Chemistry Reference
In-Depth Information
acid substitutions. Disulfide capture has recently been used to evaluate the binding of these
ligands to the C5a receptor.
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Cysteine residues were inserted into peptide ligands and in
various positions of the C5a receptor and tested for activity. Binding and receptor activation
studies showed that the disulfide-captured ligands maintained the agonism/antagonism of
their noncovalent parents and confirmed the location of the activation site to within a hel-
ical triad previously identified through site-directed mutagenesis. Since disulfide capture
compensates for reduction in binding affinity, the size and complexity of ligands can be
greatly reduced, allowing a high-resolution mapping of key agonistic/antagonistic motifs
and complementing mapping by traditional mutational analysis. Four cysteine insertions
in the C5a receptor were subsequently screened by Tethering to identify small-molecule
ligands.
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Asubset of these ligands showed agonism or antagonismwhen tethered to either
of two cysteines within the seven-helix bundle. Mutation of Ile116, which is located within
10 Å of the Tethering site, had a significant effect on receptor activation for some ligands.
Interestingly, replacing Ile116 with Ala increased the degree of agonism for many ligands,
whereas replacing it with a larger Trp residue led to a decrease in agonism and also the
number ligands that could serve as agonists. This study illustrates the use of Tethering as
a capture method to characterize functionally small ligands prior to selecting the preferred
fragments for optimization.
10.3.4 Tethering with Extenders
The examples listed above illustrate the power of Tethering to identify readily a variety
of ligands, but, as with other fragment discovery methods, linking and/or expanding these
ligands to increase affinity often requires extensive crystallography, molecular modeling
or trial and error. A second-generation version of Tethering, Tethering with extenders,
provides a powerful solution to this problem by exploiting a known fragment as the basis
for capturing a new fragment, essentially allowing the protein to guide the assembly of
the two fragments.
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In this method (Figure 10.2B), the protein is modified with a known
ligand referred to as an 'extender,' which is a small fragment that contains both a reactive
functionality and a thiol: the reactive functionality covalently labels the protein and the
thiol is used for Tethering to capture a fragment that binds nearby. The resulting molecule
contains two fragments connected with a disulfide linker that is subsequently replaced
with a variety of other linkers and tested for activity in a bioassay. In effect, Tethering
with extenders is an
in situ
assembly process that greatly accelerates and simplifies hit
identification and permits rapid expansion from a simple fragment to a more elaborate
molecule. Moreover, the extender itself can be derived from Tethering.
Sunesis researchers used this approach to discover low micromolar inhibitors of the
pro-apoptotic cysteine protease caspase-3. Simple rigidification of the flexible linker con-
necting the ligand and extender improved potency by more than an order of magnitude, to
K
i
=
200 nM,
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as did decoration of the linker.
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Further medicinal chemistry achieved
another order of magnitude improvement in potency
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(Figure 10.3B). The same approach
was applied to the related protease caspase-1 (interleukin-1 converting enzyme or ICE) and
a series of low nanomolar inhibitors was discovered.
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Avariant of Tethering with extenders, termed 'breakaway Tethering,'has been developed
for enzymes with catalytic sites that do not tolerate the insertion of a cysteine residue due
to disruption of structural or functional integrity. Protein tyrosine phosphatases (PTPs)