Biomedical Engineering Reference
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papers that describe synthesis of sets of molecules as potential inhibitors and a
complete description of these important efforts is beyond the scope of this
chapter. Instead, this section will focus on the work of several groups that have
published extensively on inhibitor development for various proteolytic
enzymes. In the following paragraphs, the narrative will be divided up by the
different groups and will not, therefore, be in chronological order overall.
It is interesting that efforts to identify strong inhibitors of the plasmepsins
illustrate several important thrusts in current drug discovery. First, two early
papers from Pharmacopeia in the Princeton, New Jersey area 75,76 described the
use of combinatorial libraries of compounds in such a search. While the syn-
thetic and analytical efforts were led by Carolyn Dianni Carroll and her col-
leagues of Pharmacopeia, the Daniel E. Goldberg's laboratory at Washington
University, St. Louis was involved to provide the plasmepsin II for the tests.
The strategy for preparing the compounds was to utilize the amino acid sta-
tine, 77,78 which contains the -CH 2 -CHOH- insertion between what would be
the alpha carbon and the carbonyl carbon of the amino acid leucine. When a
peptide containing this group binds to an aspartic proteinase with the Leu-like
side chain in the S1 subsite, this effectively places the hydroxyl group into a
position where it can interact with the two catalytic Asp residues of the active
site. The resulting hydrogen bonding and optimal fit leads to very tight binding
inhibitors. Carroll et al. prepared a combinatorial library in which five different
groups around the statine core were varied to yield 18 900 members. Out of the
analysis of binding to both plasmepsin II and human cathepsin D (as a counter
screen), the authors found at least one compound with a K i of 490 nM for PM
II and
45 000 nM for cathepsin D. 75 The selectivity observed is excellent;
however, a full evaluation of the compound against all possible human aspartic
proteinases was not reported, and further studies with other plasmepsins have
not appeared subsequently. In 1999, Haque et al. 79 described the synthesis of a
different library of non-peptide compounds and reported the discovery of
inhibitors in the low-nanomolar range with favorable hydrophobic properties
and lack of binding to human serum albumin (Figure 11.6).
A strong collaboration between the laboratories of Ernesto Freire at Johns
Hopkins and Yoshiaki Kiso at the Kyoto Pharmaceutical University has
produced significant advances in our understanding of the critical elements for
drug discovery in the plasmepsin area, 80-87 plus many other papers. Freire and
colleagues developed the theory of 'adaptive inhibitors', where flexibility in the
designed compounds is expected to permit the molecule to try out various
binding modes in order to find the optimal fit. This theory is in opposition to
the theory that adding conformational restriction to a molecule could reduce
flexibility and permit only the optimal binding. The latter theory would work if
one knew exactly the conformation of the binding site of an enzyme; however,
as we now have a greater appreciation for the inherent flexibility of enzyme
active sites under different conditions 34 , current thinking favors the adaptive
inhibitor concept. There are many examples where there is a significant tran-
sition between the conformation of the apo-enzyme, where no ligand is bound,
and the structure of the inhibitor-bound enzyme. Freire and colleagues also
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