Chemical synthesis and characterizations of wild-type and disulfide modifications of psalmopeotoxin I-an anti-malarial cysteine knot peptide natural product extracted from Psalmopoeus cambridgeiPacharin Kamolkijkarn
( M.Sc. )
There are a number of small molecule natural products that have anti-malarial activity, such as artemisinin and quinine. The need for new anti-malarial drugs was driven by the increasing resistance of parasites to classical anti-malarial agents. An alternative to small molecule was the application of biologics, as therapeutic agents. Recently, Camadro et al identified two peptides, Psalmopeotoxin I and II (PcFK1 and 2), extracted from Psalmopoeus cambridgei. These peptides have antiplasmodial activity against the intraerythrocyte stage of P. falciparum in vitro (IC50 ~ 1 8M), but did not exhibit any toxicity usually associated with other structurally similar neurotoxins. The purposes of this research were to chemically synthesize PcFK1-an anti-malarial peptide, both native and relevant mutations, and to study the structure, biophysical characteristics, and biological activities of PcFK1 and its mutants. PcFK1 is a 33-residue peptide containing three disulfide bridges and belongs to the Inhibitor Cysteine Knot (ICK) superfamily. The presence of multiple disulfide bonds could be synthetically challenging. The linear PcFK1 was first synthesized by SPPS. Then, the three disulfide bonds were formed using these two disulfide bond formation strategies: regioselective and random oxidative. The resulting peptides from both synthetic strategies were compared based on physical, biophysical, and biological properties. The physical properties included chromatography and peptide mapping. The biophysical characterizations included 2O and 3O structure study by circular dichroism (CD) and fluorescence spectroscopy. The biological activity was antiplasmodial assay, which assessed the development of P. falciparum (K1, MDR strain) in erythrocytes in vitro. Moreover, it was not known whether these types of peptides needed multiple disulfide bonds for both activity and stability. For this reason, each disulfide bond was removed for each mutant. Both wild type and mutants were subjected to CD, fluorescence, thermal unfolding, and antiplasmodial assay. Afterwards, the analysis of the structures, biophysical characteristics, and biological activities were discussed in terms of biophysical-biological activity relationship, aiming to elucidate the mechanism of PcFK1 antiplasmodial activity. Overall, our study revealed that PcFK1 and its mutants should be synthesized by regioselective disulfide bond formation. The biophysical results suggested that the compactness played an important role on stability. Each disulfide bond in PcFK1 helped stabilizing secondary structure in PcFK1 wild-type, particularly Cys9-Cys24. On the other hand, the anti-malarial activity data showed that the impact of Cys9-Cys24 and Cys2-Cys19 was less than the impact of Cys18-Cys29. Cys18-Cys29 connected the 3rd β-strand to the rest of PcFK1, thus the 3rd β-strand was important for the biological activity of PcFK1.