Conformational Analysis of Human Dihydrofolate Reductase Inhibitor Complexes: Crystal Structure Determination of Wild Type and F31 Mutant Binary and Ternary Inhibitor Complexes

Since dihydrofolate reductase (DHFR) is responsible for maintaining intracellular folate pools in their biochemically active reduced state, its inhibition has been the target of continuing antifolate development to improve selectivity, penetration into target cells, and effectiveness against tumors with either intrinsic or acquired antifolate resistance1. For example, investigation of patients with methotrexate (MTX)-resistance has shown that in some tumors the resistance is due to defects in cellular transport or polyglutamation and that new, second generation, antifolate drugs such as 10-ethyl-10-deazaminopterin (10-EDAM) or the lipophilic antifolates piritrexim (PTX) and trimetrexate (TMQ), are more effective than methotrexate (MTX)2 . In other tumors it was found that DHFR encoded mutations which were responsible for the MTX resistance. Studies of a highly MTX-resistant subline of the HCT-8 human colon carcinoma cell line showed that DHFR possessed a phenylalanine to serine mutation at residue 313–5 . Kinetic studies further showed that the Ki for MTX was 100-fold higher than wild type DHFR while a mutation of phenylalanine 34 to serine resulted in over 20,000-fold increase in the Ki for MTX. Similar changes in Ki were noted for trimetrexate as well.