Biomedical Engineering Reference
In-Depth Information
plasmepsins. Zu¨ rcher et al. tested the '55% Rule' in binding to active site
pockets in the plasmepsoin II and IV.
108
Mecozzi and Rebek
109
stated that
binding is optimal when 55% of the available volume is occupied in a host-
guest complex. Zu¨ rcher et al. applied this rule to an enzyme to see if it applied
as well as it did for host-guest chemistry. Indeed, the results confirmed that the
rule is generally applicable. Two papers have already been cited in which Bur
and colleagues created potent achiral compounds that bound well to plas-
mepsin II.
35,110
In Prade et al.'s study, a new binding pocket was observed
following binding to plasmepsin II. This is among the observations confirming
that conformational changes occur upon binding, thus making the 'Adaptive
Inhibitor' concept highly viable.
The group of Sergio Romeo at the University of Milan has developed a
concept they call the 'Primaquin-Statine ''double-drug''' approach.
111-114
In this
effort, they used the amino acid statine as a group to induce strong binding at
the active site, as others have done. In addition, they designed linker sequences
to be able to couple the statine portion to the molecule primaquine, which is a
known anti-malarial agent. Their concept was that binding to plasmepsins
would help get the compounds into the parasite, where cleavage of the linkage to
the primaquine by host cell enzymes would allow the primaquine to kill the
parasite. Any effect of inhibiting the plasmepsins would be an added bonus.
Interestingly, in vitro experiments demonstrated that some of the compounds
synthesized by Romeo's group are powerful inhibitors of plasmepsin IV.
112
In the author's laboratory at the University of Florida, a substrate-based
strategy was employed to discover information on the preferences of various
plasmepsins for binding in six subsites along the active site cleft.
25,51
Combi-
natorial libraries of peptide substrates were designed to probe, first, the half of
the active site cleft that interacts with the P3, P1, and P2
0
amino acids of an
octapeptide substrate and, second, the half of the active site cleft that interacts
with the P2, P1
0
, and P3
0
amino acids. Binding of ligands along the active site
cleft was known from earlier crystallographic work to utilize the N-terminal
half of the enzyme to bind to the P3, P1, and P2
0
amino acids and the C-
terminal half of the enzyme to bind to the P2, P1
0
,andP3
0
amino acids. Because
a chromogenic amino acid was placed in the P1 position in the first library and
in the P1
0
position in the second library, the selectivity of those two positions
could be determined by analyzing the eciency of cleavage as a function of the
amino acid substituted in the P1 position for the first library and for sub-
stitution in the P1
0
position for the second library. Specificity at the flanking
positions was determined by analysis of the products of cleavage by LC-MS
analysis. The assumption in this work is that the more product that is formed,
the better the interaction in a particular subsite of the enzyme. The libraries
were designed to probe binding specifically in plasmepsins; however, these
combinatorial libraries have also been useful for analysis of interactions of
other aspartic protease active sites.
Once the data were collected for a particular plasmepsin, an inhibitor could
be constructed by choosing the best amino acid substitution for a particular
sequence position based on the results of the screening. By replacing the peptide
