Chemistry Reference
In-Depth Information
among different cysteine residues. This method was originally developed and validated
with the discovery of nanomolar inhibitors of the enzyme thymidylate synthase
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and
it has since been applied to many other targets, including protein-protein interactions,
catalytic sites, inactive conformations of enzymes or even regions outside a catalytic site.
Some of these applications are described in the following section.
10.3.2 Tethering at Protein-Protein Interfaces
The binding of interleukin-2 (IL-2) to its receptor constitutes an early event in T-cell activ-
ation and has been pursued as a target for novel immuno-modulators.
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A small-molecule
antagonist of IL-2 (Ro26-4550) was first reported by researchers at Hoffman-LaRoche,
who identified a compound that binds to IL-2 with low micromolar potency and blocks the
binding to the receptor.
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This small-molecule binding site was used as a starting point for
designing a series of Tethering screens for IL-2. Researchers at Sunesis solved the crystal
structures of IL-2 in the absence or presence of Ro26-4550, revealing an elongated bind-
ing site composed of a highly adaptive hydrophobic subsite and a rigid polar subsite.
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This structural information was then used to guide the engineering of a series of cysteine
residues flanking the Ro26-4550-binding site. Tethering was then used to interrogate the
small-molecule binding sites and revealed a number of fragments that can be divided into
two classes. Hydrophobic ligands constituted the majority of hits and were identified from
a variety of positions around the adaptive subsite. A smaller set of hits bound around a
polar subsite, many of which shared similarity with the polar guanidine pharmacophore
in Ro26-4550. This is consistent with the notion that hydrophobic interactions are more
promiscuous, whereas highly directional polar interactions require precise positioning and
produce fewer hits.
Several of the ligands selected around the adaptive subsite of IL-2 were small aromatic
carboxylic acids, a pharmacophore that had not previously been explored for this site.
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Using molecular modeling to guide compound design, aromatic carboxylic acids were
fused to a distant relative of Ro26-4550.
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A small library of 20 compounds yielded eight
compounds with sub-micromolar affinity and the most potent compound inhibited the
binding of IL-2 to IL-2R with an IC
50
of 60 nM, 50-fold more strongly than the parent
compound (Figure 10.3A). The structure of this compound bound to IL-2 was subsequently
determined, revealing further movements around the adaptive subsite to accommodate
the aromatic acid.
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Together, these results illustrate the utility of site-directed ligand
discovery to identify ligands to a highly flexible protein site that is not amenable tomolecular
modeling, and the ability to use these ligands to advance medicinal chemistry.
10.3.3 Probing a Ligand Binding Site Using Disulfide Capture and
Tethering
G-protein-coupled receptors (GPCRs) constitute a large class of transmembrane receptors
that mediate signaling by a plethora of extracellular ligands. GPCRs are unique in their sens-
itivity to even subtle changes in ligand structure, allowing an agonist to be readily converted
into an antagonist. The basis for this triggering mechanism is only beginning to become
understood at the molecular level. The complement factor 5a GPCR (C5aR) belongs to this
category and its peptide ligands can switch from agonism to antagonism with single amino
