Consequently over-expression studies of ThyA and DHFR in bov

Consequently over-expression studies of ThyA and DHFR in bovis BCG were performed to confirm the target of THT1 and to determine the impact on the MIC of the remaining in silico identified compounds. There was no increase in resistance upon over-expression of DHFR or ThyA on the negative control, isoniazid. Only the DHFR over-expresser strain exhibited an increase in resistance when tested on the positive control, PAS as shown by the MICs given in Fig. 3. Chemogenomics approaches have provided fast and cheap utilization of the chemical and genomic space in identification of target-ligand pairs that were 273404-37-8 confirmed by using WGS methods, followed by over-expression of ThyA and DHFR in M. bovis BCG. To our knowledge, this is the first time computationally predicted mycobacterial target-ligand pairs have been phenotypically validated. Compounds S4 and THT2 have been reported to potentially modulate the folate pathway. Here, compounds THT1 and THT2 have been confirmed to target mycobacterial DHFR. Three distinct, yet complementary, in silico methods independently predicted the two compounds. In docking calculations involving Mtb DHFR, the two compounds have PD 151746 similar orientation in the binding pocket, similar to the binding modes of cycloguanil, methotrexate, trimethoprim and Br-WR99210 previously reported. The THT moiety in THT1 and THT2 occupied the inner hydrophobic binding site bordered by, amongst other residues, Phe31 and forms H-bonds with Ile5 and Asp27 and Ile94 as well as hydrophobic interactions. The ortho-substituted phenyl ring occupies the outer hydrophobic binding site close to the entrance of the pocket and form van der Waals forces with these residues with residues Gly18, Ile20, Thr46, Ser49, and Leu50. In this site there are differences in orientation where the phenyl ring in THT1 is drawn closer to Il320 and closest distance between them whilst the tert-butyl fragment interacts more with Leu50. In contrast, the ethyl-phenyl- moiety of THT2 is closer to Leu50 and there is minimum contact with Ile20. Largely, both molecules are stabilized by hydrophobic and polar interactions. DHFR is essential for the production of tetrahydrofolate that is crucial for the synthesis of DNA and proteins. Inhibition of this enzyme could lead to cell death and therefore inhibit the growth of Mtb. It is important to note that THT2 was also predicted to target InhA, Phenylalanine tRNA ligase alpha subunit, and Fibronectin-binding protein C. On the other hand THT1 was also predicted to target dihyropteroate synthase 1 and Phenylalanine tRNA ligase alpha subunit. In our predictions the Mtb DHFR was inferred from its orthologous genes that included DHFR from Homo sapiens, Bacillus anthracis, Escherichia coli, Lactococ