Thymidylate Synthase

Case study 5 The design of an inhibitor for the enzyme thymidylate synthase is a representation for how de novo drug design and structure-based drug design can go hand-in-hand. In the human body, this enzyme uses the coenzyme, 5,10-methylentetrahydrofolate, to catalyze the addition of a methyl group to the substrate deoxyuridylate monophosphate (dUMP). The product of this reaction is deoxythymidylate monophosphate (dTMP). Because molecules that inhibit thymidylate synthase tend to display anti-tumor properties, drugs that are similar to the substrate and cofactor could have significant medical potential. However, drugs that are similar to dUMP and 5,10-methylenetetrahydrofolate have a tendency to cause problems because they can also inhibit

The bacterial form of the enzyme was similar enough in structure to the human one that its active site could be used to determine the binding regions necessary for the new drug. Along with the bacterial thymidylate synthase, the inhibitors of the bacterial enzyme were crystallized, and their structure was established by X-ray crystallography (Patrick, 2013, p. 407). The binding site and the inhibitors were examined together, in order to obtain a better idea of the binding interactions that were utilized by the inhibitors. One of the inhibitors, CB 3717, was shown to form hydrogen bonds with Asp-169 and Ala-263 within the binding site of the enzyme via its pteridine ring system. Likewise, a water molecule was utilized by the inhibitor to mediate a hydrogen bond with Arg-21. To further evaluate the binding regions within the binding site, an aromatic CH probe was used to locate hydrophobic regions (Patrick, 2013, p. 407). It was found that the pteridine ring of CB 3717 formed its hydrogen bond within a hydrophobic region. Based on this, the researchers determined that a naphthalene ring could be a reasonable replacement for the pteridine group, with ample room to include a substituent that could recreate the hydrogen bonding

408). These new structures would accomplish the hydrogen bonding that took place on Asp-169 and the mediating water molecule. A group was now needed that could take the place of the benzene ring on the original inhibitor. This group, a dialkylated amine, was added to position 5 on the naphthostryl scaffold. An amine in position 5 was selected due to an easy synthesis, easy adjustability, and the lack of a chirality center which would have given more than one final product of synthesis. The amino group was alkylated with a benzene ring in order create one molecule that could occupy all the binding regions of both the natural substrate and the cofactor. For future crystallization and X-ray crystallography to occur, the drug needed to become more soluble in water. In order to achieve this, the researchers added a phenylsulphonyl piperazine substituent to the benzene ring. This new addition was placed in the para position so that it would stick out of the binding site and eliminate the need to remove the water from the binding site (Patrick, 2013, p.