4.2 Trimethoprim
Trimethoprim is a widely used antibacterial drug that is often used
in combination with the antibiotic sulfamethoxazole to treat
numerous bacterial infections including E. coli, Staphylococcus
aureus, Shigella species, Streptococcus pneumoniae, and many more
[62]. TMP is a potent inhibitor of bacterial DHFRs but a much
weaker inhibitor of vertebrate DHFRs. In fact, TMP binds to
ecDHFR about 10,000 times stronger than it does hDHFR (IC 50
values against E. coli and human enzymes are approximately
5 Â 10
À9 M and 3 Â 10
À4 M, respectively) [63]. The strong
selectivity for the bacterial enzyme is what allows TMP to be an
effective antibiotic, because inhibition leads to cell death in the
pathogenic bacterial cells, but not in the human host cells
[64]. In contrast, MTX is a potent inhibitor of both bacterial and
human DHFR, therefore it is too toxic to be used as an antibiotic
[65]. The main contribution to the selectivity of TMP binding is
the large positive cooperative effect between TMP and the
NADPH cofactor during the formation of the ternary complex
with the bacterial DHFR. TMP binds to bacterial DHFR
135 times more tightly in the presence of NADPH [66]. There is
a large positive cooperative effect because the binding of one ligand
(NADPH) greatly increases the affinity of DHFR for the second
ligand (TMP). This large cooperative binding effect is not observed
in the complex of TMP with human DHFR [67, 68]. Several
explanations have been proposed for the cooperative binding effect
including: direct interaction of the ligands with each other, allosteric effects due to conformation change of the protein upon ligand
binding, resonance effects that strengthen networks of hydrogen
bonds and electrostatic interactions in the ternary complex relative
to the binary complexes, and correlated movements of the ligands
[64]. There is also a direct hydrophobic interaction between TMP
and NADPH in the ternary complex [69]. The analogous interaction in hDHFR is much weaker because there is greater separation
between the ligands [67]. However, it is unlikely that the direct
contact between ligands alone is enough to explain the large cooperative effect. The additional free energy change may be due to a
conformational change in the enzyme resulting in a more favorable
interaction of DHFR with both ligands [64].
Another interesting aspect of TMP binding to bacterial DHFR
is the observation of two coexisting conformational states in the
ternary complex of the Lactobacillus casei enzyme [70, 71]. The
complex of lcDHFR with TMP and NADP
+ exists in solution as a
mixture of approximately equal amounts of two slowly interconverting conformational states [72, 73]. NMR experiments were
used to characterize the conformational equilibrium of the
L. casei enzyme complexes, which showed that the active site is
clearly involved. There are significant differences in the environment of the bound ligands between the two conformations but
conformational effects are not restricted to the active site
200
Melanie Goldstein and Nina M. Goodey
Trimethoprim is a widely used antibacterial drug that is often used
in combination with the antibiotic sulfamethoxazole to treat
numerous bacterial infections including E. coli, Staphylococcus
aureus, Shigella species, Streptococcus pneumoniae, and many more
[62]. TMP is a potent inhibitor of bacterial DHFRs but a much
weaker inhibitor of vertebrate DHFRs. In fact, TMP binds to
ecDHFR about 10,000 times stronger than it does hDHFR (IC 50
values against E. coli and human enzymes are approximately
5 Â 10
À9 M and 3 Â 10
À4 M, respectively) [63]. The strong
selectivity for the bacterial enzyme is what allows TMP to be an
effective antibiotic, because inhibition leads to cell death in the
pathogenic bacterial cells, but not in the human host cells
[64]. In contrast, MTX is a potent inhibitor of both bacterial and
human DHFR, therefore it is too toxic to be used as an antibiotic
[65]. The main contribution to the selectivity of TMP binding is
the large positive cooperative effect between TMP and the
NADPH cofactor during the formation of the ternary complex
with the bacterial DHFR. TMP binds to bacterial DHFR
135 times more tightly in the presence of NADPH [66]. There is
a large positive cooperative effect because the binding of one ligand
(NADPH) greatly increases the affinity of DHFR for the second
ligand (TMP). This large cooperative binding effect is not observed
in the complex of TMP with human DHFR [67, 68]. Several
explanations have been proposed for the cooperative binding effect
including: direct interaction of the ligands with each other, allosteric effects due to conformation change of the protein upon ligand
binding, resonance effects that strengthen networks of hydrogen
bonds and electrostatic interactions in the ternary complex relative
to the binary complexes, and correlated movements of the ligands
[64]. There is also a direct hydrophobic interaction between TMP
and NADPH in the ternary complex [69]. The analogous interaction in hDHFR is much weaker because there is greater separation
between the ligands [67]. However, it is unlikely that the direct
contact between ligands alone is enough to explain the large cooperative effect. The additional free energy change may be due to a
conformational change in the enzyme resulting in a more favorable
interaction of DHFR with both ligands [64].
Another interesting aspect of TMP binding to bacterial DHFR
is the observation of two coexisting conformational states in the
ternary complex of the Lactobacillus casei enzyme [70, 71]. The
complex of lcDHFR with TMP and NADP
+ exists in solution as a
mixture of approximately equal amounts of two slowly interconverting conformational states [72, 73]. NMR experiments were
used to characterize the conformational equilibrium of the
L. casei enzyme complexes, which showed that the active site is
clearly involved. There are significant differences in the environment of the bound ligands between the two conformations but
conformational effects are not restricted to the active site
200
Melanie Goldstein and Nina M. Goodey