Chemistry Reference
In-Depth Information
X is known, information about binding location. This can be exploited to direct the screen
to a particular site on a protein. This location is typically the active site, but the method
can also be used to investigate allosteric sites or protein-protein binding interfaces. In the
following, we will describe various practical approaches to covalent capture and provide
examples of their application in studying protein-ligand interactions and drug discovery.
10.3 Reversible Capture Methods
10.3.1 Tethering
10
Tethering is a capture method that is based on thiol-disulfide exchange, where a free thiol
presented by a cysteine residue on a protein surface can react to form a disulfide with a
disulfide-containing small fragment.
[
11, 12
]
In this method, X is a cysteine residue and Y
is a disulfide (Figures 10.1 and 10.2A) and if Y binds close to X, a disulfide bond will
form between X and Y. The resulting protein-small molecule conjugate can be detected
by mass spectrometry (MS) and the identity of the conjugated fragment can be confirmed
based on the mass shift relative to unmodified protein. For screening purposes, fragments
of different molecular masses can be pooled and captured hits can be identified based
on their unique mass shifts. Tethering is well suited for site-directed ligand discovery,
as new cysteine residues can be engineered into the target protein to direct the screen
towards a site of interest. Importantly, the sensitivity of the Tethering reaction can be
adjusted by addition of a reducing agent, thereby compensating for variability in reactivity
NH
2
NH
2
NH
2
NH
2
S
S
S
S
S
S
S
S
S
S
SH
SH
(a) Tethering
NH
2
NH
2
S
S
S
S
S
SH
S
SR
SH
S
S
LG
(b) Tethering with extenders:
In situ
fragment assembly
NH
2
NH
2
S
S
S
R-S-X
R-SH
S
R-S
S
LG-R-S-X
SH
S
S
S
H
2
NOH
(c) Tethering with breakaway extenders: Preserving catalytic site integrity
Figure 10.2
Different versions of Tethering that have been used for covalent capture.
