Chemistry Reference
In-Depth Information
acrylamide, they were able to capture ligands even in very complex mixtures such as cell
media. In a separate example, Belshaw and colleagues converted a potent reversible inhib-
itor into an irreversible one.
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They added acrylamide moieties to analogs of cyclosporin
A, which allowed cysteine-modified cyclophilin A to capture these moieties. As expected,
the acrylamide-containing cyclosporin A irreversibly modified the engineered cyclophilin
A but not wild-type protein lacking the additional cysteines.
Importantly, less reactive ligandsmay bemore selective in complex systemswhere highly
reactive ligands may react with a variety of proteins. Sames and colleagues showed this by
attaching more than a dozen different electrophiles to benzenesulfonamide, a nanomolar
inhibitor of the enzyme carbonic anhydrase.
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Probes containing electrophiles such a vinyl
sulfones bound to many different proteins, but probes containing a less-reactive epoxide
reacted only with carbonic anhydrase, forming a one-to-one adduct with the enzyme. Fur-
thermore, the reaction did not occur in the presence of other ligands that bind in the active
site, demonstrating that the labeling was specific in crude cell extracts. Thus, if epox-
ides were used in a fragment library, epoxide opening could, like disulfides, be useful for
site-directed ligand discovery.
Even disulfide bonds are not always reversible: use of a highly reactive methanethiosulf-
onate (MTS) reagent under nonreducing conditions gives complete disulfide formation with
cysteine-containing proteins. Rosenberry and colleagues introduced a cysteine residue just
outside the active site of acetylcholinesterase and functionalized it with several different
MTS-derivatized ligands.
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44
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The length of the linker and the nature of the inhibitor resulted
in different activity profiles for the modified enzyme and molecular modeling suggested
that the different ligands bind to different areas of the large active site.
The reactivity of natural cysteines has recently been exploited to discover an allosteric
site in the phosphatase PTP1B. While evaluating a series of cysteine-reactive probes for
their potential to selectively label the active site cysteine, Hansen
et al
. discovered that the
small molecule 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (ABDF) reacted select-
ively and quantitatively with a single cysteine residue.
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45
]
Surprisingly, although the
V
max
value of the enzyme was reduced, the
K
m
value was not substantially affected and the
enzyme still retained some activity, suggesting that the modification could not be at the
active-site cysteine. Proteomics experiments subsequently revealed that the modification is
in fact on cysteine-121, which is located approximately 8 Å from the active-site cysteine.
Although crystallization of the modified protein was not successful, it is clear that ABDF
selectively targets a previously cryptic allosteric site (Figure 10.4A).
Finally, several groups have reported irreversible chemistries that work selectively in the
presence of proteins or even whole cells. These include the Huisgen cycloaddition recently
improved by Sharpless and others,
[
46 48
]
chemical ligation,
[
49
]
the formation of oximes or
hydrazones and the Staudinger ligation.
[
50
]
Until recently, irreversible chemistries have not been applied to the systematic discovery
of novel ligands. Instead, such methods have proven to be more useful for ligand-directed
protein discovery, in which ligands specific to a certain protein or class of proteins are
used to identify related proteins from a crude cell extract.
[
4, 51
]
Irreversible site-directed
ligand discovery forms a continuum with affinity labeling and activity-based protein pro-
filing, in which a reactive (often substrate-like) molecule binds covalently to a protein of
interest.
[
52, 53
]
These techniques are typically used to probe enzyme mechanism or function
or to identify a specific protein or class of proteins from a complicated mixture such as
