complexes examined were DHFR:folate, DHFR:NADP
+
, and
DHFR:NADP
+ :folate, which are models for the binary product
complex, the holoenzyme, and the Michaelis complex, respectively.
They observed two kinetic events in the temperature-jump transients of each of the complexes: a fast relaxation event and a slow
relaxation event. The slow off-pathway conformational rearrangement observed can be interpreted as evidence for conformational
selection as a mechanism for ligand binding. The millisecond conformational rearrangements observed using Trp fluorescence are
not coupled to a binding event as would be expected for an induced
fit model of ligand binding. The presence of the slow relaxation
event regardless of the ligand state suggests that it corresponds to
fluctuations of the protein that would be necessary for the conformational selection process. In addition, the dependence of the rate
of the slow event on the ligand identity is consistent with a liganddependent population shift to a favored conformation, as is
expected in the conformational selection model [46].
4 Drug Binding
The role of enzyme dynamics in the binding of three DHFR
inhibitors will be discussed here. Methotrexate (MTX), trimethoprim (TMP), and pyrimethamine (PYR) were the first DHFR inhibitors in clinical use (Fig. 7) [47]. While MTX is a potent inhibitor
of essentially all DHFRs (most likely due to its similarity to the
natural substrate), TMP and PYR show strong selectivity for bacterial and protozoal enzymes, respectively [48]. For example, TMP
shows 14- and 6000-fold selectivity for ecDHFR over P. berghdi
and rat liver DHFR, respectively. In contrast, PYR displays 1400and 5000-fold selectivity for P. berghdi over rat liver DHFR and
ecDHFR, respectively [48]. Studying the differences in binding of
these small molecules to DHFR from different species and the
structural features that drive selectivity can provide insight into
the role of conformational dynamics in inhibitor binding. Furthermore, understanding the role of conformational selection in the
binding of MTX, TMP, and PYR will guide development of novel
DHFR inhibitors with improved potency and selectivity.
4.1 Methotrexate
Methotrexate (MTX) was first introduced as an effective treatment
for acute leukemia in 1950 and for other solid tumors during the
1950s and 1960s [49]. It is a folic acid analogue and an extremely
potent competitive inhibitor of all DHFRs, including the human
enzyme. Rapidly proliferating cancer cells need a continuous supply
of THF for nucleic acid synthesis and replication. Inhibition of the
folate pathway eventually results in cell death. In addition to cancer
chemotherapy, MTX has been successfully used to treat rheumatoid
arthritis, juvenile idiopathic arthritis, uveitis, graft vs. host disease
Distal Regions Regulate Dihydrofolate Reductase-Ligand Interactions
197
+
, and
DHFR:NADP
+ :folate, which are models for the binary product
complex, the holoenzyme, and the Michaelis complex, respectively.
They observed two kinetic events in the temperature-jump transients of each of the complexes: a fast relaxation event and a slow
relaxation event. The slow off-pathway conformational rearrangement observed can be interpreted as evidence for conformational
selection as a mechanism for ligand binding. The millisecond conformational rearrangements observed using Trp fluorescence are
not coupled to a binding event as would be expected for an induced
fit model of ligand binding. The presence of the slow relaxation
event regardless of the ligand state suggests that it corresponds to
fluctuations of the protein that would be necessary for the conformational selection process. In addition, the dependence of the rate
of the slow event on the ligand identity is consistent with a liganddependent population shift to a favored conformation, as is
expected in the conformational selection model [46].
4 Drug Binding
The role of enzyme dynamics in the binding of three DHFR
inhibitors will be discussed here. Methotrexate (MTX), trimethoprim (TMP), and pyrimethamine (PYR) were the first DHFR inhibitors in clinical use (Fig. 7) [47]. While MTX is a potent inhibitor
of essentially all DHFRs (most likely due to its similarity to the
natural substrate), TMP and PYR show strong selectivity for bacterial and protozoal enzymes, respectively [48]. For example, TMP
shows 14- and 6000-fold selectivity for ecDHFR over P. berghdi
and rat liver DHFR, respectively. In contrast, PYR displays 1400and 5000-fold selectivity for P. berghdi over rat liver DHFR and
ecDHFR, respectively [48]. Studying the differences in binding of
these small molecules to DHFR from different species and the
structural features that drive selectivity can provide insight into
the role of conformational dynamics in inhibitor binding. Furthermore, understanding the role of conformational selection in the
binding of MTX, TMP, and PYR will guide development of novel
DHFR inhibitors with improved potency and selectivity.
4.1 Methotrexate
Methotrexate (MTX) was first introduced as an effective treatment
for acute leukemia in 1950 and for other solid tumors during the
1950s and 1960s [49]. It is a folic acid analogue and an extremely
potent competitive inhibitor of all DHFRs, including the human
enzyme. Rapidly proliferating cancer cells need a continuous supply
of THF for nucleic acid synthesis and replication. Inhibition of the
folate pathway eventually results in cell death. In addition to cancer
chemotherapy, MTX has been successfully used to treat rheumatoid
arthritis, juvenile idiopathic arthritis, uveitis, graft vs. host disease
Distal Regions Regulate Dihydrofolate Reductase-Ligand Interactions
197