Vertebrate DHFR

Dihydrofolate reductase (DHFR) is by far the most extensively investigated enzyme. 3D structures of binary and ternary DHFR complexes from different bacteria and vertebrates have been published and an extremely large numter of QSAR equations have been derived, both for the isolated enzyme and for growth inhibition of whole cells [288, 396, 431, 432, 671, 677 — 691]. Due to the central role of DHFR in purine biosynthesis, DHFR inhibitors are therapeutically important as highly selective antibacterial (trimethoprim), antimalarial, and antitumor agents (methotrexate). 

While the equations look the same at a first glance, some striking differences can be seen on a closer inspection. First, the vertebrate, but not the bacterial DHFR equations contain an electronic parameter in addition to lipophilicity and molar refractivity terms. Second, in the case of L. casei (eq. 137) the 5-position of the benzyl group does not at all contribute to biological activities. An explanation could be derived by a comparison of the 3D structure of L. casei DHFR with the E. coli DHFR structure. The active sites of both enzymes are more or less identical in the geometries of the protein backbone and the amino acid side chains. However, there is one significant difference E. coli DHFR contains a methionine side chain in the area where the 5-substituents bind, while there is a relatively rigid leucine side chain in the L. casei DHFR which obviously interferes with the 5-substituents. Therefore, the active site of L. casei DHFR is sterically more constrained and the positive lipophilicity and polarizability contributions of the 5-substituents are counterbalanced by their steric hindrance [432, 682]. 

X-ray diffraction analysis of dihydrofolate reductase (DHFR), co-crystal-lized with methotrexate, has shed much light on the action of this inhibitor. This work, one of the earliest visualizations of a drug interacting with its receptor (Matthews etal., 1977), has since been refined to the remarkably clear resolution of 1.7 A (Bolin et al., 1982). A typical diagram of DHFR, its coenzyme (NADPH), and methotrexate is shown in Fig. 9.4. The enzyme depicted there is from Lactobacillus casei and the same authors also report on DHFR (with cocrystallized methotrexate) from the bacterium E. colt. However, they have not been able to co-crystallize methotrexate with DHFR from any vertebrate source. 

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