Design, Preparation And Function Of Active Peptide Analogues

This study is design and preparation of active peptide analogues of mung bean trypsin inhibitor Lys fragment, Trichosanthes trypsin inhibitor I, carcinoembryonic antigen peptide and aspartame using solid and liquid phase peptide synthesis methods. Based on the activities of the synthtic analogues, the ralationship of structure and activity was discussed.Chapter one is template-assisted rational design of peptide inhibitors of furin using the lysine fragment of the mung bean trypsin inhibitor. Highly active, small-molecule furin inhibitors are attractive drug candidates to fend off bacterial exotoxins and viral infection. Based on the 22-residue, active Lys fragment of the mung bean trypsin inhibitor, a series of furin inhibitors were designed and synthesized, and their inhibitory activity toward furin and kexin was evaluated using enzyme kinetic analysis. The most potent inhibitor, containing 16 amino acid residues with a Ki value of 2.45×10^-9 M for furin and of 5.60×10^-7 M for kexin, was designed by three incremental approaches. First, two non-essential Cys residues in the Lys fragment were deleted via a Cys-to-Ser mutation to minimize peptide miss-folding. Second, residues in the reactive site of the inhibitor were replaced by the consensus substrate recognition sequence of furin, namely, Arg at P₁, Lys at P₂, Arg at P₄ and Arg at P₆. In addition, the P₇ residue Asp was substituted with Ala to avoid possible electrostatic interference with furin inhibition. Finally, the extra N and C terminal residues beyond the doubly conjugated disulfide loops were further truncated. However, all resultant synthetic peptides were found to be temporary inhibitors of furin and kexin during a prolonged incubation, with the scissile peptide bond between P1 and P'1 cleaved to different extents by the enzymes. To enhance proteolytic resistance, the P'1 residue Ser was mutated to D-Ser or N-methyl Ser. The N-methyl Ser mutant gave rise to a Ki value of 4.70x10^-8 M for furin, and retained over 80% inhibitory activity even after a 3-h incubation with the enzyme. By contrast, the D-Ser mutant was resistant to cleavage, although its inhibitory activity against furin drastically decreased. Our findings identify a useful template for the design of potent, specific and stable peptide inhibitors against furin, shedding light on the molecular determinants that dictate the inhibition of furin and kexin.Charpter two studies on the two disulfide bonds (Cys14- Cys26 or Cys8- Cys20)of Trichosanthes trypsin inhibitor I. Disulfide bridges between cysteine residues are the key structural and functional elements of protein. Could theÏ€stacking made by two aromatic residues partially compensate for the loss of the disulfide bridge? Two analogues of Trichosanthes trypsin inhibitor I with the Cys14- Cys26 or Cys8- Cys20 disulfide bonds replaced by Tys or Phe and two negative control analogues substitution serine for cysteine at 14 and 26 or 8 and 20 positions were synthesized, respectively. The C14F-C26Y mutant had 1/140 inhibitory activity of native Trichosanthes trypsin inhibitor I to trypsin. But the negative control the C14S-C26S mutant had no activity ( Ki > 0.4 mM) indicating that theÏ€stacking made by C14F and C26Y may partially compensate for the action of the disulfide bond. However, different results obtained from the Cys14- Cys26 mutants that both the C8Y-C20Fand C8S-C20S mutants had no activity( Ki > 0.4 mM) . So theÏ€stacking made by two aromatic residues may partially compensate for the loss of the disulfide bridge in the some conditions.Charpter three studies on the action of the five amino acid residues (position 1, 2, 3, 4, 5) of carcinoembryonic antigen peptide-1 (CAP-1, YLSGANLNL). CAP-1 is an HLA-A2 resticted epitope of tumor antigen carcinoembryonic antigen (CEA). The epitope CAP-1 is an attractive drug target because more than 40% Chinese patients are HLA-A2 positive and more than 90% of gastric cancer, colon cancer and rectum cancer patients are carcinoembryonic antigen expressing positive. Autologous human cultured dendritic cell, loaded with CAP-1 was used for the treatment of the patients with advanced CEA-expressing malignancies in clinic. The analogues with five amino acid residues (position 1, 2, 3, 4, 5) were replaced by other residues were synthesized and tested for the activities of binding to HLA-A2 and generating cytotoxic T Lymphocyte. The analogue with the Gly4 mutation to Pro had a higher activity for binding to HLA-A2 and lower activity for generating cytotoxic T Lymphocyte. Our findings indicate the Gly4 influence the binding of CAP-1 to HLA-A2 and T cell receptor, shedding light on the molecular design of new epitope analogues.Charpter four studies on the relationship of structure and activity of aspartame. An aspartame analogue, L-asparaginyl L-3-phenyllactic acid methyl ester was synthesized with aspartic acid replaced by asparagine and peptide bond replaced by ester bond. The aspartic acid of aspartame could be replaced by asparagine as reported in the literature. In this analogue, the hydrogen of amide group could still form a hydrogen bond with the oxygen of ester bond and the ester bond was isosteric with peptide bond. However, the product was not sweet, showing that the peptide bond could not be replaced by ester bond. The peptide C-N bond behaves as a double bond that is not free to rotate and the C, O, N and H atoms are in the same plane. The replacement of peptide bond by ester bond destroyed the unique conformation of peptide bond, resulting in the loss of sweet taste.