David J. S. Guthrie

Identification of the region of non‐Aβ component (NAC) of Alzheimer's disease amyloid responsible for its aggregation and toxicity

The non‐beta‐amyloid (Aβ) component of Alzheimers disease amyloid (NAC) and its precursor α‐synuclein have been linked to amyloidogenesis in several neurodegenerative diseases. NAC and α‐synuclein both form β‐sheet structures upon ageing, aggregate to form fibrils, and are neurotoxic. We recently established that a peptide comprising residues 3–18 of NAC retains these properties. To pinpoint the exact region responsible we have carried out assays of toxicity and physicochemical properties on smaller fragments of NAC. Toxicity was measured by the ability of fresh and aged peptides to inhibit the reduction of the redox dye 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5 diphenyltetrazolium bromide (MTT) by rat pheochromocytoma PC12 cells and human neuroblastoma SHSY‐5Y cells. On immediate dissolution, or after ageing, the fragments NAC(8–18) and NAC(8–16) are toxic, whereas NAC(12–18), NAC(9–16) and NAC(8–15) are not. Circular dichroism indicates that none of the peptides displays β‐sheet structure; rather all remain random coil throughout 24 h. However, in acetonitrile, an organic solvent known to induce β sheet, fragments NAC(8–18) and NAC(8–16) both form β‐sheet structure. Only NAC(8–18) aggregates, as indicated by concentration of peptide remaining in solution after 3 days, and forms fibrils, as determined by electron microscopy. These findings indicate that residues 8–16 of NAC, equivalent to residues 68–76 in α‐synuclein, comprise the region crucial for toxicity.

Inhibition of fibril formation and toxicity of a fragment of α-synuclein by an N-methylated peptide analogue

Alpha-synuclein has been linked to amyloidogenesis in Parkinsons disease and other neurodegenerative disorders. We have previously shown that a peptide comprising residues 68-78 of alpha-synuclein is the minimum fragment that, like alpha-synuclein itself, forms amyloid fibrils and exhibits toxicity towards cells in culture. Hughes et al. [J. Biol. Chem. 275 (2000) 25109] showed that an N-methylated derivative of Abeta(25-35) inhibited the formation of fibrils by Abeta(25-35) and reduced its toxicity. We have now extended this concept to an amyloidogenic alpha-synuclein-based peptide. Alpha-synuclein(68-78), N-methylated at G1y73, was compared to non-methylated peptide. Whereas alpha-synuclein(68-78) formed fibrils and was toxic to cells, the N-methylated analogue had neither of these properties. Moreover, an equimolar mixture of the non-methylated and methylated peptides formed very few fibrils and toxicity was markedly reduced.

The synthesis of some peptides related to the amyloid ? peptide 25?35: Use of N-(2-hydroxy-4-methoxybenzyl) protection

The sequence from residues 25–35 of the amyloid β peptide has been identified as an important factor in the neurotoxicity displayed by this peptide. Attempts to synthesize analogues of this sequence using Fmoc-protected amino acids in solid phase synthesis were initially unsuccessful, with coupling stopping after Ala30. By incorporating N-(2-hydroxy-4-methoxybenzyl)alanine at this position, total synthesis of the undecapeptide was accomplished. Success was also achieved by switching the synthesis to a commercial synthesiser which uses a flow system and improved solvent conditions.

An enzyme-scissile linker for solid-phase peptide synthesis

We describe a new linker that contains a phosphodiester moiety for solid-phase peptide synthesis; after completion of the cycle of coupling and deprotection steps, the phosphodiester can be cleaved with phosphodiesterase.

Effects of some neurokinin A analogues on tachykinin-induced contraction of guinea pig trachea.

A series of analogues of neurokinin A (NKA) has been synthesized and characterized by testing for their abilities, in vitro, to contract guinea pig tracheal smooth muscle or to antagonize NKA-, NKB- and SP-induced contraction of this tissue. Substitution of NKA residues Gly8 or Leu9 by conformationally restricting amino acids produced peptides that were antagonists of NKA action, but the type and specificity of the antagonism depended on the size of the peptide. Thus, while [Ala5, Aib8, Leu10]NKA(2-10) showed no agonism and was a specific, competitive antagonist of NKA, [Ala5, Aib8, Leu10]NKA(4-10) was a noncompetitive antagonist of NKA and substance P (SP) and was itself a weak agonist at concentrations above 10(-7) M.

Thioesters of amino acid derivatives as thioacylating agents in thiopeptide synthesis

Thioesters, mainly of N-substituted glycine, have been synthesized and examined for their ability to thioacylate amino acids, peptides, and esters and amides of amino acids and peptides. Methyl, ethyl, and benzyl thioesters have been obtained in high yield by thiohydrolysis of the corresponding imino esters. All were found to be excellent thioacylating agents for amino acids, though under the conditions used, they reacted much less readily with amino acid esters and amides including peptides, except where the reacting nucleophile was the amino group of Gly or the imino group of Pro. Attempts to improve the leaving properties of the alkoxy group (OR′) in O-alkyl thioesters (RCSOR′) by introduction of a 2-nitro substituent result in poorer yields of the thioester, probably because of competing elimination between the iminium group and OR′ on treatment of the imino ester with H2S, although the nitro substituted thioesters are slightly more reactive than simple alkyl derivatives. The reaction of N-benzyloxycarbonylaminoacetonitrile with phenol gives a 30% yield of a product which failed to yield any thioester on reaction with H2S.

Conformational studies on analogues of the invertebrate peptide pyroGlu-Asp-Pro-Phe-Leu-Arg-Phe-NH2 using 1H NMR

The peptide pQDPFLRFamide is one of several closely related heptapeptides found in invertebrates, including molluscs. Electrophysiological findings and ligand binding studies have both suggested that these heptapeptides probably interact with receptors distinct from those that are activated by the related tetrapeptide FMRFamide (also found in the same group of invertebrates), despite the fact that some synthetic N-terminally extended analogues of the latter show marked tetrapeptide-like activity. We have carried out structural studies, using 1- and 2-dimensional 1H NMR, on pQDPFLRFamide and some synthetic analogues in which Asp-2 was replaced by Asn and Pro-3 by either Aib (α-amino isobutyric acid) or by Gly. The results are consistent with the suggestion that pQDPFLRFamide can adopt a bent conformation, which might form the basis of the selectivity of this and related heptapeptides.