These agents inhibit the enzyme dihydrofolate reductase, an important rate-limiting step in the de novo folate synthesis pathway. Methotrexate is a cancer chemotherapeutic agent, whilst trimethoprim is an antibiotic. The reason for this disparity, despite both being dihydrofolate reductase inhibitors, is that methotrexate has a selective affinity for mammalian dihydrofolate reductase, while trimethoprim has a selective affinity for the bacterial enzyme.

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