Solid Phase Synthesis And Immunosuppressive Effects Of Hymenistatin-1 And Its Analogs

Survival of transplanted organ is a critical problem in transplantation. Cyclosporin A and FK-506, although be used as immunosuppressants in many fields, its application is limited for the side effects. So, it is necessary to search for a new, effective and nontoxic immunosuppressant for the transplantation.Hymenistatin-1 (HS-1), isolated by Petit in 1990, is a natural cyclooctopeptide. Its sequence is cyclo(-Ile¹-Ile²-Pro³-Pro⁴-Tyr⁵-Val⁶-Pro⁷-Leu⁸-). Siemion and his colleagues found that HS-1 could inhibit the humoral(PFC) and cellular(DTH) immunity. Dissolved in CHCl₃ or DMSO, there is a cis-amide bond between Pro³-Pro⁴ residues, aβⅥa turn in the Ile²-Tyr⁵ region andαβⅠorβⅡturn in the Val⁶- Ile¹ region.To evaluate the significance of the Ile²-Pro³ , Pro³-Pro⁴ and Val⁶-Pro⁷ peptide bonds for HS-1 biological activity, we have synthesized and screened four cyclic analogs of HS-1, with the dipeptide segments Ile²-Pro³ , Pro³-Pro⁴ , Val⁶-Pro⁷ and Pro³-Pro⁴ replaced by Ile²-statine³, Pro³-statine⁴, Val⁶-statine⁷ and statine³-statine⁴, respectively.The 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA) inhibitors (statins) have been proved to have a variety of inmunomodulating and other cellular effects, independent of their cholesterol lowering. The first new immune effect of statins is that statins inhibit the expressin of classâ…¡major histocompatibility antigens (MHC-â…¡) on human macrophages, endothelial cells and smooth muscle cells stimulated by interferonγ(IFN_γ). The second recently discovered immunosuppressive effect of statins is a selective blocking ofβ2 integrin, leukocyte function antigen-1(LFA-1). These two effects were unrelated to the statins' inhibition of HMG-CoA. These reports suggested that statins was a protential immunosuppressants. In this study, the statine is a special amino acid. Its chemical name is (3s, 4s)-4-amino-3-hydroxyl-6- methylheptanoic acid. All the statins are derivates of statine, and generaly, most peptides containing statine have significant physiological functions.Because of the lepto-bioactivity of HS-1, we tried to modify HS-1 with statine to enhance the immunosuppressive activity of HS-1 and synthesized natural HS-1 and some analogs with statine by solid-phase peptide synthesis(SPPS) methods.The crictical problem related to the success of the design, synthesis and purification of polypeptide is the optical purity. The products of SPPS are easy to purify and have high optical purity. With Fmoc or Boc method, we have synthesized HS-1 and four analogs with higher purity and yields.Through large scale of synthesis and screening, we have given up some analogs difficult to synthesize or purify and selected these four modified HS-1 to investagate.â‘ cyclo(-Ile¹-Ile²-statine³-Pro⁴-Tyr⁵-Val⁶-Pro⁷-Leu⁸-); (A1HS1)â‘¡cyclo(-Ile¹-Ile²-Pro³-statine⁴-Tyr⁵-Val⁶-Pro⁷-Leu⁸-); (A2HS1)â‘¢cyclo(-Ile¹-Ile²-Pro³-Pro⁴-Tyr⁵-Val⁶-statine⁷-Leu⁸-); (A3HS1)â‘£cyclo(-Ile¹-Ile²-statine³-statine⁴-Tyr⁵-Val⁶-Pro⁷-Leu⁸-); (A4HS1)Purified by RP-HPLC and identified by MS, we synthesized these cyclopeptides successifully. The purity of all the products has exceeded 90%.Bioactivity test displayed that:(1)LPT:The inhibition ratio of A1HS1-A4HS1 high dose groups for lymphocyte proliferation are significantly low, compared 0with CsA high dose group(every one is P＜0.01). The inhibition ratio of A1HS1-A4HS1 low dose groups are significantly higher than CsA low dose group (every one is P＜0.01). A3HS1 low dose group is the highest.(2)DTH test:For high dose groups, the swelling degrees of CsA, HS-1, A1HS1 and A3HS1 aresignificantly lower than negative group (P< 0.01, P< 0.01, P< 0.01, P< 0.01) . For A2HS1 and A4HS1, there are no significant differences with negative group. Compared with CsA, there are no significant differences for HS-1, A1HS1 and A3HS1 groups. But A2HS1 and A4HS1 are significantly higher than CsA(P< 0.05, P< 0.05). A2HS1 and A4HS1 are also significantly higher than HS-1(P< 0.05, P< 0.05).For low dose groups, the swelling degrees of CsA, HS-1, A1HS1-A4HS1 are significantly lower than negative group(every one is P< 0.01). Compared with CsA, there are no significant differences for HS-1 and A1HS1-A4HS1. And there are no significant differences between A1HS1-A4HS1 and HS-1.For a compound, the high dose groups and low dose groups of CsA, HS-1, A1HS1 and A3HS1 have no significant differences. But for A2HS1 and A4HS1, their high dose groups are significantly higher than their low dose groups , respectively (P< 0.05, P< 0.05).(3)Carbon clearance test:Compared with the negative group, the phagocytic index of CsA(high and low dose groups), A2HS1(high dose) and A4HS1(low dose) groups are all significantly lower (P＜0.05, P＜0.05, P＜0.05, P＜0.01). The phagocytic index of other peptides has no significant difference with negative group.For a compound, the phagocytic index of A4HS1(low dose) is significantly lower than its high dose group(P<0.01). There are no significant differences between high and low dose groups of other peptides.The phagocytic index of A2HS1(high dose) is significantly lower than that of CsA(high dose) and HS-1(high dose)(P<0.05, P＜0.01).The phagocytic index of A4HS1 (low dose) is significantly lower than that of CsA(low dose) and HS-1(low dose)(P<0.01, P<0.01).In clusion, these four analogs of HS-1 can inhibit the proliferation of lymphocytes of mice better than CsA and HS-1. The activity of A3HS1 is highest. The immunosuppressive effect of A1HS1 on DTH of mice is almost same to CsA, but better than natural HS-1. The immunosuppressive effects of A2HS1 and A4HS1 on phagetrophy of the macrophage are stronger than CsA and natural HS-1. So that, the statine analogs of HS-1 have higher immunosuppressive effects than HS-1.The different modifications may produce different influences on functional group of HS-1, and then affect the immunosuppressive mechanism of HS-1. These methods of modification may provide a useful strategy for specfic immunosuppressant development. The hypothesis , -Pro⁴-Tyr⁵-Val⁶- is the active center of HS-1 , was also proved indirectly.These four analogs also showed some specificities initially. They may be used as a kind of new immunosuppressant for the organ-graft refection in future.