A molecular dynamics simulation of the folding of a short alanine-based helical peptide of 17 residues with three Glu...Lys (i, i + 4) salt bridge pairs, referred to as the AEK17 peptide, was carried out. The simulation gave an estimated simulation folding time of 2.5 ns, shorter than 12 ns for an alanine-based peptide of 16 residues with three Lys residues only, referred to as the AK16 peptide, simulated previously. After folded, the AEK17 peptide had a helical content of 77%, in excellent agreement with the experimentally determined value of 80%. An examination of the folding pathways of AEK17 indicated that the peptide proceeded via three-turn helix conformations more than the helix-turn-helix conformation in the folding pathways. An analysis of interactions indicated that the formation of hydrogen bonds between Lys residue side chains and backbone carbonyls is a major factor in the abundant conformation of the three-turn helix intermediate. The substitution of three Ala with Glu residues reduces the extent of hydrophobic interaction in alanine-based AK peptides with the result that the breaking of the interactions of Lys epsilon-NH3+(side chain)...C=O(backbone) is a major activation action for the AEK17 to achieve a complete fold, in contrast to the AK16 peptide, in which breaking non-native hydrophobic interaction is the rate-determining step.

Purpose: Most lung cancer patients have some resistance to and suffer from side effects of conventional chemotherapy. Thus, identification of a novel anticancer drug with better target selectivity for lung cancer treatment is urgently needed. Experimental Design: In order to investigate whether OSU03013, a derivative of celecoxib, can be a potential drug for lung cancer treatment, we examined its cytotoxicity mechanisms by flow cytometry and phosphatidylserine staining in A549, CL1-1, and H1435 lung cancer cell lines, which are resistant to the conventional drug, cisplatin. In addition, we identified the affected proteins by proteomics and confirmed the selected proteins by Western blot analysis. We examined the interaction between OSU03013 and potential target protein by molecular modeling. Results: Our results indicated that OSU03013 had low-dose (1f4 AM) cytotoxicity in all lung cancer cell lines tested 48 hours posttreatment. OSU03013 caused cell cycle G1phase arrest and showed phosphatidylserine early apoptosis via endoplasmic reticulum stress. Several proteins such as heat shock protein 27, 70, and 90, CDC2, a-tubulin, annexin A3, cAMP-dependent protein kinase, glycogen synthase kinase 3-beta, and h-catenin were identified by proteomics and confirmed by Western blot. In addition, molecular modeling showed that OSU03013 competes with ATP to bind to cAMP-dependent protein kinase. Conclusions: We identified for the first time that OSU03013 inhibits cAMP-dependent protein kinase activity and causes dephosphorylation of glycogen synthase kinase 3-beta leading to h-catenin degradation, which is often overexpressed in lung cancer. Our molecular and proteomic results show the potential of OSU03013 as an anticancer drug for lung cancer.

A molecular dynamics simulation of the folding of a short helical toxin peptide was carried out. The simulation gave a folding time of ~10 ns, which is longer than typical time of ~1 ns for the formation of 1–2 helical turns. The simulation demonstrates that a helical peptide with disulfide bonds, which may encounter extra steric hindrance compared with the peptide without disulfide bonds, can fold in nanosecond timescale. An analysis shows that this folding time should correspond to the folding time in weak denaturation condition in experiment. Interactions and factors affecting folding pathways are analyzed and discussed.