Carvell H. Williams

Aggregation and metal-binding properties of mutant forms of the amyloid Aβ peptide of Alzheimer's disease

Abstract: The fibrillogenic properties of Alzheimers Aβ peptides corresponding to residues 1–40 of the normal human sequence and to two mutant forms containing the replacement Ala21 to Gly or Glu22 to Gln were compared. At pH 7.4 and 37°C the Gln22 peptide was found to aggregate and precipitate from solution faster than the normal Aβ, whereas the Gly21 peptide aggregated much more slowly. Electron microscopy showed that the aggregates all had fibrillar structures. Circular dichroism spectra of these peptides revealed that aggregation of the normal and Gln22 sequences was associated with spectral changes consistent with a transformation from random coil to β sheet, whereas the spectrum of the Gly21 peptide remained almost unchanged during a period in which little or no aggregation occurred. When immobilised by spotting onto nitrocellulose membranes the peptides bound similar amounts of the radioisotope 65Zn2+. Of several competing metal ions, tested at 20× the concentration of Zn2+, Cu2+ displaced >95% of the radioactivity from all three peptides and Ni2+ produced >50% displacement in each case. Some other metal ions tested caused lesser displacement, but Fe2+ and Al3+ were without effect. In a saturation binding assay, a value of 3.2 µM was obtained for the binding of Zn2+ to Aβ but our data provided no evidence for a reported higher affinity site (107 nM). The results suggest that the neuropathology associated with the Gly21 mutation is not due to enhanced fibrillogenic or different metal‐binding properties of the peptide and that the binding of zinc to amyloid peptides is not a specific phenomenon.

Effects of the mutations Glu22 to Gln and Ala21 to Gly on the aggregation of a synthetic fragment of the Alzheimer's amyloid β/A4 peptide

We assessed the fibrillogenic properties of synthetic peptides corresponding to residues 13-26 of beta/A4 amyloid, containing either the normal sequence (beta 13 26) or the mutations Glu22 to Gln (beta 13-26Q22) and Ala21 to Gly (beta 13-26G21). The kinetics of aggregation were monitored at 37 degrees C and pH 7.4 by measuring the amount of peptide remaining in solution, using reverse-phase high performance liquid chromatography. Negative stain electron microscopy revealed that all of the peptides formed fibrils. However, beta 13-26Q22 showed greatly accelerated fibril formation compared to the other two. The results suggest that the Q22 mutation confers increased amyloidogenic properties on the beta/A4 peptide, whereas the G21 mutation acts by a different pathogenic mechanism.

Monoamine oxidase—I: Specificity of some substrates and inhibitors

Abstract A number of synthetic substrates and inhibitors of monoamine oxidase have been studied, using the enzyme from porcine brain. Km and Vmax values have been calculated for substrates using Lineweaver-Burk plots. In many cases large variations in Km and Vmax were observed for relatively small changes in the structure of substrates. Similar observations were made concerning Ki values for some competitive inhibitors. The effects of hydrogen-ion concentration on enzyme activity are consistent with the view that unprotonated amines are the species which bind to the enzyme. This finding, together with the observation that tertiary amines can act as substrates has led to formulation of a proposed mechanism of dehydrogenation which does not depend upon intermediate formation of Schiff base from the substrate amine.

Changes of angiotensin-converting enzyme activity in the pancreas of chronic hypoxia and acute pancreatitis.

Emerging data have provided evidence for the presence of a local renin-angiotensin system (RAS) in the pancreas, which play a role in the regulation of pancreatic microcirculation, thus affecting islet hormonal secretion. The present study aimed, therefore, at elucidating the presence and changes of angiotensin-converting enzyme (ACE) using reverse transcription-polymerase chain reaction (RT-PCR) and a specific assay for ACE activity using the internally quenched fluorogenic substrate Meoc-DL-Amp-Gly-Lys(epsilon -DNP)-Gln-OH. RT-PCR clearly demonstrated the expression of ACE mRNA in the pancreas. ACE activity was markedly and significantly increased by chronic hypoxia and by acute pancreatitis when compared with that of their respective control pancreas. Addition of captopril, a specific inhibitor for ACE, completely blocked the ACE activity both in the control and experimental groups. All these data suggest that increased activity of pancreatic ACE in chronic hypoxia and acute pancreatitis could have implications for pancreatic physiology and pathophysiology.

