An analysis of hydrophobic interactions of thymidylate synthase with methotrexate: free energy calculations involving mutant and native structures bound to methotrexate.

Since the human body for many reasons can adapt and become resistant to drugs, it is important to develop and validate computer aided drug design (CADD) methods that could help predict binding affinity changes that can result from these resistant enzymes. The free energy perturbation (FEP) methodology is the most accurate means of estimating relative binding affinities between inhibitors and protein variants. In this paper, we describe the role played by hydrophobic residues lining the active site region, particularly (79)Ile and (176)Phe, in the binding of methotrexate to the Escherichia coli (E. coli) thymidylate synthase (TS) enzyme, using the thermodynamic cycle perturbation (TCP) approach. The computed binding free energy differences on the binding of methotrexate to the native and some mutant E. coli TS structures have been compared with experimental results. Computationally, four different 'mutations' have been simulated on the TS enzyme with methotrexate (MTX): (79)Ile --> (79)Val; (79)Ile --> (79)Ala; (79)Ile --> (79)Leu; and (176)Phe --> (176)Ile. The calculated results indicate that in each of these cases, the native residues ((79) Ile and (176) Phe) interact more favorably with methotrexate than the mutant residues and these results are corroborated by experimental measurements. Binding preference to wild type residues can be rationalized in terms of their better hydrophobic contacts with the phenyl ring of methotrexate.

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