Study On Immune Active Peptidomimetics For Their Synthesis, Bioactivity And Stability

Peptides that widespread in the nature are polymer of amino acid, most of which have various biological functions. Bioactive peptides possess many functions of nerve, hormone, immune regulation, anti-thrombus, anti-hypertension, anti-cholesterol, anti-bacteria, anti-cancer, anti-oxidation and cleaning free radical, etc. With the development of isolation and identification technique, more and more natural bioactive and medicine peptide compounds increasingly draw attention. Although many peptide sequences have been identified as potent bioactive agents, there are fundamental limitations associated with the development of peptides as therapeutics. The inherent conformational flexibility of a small peptide results in a myriad of conformations adopted by the peptide. In addition, there are a number of metabolic limitations restricting the use of peptides as therapeutics. Poor permeability across membranes, proteolytic degradation, rapid clearance, poor solubility in some cases, and a tendency to aggregate, all of which contribute to low oral availability of peptide for therapeutics. The susceptibility of externally administered peptides to proteolytic degradation in the gastrointestinal tract, blood and other tissues results in rapid clearance, thus significantly reduce the effectiveness of these peptides in inducing a satisfactory response. The peptide must therefore survive in thepharmacologically active form under conditions of exposure to various proteolytic enzymes in the digestive and circulatory systems. Given that therapeutics is usually administered far from the site of action, there is a clear need for enhanced metabolic stability.The use of peptidomimetics to overcome the limitations inherent in the physical characteristics of peptides has become an important strategy for improving the therapeutic potential of peptides. The term peptidomimetic is a broad term referring to any compound designed to perform the function of a peptide. Such functions include eliciting a specific biological effect through inhibition or agonist/antagonist activity. Generally, peptidomimetics are derived from a lead peptide sequence where structural modifications have been incorporated to improve binding affinity and/or metabolic resilience. Today, the method of designing peptidomimetics includes reform, circuit of amide-band and incorporation of non-protein amino acid, such as D-amino acid. The peptidomimetics incorporated D-amino acid can be classed into containing parts of D-amino acid and entire D-amino acid peptide in peptide. The peptides consisted entirely of D-amino acid have normal amino acid sequence and reverse amino acid sequence peptidomimetics. The activity of those peptidomimetics have many limitations, for instance, some have higher enzyme stability, but lower activity, even totally lost bioactivity. There are some mimetics difficult to synthesize, while the cost of synthesis materials of others mimetics is too high.A peptidomimetic approach with significant potential that has emerged in recent years is the use of B-amino acids. B-Amino acids are similar to a-amino acids in which they contain an amino terminus and a carboxyl terminus. However, these functional terminuses were separated by two carbon atoms. The peptides containing B-amino acid have been named as B-peptide, which can form definite secondary structure that resembles the natural a-peptide,such as spiral, folder and corner, so 6-peptide possessed similar bioactivity to a-peptide, and have excellent stability against most protease.We can learn from the summary of recent relative reports that 6-amino acid has been known very early, and along with relative research launching, researchers have learned that fi-amino acid has immense potent of biological molecular design. At present, 6-amino acid was utilized to developing researches of protease inhibitors, receptor Agonists and antagonists, 6-peptide vaccine and T cell receptor antagonist, etc. The method of utilizing 6-amino acid to prepare peptide and protein mimetics provide a new technique platform for drug design.Tal and TP5 are very excellent immune regulators and therapeutics drugs; however, they lack ideal enzyme stability and bioavaiability as oral drugs. As external administration of active peptide, it was cleared quickly for its sensitivity to protease, so its response function is decreased significantly, which limits clinical application of Tal and TP5. Researchers have to rerform structure to overcome the limitations, so many peptidomimetics were designed.Hitherto, researches of the relationship among structure, activity and stability of TP5 did not present the reports of utilizing 6-^ y-amino acid to substitute a-amino acid for reforming immune active peptide structure. According to the chemical structure of TP5, TP3 and Tal, we have designed TP5> Tal and TP3 mimetics containing 6-% y-amino acid. Those compounds were synthesized by solid phase synthesis strategy, and their activity and stability in vivo and vitro were researched.According to the possible active site in TP5 and structure characteristic of Tal and TP5, we designed and screened three immune active peptidomimetics from such synthesized compounds as LW501 > LW502-. TARE(l) > TARE (4) ^ 228-134 and LW301. As for the synthesis of TP3, we improveone-pot peptide synthesis method which is a convenient to perform, lower cost and high productivity. This innovation provides a new way for the improvement of producing short peptide technique and decreasing production cost.We studied immune activity of the designed peptidomimetics through external rosette, cell proliferation and inducing secretive IL-2. The results showed that all the peptidomimetics we designed still maintain bioactivity stimulating porcine thymus T cell E receptor generation except the TARE(4).We detected stimulating mice spleen lymph T cell proliferation assay in vivo and vitro and inducing secretive IL-2 activity, and made a research of effect mechanism in vitro of TP5 and Tal peptidomimetics. The experimental results showed that the immune active peptidomimetics that we designed have similar bioactivity to TP5 and Tal. Those results demonstrated that the peptidomimetics and TP5% Tal perhaps couple the same site of the receptor on T cell surface, and possessed the same effect mechanism.To detect peptidomimetics stability, we detected stability of both TP5 and their peptidomimetics by utilizing human plasma in vitro. The results showed that TP5 and their peptidomimetics were broken at Arg-Lys band on the N-terminal, the hydrolysis products were Lys-Asp-Val-Tyr. The half life of TP5 and peptidomimetics in vitro human plasma is as follows: 1.077min (TP5), 2.962min (LW501) and 12.499min (LW502), which demonstrated that two TP5 mimetics LW501 and LW502 have higher plasma stability than TP5.To research the stability of Tal mimetics, we used rats as animal model to detect stability of Tal and mimetics in vivo of rats. The results showed that the half life of mimetics TARE (4) is 89.12h, far higher than Tal and other mimetics, almost twelve times of Tal and other mimetics in half life. The half life of other mimetics was lower slightly than 7.02 h(T a 1), 4.13h (TARE(1))> 4.05h (228-134).