Design, synthesis, and biological evaluation of new cyclic disulfide decapeptides that inhibit the binding of AP-1 to DNA.

The transcription factor activator protein-1 (AP-1) is an attractive target for the treatment of immunoinflammatory diseases, such as rheumatoid arthritis. Using the three-dimensional (3D) X-ray crystallographic structure of the DNA-bound basic region leucine zipper (bZIP) domains of AP-1, new cyclic disulfide decapeptides were designed and synthesized that demonstrated AP-1 inhibitory activities. The most potent inhibition was exhibited by Ac-c[Cys-Gly-Gln-Leu-Asp-Leu-Ala-Asp-Gly-Cys]-NH2 (peptide 2) (IC50 = 8 microM), which was largely due to the side chains of residues 3-6 and 8 of the peptide, as shown by an alanine scan. To provide structural information about the biologically active conformation of peptide 2, the structures of peptide 2 derived from molecular dynamics simulation of the bZIP-peptide 2 complex with explicit water molecules were superimposed on the solution structures derived from NMR measurements of peptide 2 in water. These showed a strong structural similarity in the backbones of residues 3-7 and enabled the construction of a 3D pharmacophore model of AP-1 binding compounds, based on the chemical and structural features of the amino acid side chains of residues 3-7 in peptide 2.

Eight-residue Abeta peptides inhibit the aggregation and enzymatic activity of Abeta42.

Insoluble Abeta1-42 is the main component of the amyloid plaque. We have previously demonstrated that exposure to low pH can confer the molten globule state on soluble Abeta1-42 in vitro [Biochem. J. 361 (2000) 547] and unfolding experiments with guadinine hydrochloride (GdnHCl) have now confirmed this observation. The molten globule state of the protein has many biological properties and understanding the mechanisms of its formation is an important step in devising a therapeutic strategy for Alzheimer's disease (AD). We therefore investigated the ability of a series of synthetic eight-residue peptides derived from Abeta1-42 to inhibit the acid-induced aggregation of Abeta1-42 and identified the potent peptides to be Abeta15-22, Abeta16-23 and Abeta17-24. A1-antichymotrypsin, a member of the serine proteinase inhibitor (serpin) family is another major component of the amyloid plaque. In the present study, we investigated the proteolytic activity of Abeta1-42 against casein at different pHs. Chemical modification of amino acid residues in Abeta1-42 indicated that serine and histidine residues, but not aspartic acid, are necessary for enzymatic activity, suggesting that it is a serine proteinase. Amino acid substitution studies indicate that glutamic acids at positions 11 and 22 participate indirectly in proteolysis and we surmise that amino acid residues 29-42 are required to stabilize the conformer. A study of metal ions suggested that Cu2+ affected the enzymatic activity, but Zn2+ and Fe2+ did not. Interestingly, Abeta14-21 and Abeta15-22 were the only peptides that inhibited the proteolytic activity of Abeta42. Therefore, Abeta15-22 may control both aggregation of Abeta1-42 at acidic pH and its proteolytic activity at neutral pH. Consequently, we suggest that it may be of use in the therapy of Alzheimer's disease.

A pH-dependent conformational transition of Abeta peptide and physicochemical properties of the conformers in the glial cell.

In the present study we identified the epitopes of antibodies against amyloid beta-(1-42)-peptide (Abeta1-42): 4G8 reacted with peptides corresponding to residues 17-21, 6F/3D reacted with peptides corresponding to residues 9-14, and anti 5-10 reacted with peptides corresponding to residues 5-10. The study also yielded some insight into the Abeta1-42 structures resulting from differences in pH. An ELISA study using monoclonal antibodies showed that pH-dependent conformational changes occur in the 6F/3D and 4G8 epitopes modified at pH 4.6, but not in the sequences recognized by anti 1-7 and anti 5-10. This was unique to Abeta1-40 and Abeta1-42 and did not occur with Abeta1-16 or Abeta17-42. The reactivity profile of 4G8 was not affected by blockage of histidine residues of pH-modified Abeta1-40 and Abeta1-42 with diethyl pyrocarbonate; however, the mutant [Gln(11)]Abeta1-40 abrogated the unique pH-dependence towards 4G8 observed with Abeta1-40. These findings suggest that these epitopes are cryptic at pH 4.6, and that Glu(11) is responsible for the changes. We suggest that the abnormal folding of 6F/3D epitope affected by pH masked the 4G8 epitope. A study of the binding of metal ions to Abeta1-42 suggested that Cu(2+) and Zn(2+) induced a conformational transition around the 6F/3D region at pH 7.4, but did not affect the region when it was modified at pH 4.6. However, Fe(2+) had no effect, irrespective of pH. Abeta modified at pH 4.6 appeared to be relatively resistant to proteinase K compared with Abetas modified at pH 7.4, and the former might be preferentially internalized and accumulated in a human glial cell. Our findings suggest the importance of microenvironmental changes, such as pH, in the early stage of formation of Abeta aggregates in the glial cell.