptive tests 10 and 30 min following intracerebroventricular (i.c.v.) injection in mice. The lowest effective peptide doses were 25 ng in the writhing test, 25–50 ng in the tail-flick test, 50–100 ng in the hot-plate test and 2000 ng in the tail electrical stimulation test. The neurotensin related hexapeptide neuromedin N also displayed antinociceptive properties but only in the writhing and tail-flick tests. Furthermore, as compared to neurotensin, the neuromedin effects required higher doses. ED 50's for neurotensin and neuromedin in the writhing test were 70 ng and 1070 ng, respectively. Separate or combined injections of the endopeptidase 24.11 (enkephalinase) inhibitor thiorphan (l0μg) and the aminopeptidase inhibitor bestatin (50μg) did not affect tail-flick latencies. In contrast, i.c.v. injection of thiorphan together with an ineffective dose of neurotensin (25 ng) resulted in a significant antinociceptive effect. Bestatin did not modify tail-flick latencies in neurotensin-treated mice whether in the absence or presence of thiorphan. On the contrary, each of these peptidase inhibitors promoted antinociceptive effects of subthreshold doses of neuromedin (lμg) in the tail-flick test. Maximal antinociception was obtained by combining both inhibitors, thus conferring antinociceptive effects to neuromedin doses that were as low as 10 ng. Naloxone (0.5–2 mg/kg, s.c.) did not significantly reduced the antinociceptive effects of combinations of neurotensin and thiorphan and of neuromedin, thiorphan and bestatin. The data show that both neurotensin and neuromedin elicit analgesia in mice through an opiate independent mechanism. Furthermore, like enkephalin, neuromedin is readily degraded by brain endopeptidase 24.11 and bestatin sensitive aminopeptidase(s), whereas the resistance of neurotensin to aminopeptidase attack confers to this peptide a broader spectrum and longer duration of action than its congener neuromedin.

Neurotensin binding sites in the forebrain and midbrain of the pigeon.

An autoradiographic method was used to assess the distribution of binding sites for [3H]neurotensin (NT) in the forebrain and midbrain of the pigeon. Within the telencephalon the highest levels of NT binding sites were observed within the hyperstriatum ventrale (HV). Moderate to high levels of NT binding were observed within the archistriatum, neostriatum intermedium, and hyperstriatum accessorium. These telencephalic regions and HV are thought to be comparable to portions of mammalian neocortex. Lower levels of binding sites were observed within the striatal complex including the laterally situated paleostriatum augmentatum and medially situated lobus parolfactorius. The lowest levels of NT binding sites in the telencephalon were observed within the paleostriatum primitivum (PP, considered comparable to mammalian globus pallidus), ectostriatum (comparable to layer IV of mammalian extrastriate visual cortex), field "L" (comparable to layer IV of mammalian auditory cortex), hippocampus, septum, and preoptic area. Despite considerable regional variation, the overall level of NT binding throughout the pigeon telencephalon appears to be significantly higher than that reported for mammals, particularly within pallial areas. Within the brainstem, moderate levels of NT binding sites were observed in the lateral habenular nuclei, the ventral tegmental area of Tsai, nucleus tegmentipedunculopontinus, pars compacta (comparable to the mammalian substantia nigra, pars compacta), locus coeruleus, and the nucleus subcoeruleus dorsalis. The latter four cell groups contain numerous catecholaminergic neurons. Corresponding catecholaminergic cell groups in mammalian forms also contain high levels of NT receptors. As in mammals, lower levels of NT binding were observed in most diencephalic nuclei. Somewhat higher levels of NT binding were observed within the pretectal nuclei spiriformis lateralis and spiriformis medialis. Moderate levels of NT binding sites were observed within the retinal terminal layers of the tectum (i.e., layers 1-7). Immunohistochemical experiments (Reiner and Carraway; Brain Res. 341:365-371, '85; Reiner: ARVO Abstracts: p. 185, '86) localizing NT and a related hexapeptide, LANT6, have shown that LANT6 is present in retinal ganglion cells, in cells of the paleostriatum, and in the striatotegmental and striatopretectal fiber pathways of the pigeon. Thus some features of the NT binding observed here in pigeon brain including the existence of substantial NT binding sites in the brainstem catecholamine nuclei, pretectum (nucleus spiriformis lateralis), and optic tectum may reflect the existence, at least in part, of functional receptors for LANT6.(ABSTRACT TRUNCATED AT 400 WORDS)

Site specificity of casein kinase-2 (TS) from rat liver cytosol. A study with model peptide substrates.

The factors determining the site recognition and phosphorylation by rat liver casein kinase-2 (CK-2) have been explored with a set of 14 related hexapeptides each including a single phosphorylatable amino acid and five acidic plus neutral residues. Such peptides are different from each other in the following features: the nature of the phosphorylatable amino acid, if any; its position relative to the critically required acidic residues; the extension and the structure of the acidic cluster. All of them were tested as substrate and/or competitive inhibitors of CK-2, and their kinetic and inhibition constants were determined. The results suggest the following conclusions. Under strictly comparable conditions Ser is by far preferred over Thr. Tyr not being affected at all. In order to carry out its role of structural determinant the critical acidic cluster must be located on the C-terminal side of the target residue, though not necessarily adjacent to it. The affinity for the protein-binding site, as deduced from Km and/or Ki values, is largely dependent on the number of acidic residues but it is also significantly enhanced if a hydroxylic residue is located on their N-terminal side. An acidic residue at position +3 relative to serine plays an especially important role for triggering phosphorylation, the peptide Ser-Glu-Glu-Ala-Glu-Glu having similar Km but negligible Vmax compared to Ser-Glu-Ala-Glu-Glu-Glu and Ser-Glu-Glu-Glu-Ala-Glu. These data provide a rationale for the substrate specificity of CK-2 and will give a helpful insight into the structure of the protein-binding site of this enzyme.