Theileria annulata peptidyl prolyl isomerase gene cloning and in silico analysis of candidate inhibitors for new anti-theilerial drug design

The development of new drugs with conventional methods is expensive and takes many years. Computer-aided drug discovery has gained great importance in the field of new drug discovery and development due to reduced cost and time requirements significantly in recent years. T. annulata causes the tropical theileriosis which threatens millions of cattle worldwide. Anti-theilerial drug buparvaquone is used to treat theileriosis. However, drug resistance reports in Theileria annulata suggest that new drug candidates need to be discovered to treat theileriosis. In the thesis study, TaPIN1 was cloned to be used for further in vitro characterization and inhibitor testing studies, inhibitor library was screened in silico for identification of candidate molecules to inhibit drug resistant TaPIN1 and the treatment of human peptidyl prolyl cis/trans isomerase enzyme derived cancers has been devised with proposed approach. In the first step of the cloning phase, peptidyl prolyl cis/trans isomerase coding sequence was amplified by PCR from T. annulata genome, then amplified gene was cloned into pLATE31 vector system and transformed into E. coli BL21 (DE3) cells. After that a non-coding region intron was removed by using Q5® Site-Directed Mutagenesis Kit and the recombinant DNA was transformed into E. coli BL21 (DE3) cells. Removal of intron and insertion of the correct gene sequence was confirmed by DNA sequencing and was made ready for further studies. In the first step of computer aided drug discovery, comparative models of the mutant and wild-type of TaPIN1 were predicted by MODELLER v9.15 software and structure validation was performed by using various types of web-servers. Library of naphthoquinone derivatives was downloaded from the PubChem and was screened by the Schrödinger Glide HTVS, SP and XP docking methodologies. The docked compounds were ranked by the Glide Extra Precision (XP) scoring function and binding free energy of the docked compounds was calculated by Prime MM-GBSA method. The two highest ranks of the docked compounds (Pubchem IDs: 5304752 and 9993782) were used for molecular dynamic (MD) simulations studies. MD simulations for ligand unbound and bound proteins were performed for 50 ns and 100 ns respectively by using AMBER software. 5304752 ligand was located in the active site of both wild type and mutant TaPIN1 enzymes, accordingly 5304752 was predicted as potential inhibitor. 9993782 ligand was located outside of the active site binding pocket of the wild-type TaPIN1 enzyme. However, the presence of the ligand near the active site of the mutant TaPIN1, suggests that ligand can be predicted as a potential inhibitor for mutant enzyme. Also, identified ligands were speculated as potential inhibitors against human peptidyl prolyl isomerase enzyme which causes cancer in humans by using the same mechanism as TaPIN1.