The Design And Synthesis And Bioactivity Studies Of Limulus Anti-lipopolysaccharide Factor Modeling Peptides

Sepsis and septic shock is the most frequent cause of death in critical ill patients with a high mortality about 40-60%. Lipopolysaccharide(LPS) usually called endotoxin, is the main initiator of sepsis. By binding with lipopolysaccharide-binding protein(LBP), a LPS-CD14 compound is formed, which together with the Toll-like receptors on the cytoplasmic membrane of mononuclear/ macrophage, thus activates the signal transduction pathway, induces cytokines such as TNF-α, IL-6 etc release, which at last results in sepsis, septic shock or even death. In patients with severe sepsis, the endotoxin level had strong correlation with prognosis, therefore neutralizing LPS would be the key step in the treatment of sepsis. The promising strategies targeting sepsis are preventing LPS from binding to its receptors, blockage of LPS intracellular signal transduction, antagonism of cytokine and application of glucocorticoids,but unfortunately all these therapies got little clinical benefits. Although polymyxin B has the effectiveness of neutralizing LPS, but the toxical and side effects forbid its wide clinical utilization. Other anti-LPS proteins such as Bactericidal/permeability increasing protein(BPI), Limulus Anti-lipopolysaccharide factor(LALF) and other peptides derived from them as well as other natural antibacterial/LPS peptides are still under experimental trials. There is no effective and safe drugs available till now. The ideal drugs with high affinity to LPS along with bactericidal activity is at the front line in contemporary medical study. How to block the LPS bioactivity at the very beginning thus to prevent the inflammatory cascade reaction from the LPS activation? The LALF and TALF from both limulus polyphemus and Tachypleus tridentatus Leach have been proved striking anti-gram negative bacillus as well as anti-LPS effects in vitro and in vivo. The LALF can bind to LPS with high affinity in a dose dependent manner and thereafter block the bioactivity of LPS. LALF demonstrated conspicuous protective effect in the sepsis mice model manifested by prolonged life span and decreased mortality. Hoess's work verify that the 31st to 52nd amino-acid residue (LALF 31-52) is the functional domain of LALF. In a model in mice with peritoneal fulminating sepsis, LALF31-52 showed both preventive and therapeutical effects which can obviously increase the survival rate of mice under LPS attack. Above studies also show that LALF31-52 is the minimum sequence .The another study proves that TALF 41-53 is also the minimum sequence and the TALF29-59 has the maximum binding potential to LPS. Previous work has laid a solid foundation for further study but unfortunately, taking advantage of nature resources have been confined by the fact that Tachypleus tridentatus Leach being on the verge of extinction. In order to circumvent this dilemma, based on the analysis of LALF and TALF structural features as well as their functional domains by way of bioinformatics methods, we designed a series of MPLALFS ( modeling peptides from Limulus anti-lipopolysaccharide factor ) by computational molecular modeling and then subjected them to chemosynthesis as well as in vitro and in vivo bioactivity studies. Two MPLALFS, the RREMP and TYS1 with optimal anti-LPS and anti-bacterial effects were obtained. Results: 1. The molecular structure, physico-chemical property and molecular dynamic parameters of LALF and TALF and their functional domains were obtained by analyzing with online PHD and GOR in combination with Antheprot 5.0 and APD, which gave abundant information for molecular modeling .Our experience indicated that PHD method was superior to GOR method. 2. The sequences of MPLALFS were successfully got by computational simulation with appropriate allostery and modification. 3. Sixteen MPLALFS were successfully synthesized by the automatic and manual combinated approach . 4. The anti-LPS efficiency of these modeling peptides were screened and tested by limulus Amebocyte Lysate test, biosense, cytokine stimulating test, MTT test, anti-bacterial test, LPS-neutralizing test and protecting test in vitro or vivo. There were three peptides, the S4, RREMP and TYS1 with ideal anti-LPS and anti-bacterial effects were obtained from 16 MPLALFS. 5. RREMP and TYS1 could effectively inhibit the TNF-αrelease induced by LPS and had a potent action against lethal dose LPS attack in mice. Both peptides at theminimum concentration of 5μM could make a averagely 60% of mice survive in seven days after LPS attack. The survival time was increased obviously. 6. Both RREMP and TYS1 showed a potent and stable anti-LPS effects both in vitro and in vivo in comparison with that of peptides reported in literatures would make them become very promising pro-drugs. 7. After changing the salt from trifluoroacetic acid to acetate in the synthesis process, S4 and S1 showed a dramatic changes in their bioactivities. S4 lost its good anti-LPS property in vitro but S1 got a wider anti-bacterial spectrum and more powerful anti-bacterial ability. This suggested that special salts could change the peptides'bioactivity. Conclusions 1. Without maxicomputer and expensive commercial software, we had successfully completed the molecular design of 15 sequences of MPLALFs by using economic, practical and timely minicomputer with common software and net servers. Of which there were 2 MPLALFs with ideal anti-LPS and anti-bacterial effects had been obtained. The methods we adopted in the study saved us a lot of time and experiment funds. 2. That the change of salt treatment in the peptide synthesis could radically affect the bioactivity of peptide is so far the first report, which suggested that the salt change may be a key factor to the maintenance of peptides'bioactivity. 3. RREMP and TYS1 had ideal and stable anti-LPS effects both in vitro and in vivo. Both peptides could effectively inhibit the TNF-αrelease induced by LPS and showed not only a powerful efficiency against LPS lethal dose attack in mice but also the bioactivity against G-and G+ bacteria in vitro. Otherwise, another peptide S4 also had a stronger anti-LPS effect in vitro. These three peptides are all the new molecules designed by ourselves and have the full intellectual property rights. The patent protection of them has been requesting. 4. As for the second structure analyzing method of protein or peptide, the PHD based on the neural network algorithm was superior to GOR based on the information theory algorithm. In the molecular design of modeling peptide, the minimum domain conservation and precise sequence arrangement of a protein or a peptide, as well as molecular cyclization may be the key to the ideal bioactivity.