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and inhibition mechanisms and provided a clarification for the mechanism by which inhibitors of both
the carboxylate [28] and spirohydantoin [29] classes bind at the active site of ALR2.
V. Aldo-Keto Reductase Family
A. Effects of Sequence on Drug Design
Page 239
The problem of structure-based drug design for ALR2 and the drug-design effort in general is
compounded by the fact that this enzymes is a member of a large family of aldo-keto reductases with
overlapping substrate specificity. In humans at least three such enzymes have been found: ALR2 [30],
aldehyde reductase (ALR1) [30], and chlordecone reductase [31]. Other members of the family that have
been isolated in other species include rat 3α-hydroxysteroid dehydrogenase [32], murine fibroblast
growth factor induced protein [33], bovine prostagladin F synthase [34], murine vas deferens protein
[35], frog ρ-crystallin [36], the P100/11E gene product in Leishmania major [37], and Corynebactium
diketogluconate reductase [38]. This large number suggests that there may be more such enzymes to be
found in humans. The similarities between the proteins with respect to both the sequence and substrate
specificity implies that the nature of the substrate binding sites are similar across the family. This has
indeed been the case in all the structures determined from this family to date (see below).
While detailed binding studies of various inhibitors with all the different enzymes have not been
conducted, it is likely that drugs intended for ALR2 are likely to “cross react” with many of the other
enzymes within the family. One such case that has recently been studied both crystallographically and
biochemically is the murine FR-1 protein described below.
The binding sites of all of these enzymes are characterized by their large size and hydrophobicity
suggesting that ideal substrates may be steroids or molecules of a similar size and nature. Sequence
comparisons of all the proteins, including those whose structure has not yet been determined, show that
there is a large amount of similarity involving residues implicated in substrate binding (see Figure 8).
One region that diverges somewhat is the 15-amino acid segment at the carboxy terminus of the protein.
This segment is likely to be responsible for what little differences in substrate specificity exhibited by
the enzymes. It is the same segment that is seen adopting a different conformation upon zopolrestat
binding to ALR2. It may then be possible that it is not only the chemical nature of this loop that—in
making positive and negative interactions with the substrate/inhibitor—modulates specificity, but also
the flexibility conferred by the amino acid sequence. Such a difference is seen when contrasting the
structures of ALR2 and FR-1 bound to zopolrestat.
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