Discovery of a dual action first-in-class peptide that mimics and enhances CNS-mediated actions of thyrotropin-releasing hormone

Thyrotropin-releasing hormone (TRH) displays multiple CNS-mediated actions that have long been recognized to have therapeutic potential in treating a wide range of neurological disorders. Investigations of CNS functions and clinical use of TRH are hindered, however, due to its rapid degradation by TRH-degrading ectoenzyme (TRH-DE). We now report the discovery of a set of first-in-class compounds that display unique ability to both potently inhibit TRH-DE and bind to central TRH receptors with unparalleled affinity. This dual pharmacological activity within one molecular entity was found through selective manipulation of peptide stereochemistry. Notably, the lead compound of this set, L-pyroglutamyl-L-asparaginyl-L-prolyl-D-tyrosyl-D-tryptophan amide (Glp-Asn-Pro-D-Tyr-D-TrpNH(2)), is effective in vivo at producing and potentiating central actions of TRH without evoking release of thyroid-stimulating hormone (TSH). Specifically, this peptide displayed high plasma stability and combined potent inhibition of TRH-DE (K(i) 151 nM) with high affinity binding to central TRH receptors (K(i) 6.8 nM). Moreover, intraperitoneal injection of this peptide mimicked and augmented the effects of TRH on behavioural activity in rat. Analogous to TRH, it also antagonized pentobarbital-induced narcosis when administered intravenously. This discovery provides new opportunities for probing the role of TRH actions in the CNS and a basis for development of novel TRH-based neurotherapeutics.

Monoamine oxidase—III: Further studies of inhibition by propargylamines

Abstract The p K a values and partition coefficients have been determined for a number of propargylamines. It is shown that in a number of cases there is a close correlation between the partition coefficient and the effectiveness of inhibition of mitochondrial monoamine oxidase as measured by I 50 values. Using 14 C-labelled pargyline and clorgyline it is shown that, in vitro , these substances fail to bind to proteins other than MAO, and that a number of other irreversible inhibitors prevent pargyline from binding to this enzyme.

Biological activity and receptor binding properties of some C-terminally modified analogues of FMRFamide.

The functional role of the C-terminal amide group (-CONH2) of the molluscan regulatory peptide FMRFamide has been examined in two sets of analogues based on FnLKFamide and FnLRFamide (nL = norleucine). In each series the amide group was replaced by -CONHCH3, -CON(CH3)2, -CONHNH2, -COOCH3, -CH2OH, and -COOH. The analogues were tested for their ability to bind to receptors in membranes from Helix aspersa circumoesophageal ganglia and for their biological effects on the isolated Helix heart. The results indicate i) that agonist activity, but not binding to the receptor, requires the presence of the amide carbonyl group; ii) the hydrogen atoms of the amide group are not essential either for binding or for agonist activity (the mono- and dimethylamides were more effective than the parent compounds on both counts); iii) the is more effective an agonist than is the amide in stimulating Helix heart.

Visual detection of peptidase activity using fluorogenic substrates in a microtiter plate assay.

A simple, inexpensive, and sensitive assay for peptidase activity has been devised. The assay was performed in a microtiter plate and was based on fluorogenic peptide substrates, many of which are commercially available. 7-Amino-4-methyl coumarin the fluorescent product liberated during an incubation period of between 1 and 16 h, was detected by inspection of the plate under ultraviolet light of wavelength 356 nm. A fluorometer was not required. Using alpha-chymotrypsin as a model enzyme, with succinyl-L-alanyl-L-alanyl-L-prolyl-L-phenylalanine 4-methyl-coumaryl-7-amide as substrate, it was shown that as little as 4 fmol of enzyme could be detected. The method was non-quantitative and was particularly suited to location of enzyme activity in fractions during a purification procedure. The validity of the assay was demonstrated by detection of activity of a known enzyme, alpha-chymotrypsin, after its purification by size-exclusion high-performance liquid chromatography. The method was used to locate two forms of aminopeptidase activity, in fractions from size-exclusion chromatography of an extract from reproductive tissue of Helix aspersa, using L-leucine 4-methyl-coumaryl-7-amide as substrate.

A new specific inhibitor of monoamine oxidase A.

A new propynylamine has been tested as an inhibitor of MAO. Loosely based upon the structure of clorgyline, it is an irreversible inhibitor of MAO A but is apparently indifferent towards MAO B. This compound, N1-(2,4-dinitrophenyl)-N2-prop-2-ynyl 1,3-diaminopropane, may be useful as a model for the design of more potent but equally specific inhibitors of MAO A.

Synthesis of ketomethylene amino pseudopeptide analogues via reductive amination of glyoxals derived from α-amino acids

The reductive amination of an amino acid derived glyoxal, with the free amino group of a protected amino acid or oligopeptide fragment, has been developed as a simple and efficient method for the preparation of ketomethylene amino pseudo-oligopeptide isosteres Aa psi(COCH2NH)Aa. Trichlorosilane-DMF is the reagent of choice for the reduction.