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for human GAPDH is better than for the parasite enzymes, namely 35 mM [13]. Despite the fact that
these IC 50 values are about ten thousand times higher than what would be considered a lead in the
pharmaceutical industry we decided to optimize the affinity and selectivity of adenosine.
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Each of the three areas where differences occur between the parasite and the human enzyme are
hydrophobic. Therefore, we modeled hydrophobic substituents at positions C2, C8, and O2' of adenosine
under the constraint that they were conformationally compatible with the C2'-endo pucker of the ribose
sugar. Designing derivatives at O2' was a problem, however. Each of the two ribosyl hydroxyls forms a
hydrogen bond with the carboxylate of Asp37. Since making direct derivatives of the hydroxyl, such as
ethers or esters, would deprive the Asp of a hydrogen-bond partner while burying the carboxylate,
resulting molecules would have a dramatically reduced affinity. Moreover, an alignment of 47 GAPDH
sequences made it clear that the Asp is highly conserved [89]. An elegant way to overcome this problem
was to replace the 2' -hydroxyl by a 2'- amino function. Moreover, coupling with carboxylic acids was
appealing from a synthetic point of view while the conformational properties of the amido-substituted
system would ensure the correct orientation of substituents into the selectivity cleft. The modeled
inhibitors were evaluated for the quality of their fit to the protein surface and subsequently synthesized.
From Table 8 it can be seen that our predictions were successful. The addition of a methyl group at C2
of the adenine ring, which is close to Val36, increased the affinity for parasite GAPDH by an order of
magnitude. The effect of a thienyl substitution on C8, targeted to Leu112, was even bigger, namely two
orders of magnitude. However, both substitutions are only mildly selective (Table 8). As expected, the
greatest gain in selectivity was obtained by modifying the 2'-position of the ribose, so that the selectivity
cleft is filled up (Figure 12). The 2'-deoxy-2'-(3-methoxybenzamido) adenosine compound (Figure 13)
bound at least 48 times better to L. mexicana GAPDH than to the human enzyme. The selectivity versus
T. brucei GAPDH appeared to be smaller. This has to be ascribed to a difference in residues contacting
the 3-methoxy moeity. The residue Asn39 of T. brucei GAPDH has a Ser equivalent in the L. mexicana
Table 8 Inhibition Gains of Designed GAPDH Inhibitors with Respect to Adenosine
C2-subst C8-subst C2'-subst T. brucei L. mexicana human
CH 3 H OH 12.5 6.25