Chemistry Reference
In-Depth Information
10.2 Principle of Capture Methods
The concept of fragment capture can be traced to the decades-old technique of affinity
labeling
[
3, 4
]
that entails modifying a ligand with a reactive functionality to label selectively
the target protein. Although historically this was often done to elucidate the biology of
protein-small molecule interactions, more recently the concept has been applied to lead
discovery.
[
5 7
]
Covalent capture methods have been particularly useful in the application
of fragment-based lead discovery by facilitating the detection of small drug fragments that
often have low binding affinity due to limited interactions with the target protein. Identi-
fying ligands with high micromolar to low millimolar binding affinities using biochemical
or binding assays can be complicated by issues such as protein denaturation, binding at
multiple sites on the protein and compound aggregation.
[
8, 9
]
Covalent capture methods
can overcome these complications and facilitate fragment discovery by forming a covalent
bond between the fragment and the protein.
The principle of covalent capture methods is illustrated in Figure 10.1. A target protein
with a native or engineered reactive functional group X is reacted with a collection of small
fragments, each of which contains a complementary reactive functional groupY. If the small
molecule binds to the protein in the vicinity of X, then the two functional groups can react to
form a covalent bond and the resulting covalent complex can be characterized by methods
such as mass spectrometry. The reaction between X and Y can be either reversible (such as
thiol-disulfide exchange or imine formation) or irreversible (such as epoxide opening or
halide displacement). The reversible reaction can be controlled thermodynamically, which
offers the advantage that fragments can be readily selected based on binding affinity. In the
case of the irreversible reaction, the control will be largely kinetic and the reactivity must
be low enough that selection is not dominated by fragment reactivity. Key advantages of
covalent capture methods are the detection of weak ligands and, assuming the position of
Non-covalent
weak binder
Drug fragments
Difficult to detect
Drug fragments
Y
Covalent bond
Y
Y
Y
Y
X
X
X
Covalent capture
Easy to detect
Figure 10.1
Principle of covalent capture methods. Drug fragments typically have weak
binding affinity and can therefore be difficult to detect. By introducing two reactive groups,
X and Y, a fragment that binds in the vicinity of X can be captured covalently by the protein
target and easily identified by mass spectrometry.
