Chemistry Reference
In-Depth Information
have relatively compact catalytic sites that catalyze the removal of phosphate groups from
tyrosine and are involved in many aspects of cell signaling. PTP-1B, for example, is a
highly attractive target for anti-diabetic and anti-obesity therapy.
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In an effort to probe
the intact active site of PTP-1B, a cysteine residue was introduced well outside the active
site and coupled to a 'breakaway extender', designed based on the insulin receptor peptide
substrate. This 'breakaway' extender contains an active site ligand, a 'breakaway' thioester
linkage and an irreversible alkylating group designed to react with the newly introduced
cysteine (Figure 10.2C). Once the protein has been reacted with the alkylating group, the
thioester linkage can be cleaved to release the active site ligand and expose a thiol group
for Tethering that is positioned just above the catalytic site. The benefit of using a substrate
mimetic 'breakaway extender' is twofold: it provides a recognition sequence to orient the
extender properly with respect to a distant cysteine, and it also protects the highly reactive
cysteine located in the active site. When this method was applied to PTP-1B, a number of
small, negatively charged ligands were identified, some of which had not been previously
reported as phosphotyrosine mimetics.
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10.3.5 Targeting the Active Site of PTP-1B with Reversible Electrophiles
The catalytic cysteine-215 of PTP-1B is highly reactive toward oxidation and electrophiles
and as such it is a tempting target for fragment discovery. Ockey and Gadek assembled
a set of 19 reversible electrophiles, such as aldehydes, nitriles and boronic acids.
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They
then used electrospray ionization mass spectrometry to look for one-to-one complexes; and
three of the compounds were found to form covalent complexes. The dissociation constants
ranged from 25 to 150 M and one of the compounds was also able to inhibit PTP-1B with
an IC
50
of 60 M.
10.3.6 Naturally Occurring Reactive Fragments
An interesting natural example of covalent capture of fragment-sizedmolecules has recently
been reported. The ion-channel protein TRPA1 responds to a variety of stimuli, including
spices and foods such as cinnamon, garlic and horseradish. Two groups independently
demonstrated that this response involves covalent modification of three cysteine residues
by electrophilic compounds contained in these foods.
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The active ingredients (cin-
namaldehyde in cinnamon, diallyl disulfide in garlic and allyl isothiocyanate in mustard
oil) are chemically distinct but react with cysteine residues (Figure 10.4A). They are also
very small molecules and likely derive most of their effects from their reactivity rather
than from noncovalent binding interactions. It would be an interesting challenge to try to
improve the binding energy while decreasing the reactivity.
10.3.7 Discovery of Peptide Ligands Using Covalent Capture
In addition to finding small organic molecules that bind to a protein, covalent capture
methods can identify peptides that interact with proteins. Kohda and colleagues used this
approach to study the mitochondrial protein Tom20, an import receptor that recognizes an
epitope on proteins targeted for the mitochondria.
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Previous work had characterized this
epitope as a five-residue peptide that assumes an amphiphilic helical conformation, and
coarse sequence preferences had been worked out. However, Tom20 has both low affinity
