Chemistry Reference
In-Depth Information
decarboxylated, indicating that they were functioning as substrates. The binding SAR was
consistent with the crystal structure of ADC.
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10.3.9 Dative Capture Using Metal Chelation
The same concept of covalent capture can also be applied to noncovalent interactions, such
as metal-ligand bonds. In an interesting application of this concept, Mallik, Srivastava
and coworkers have attached copper-chelating moieties to small ligands, such as aryl sulf-
onamides, for carbonic anhydrase.
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The resulting molecules can then present copper to
histidine residues located just outside the active site. In one example, this technique resul-
ted in taking a 1.5 M fragment to an 11 nM inhibitor
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(Figure 10.3C). Crystallographic
characterization of some of the inhibitors revealed that, in at least one case, the copper
atom does interact with the targeted histidine residue while the sulfonamide interacts with
the catalytic zinc in the active site.
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Metal chelation has also been applied in studies of the structural-functional basis for
seven-trans-membrane receptor activation. Using the
2
-adrenergic receptor as a model
system, Elling
et al
. introduced several point mutations to form a metal binding site between
trans-membrane helices III, VI and VII and showed that the receptor could be activated
by metal ions alone or metal ions chelated by phenanthroline or bipyridine.
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Based on a
homology model built over the rhodopsin receptor crystal structure, helices III, VI and VII
would have to move inwards to form a metal chelation site, suggesting that movement of
these helices is critical for receptor activation.
10.4
Irreversible Capture Methods
The reversible capture method for site-directed ligand discovery is more commonly used
than irreversible approaches. While reversible approaches select ligands on the basis of
inherent binding affinity, irreversible chemistries may select compounds with no inherent
affinity because species that react faster will out-compete slower reacting species with
higher affinity. Consider a ligand that contains two electrophiles, each of which reacts with
a protein-bound nucleophile at a different rate. If the reactivity difference between the two
electrophiles is sufficiently large, the protein is likely to react with the 'hotter' electrophile
regardless of fragment orientation or affinity.
Over a decade ago, work on the enzyme aldolase reductase elegantly demonstrated this
point. The noncovalent inhibitor alrestatin was modified to contain various electrophiles:
-chloroacetamide, -bromoacetamide or -iodoacetamide. Noncovalent interactions
between inhibitors and protein would not have changed, but molecules behaved differ-
ently based on the electrophile: the weakest showed reversible inhibition, whereas the
iodoacetamide displayed almost complete irreversible inhibition.
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These results are an
important warning: if a reaction is too facile, irreversible reactions can obscure true binding
affinities.
Nonetheless, some research indicates that irreversible chemistries can be used for site-
directed ligand discovery. Meares and colleagues constructed ligands containing metal
chelates and reactive functionalities by engineering cysteine residues into an antibody that
tightly binds metal chelates.
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Using reactive functionalities such as haloacetamides and
