Chemistry Reference
In-Depth Information
disadvantages. First, high homogeneity of the target protein is required and the method is
limited to protein targets that are happy in a volatile buffer such as ammonium acetate.
Second, the protein-ligand complex is detected in vacuum and electrostatic interactions
will be strengthened whereas hydrophobic interactions will be weakened compared with
an aqueous solution. Another approach utilizing mass spectrometry is 'tethering'.
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23
]
This
technique relies on the formation of a disulfide bond between the fragment and a cysteine
(naturally occurring or introduced bymutagenesis) at the site of interest in the target protein.
Therefore, only thiol-containing fragments can be used.
4.3.3
Surface Plasmon Resonance (SPR)
Protein-ligand interactions are detected by changes in the refractive index at the surface
of a solid support on which the target protein or the fragments are immobilized.
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55, 56
]
The analytes are then injected in a continuous flow and the real-time binding response
(sensorgram) is recorded. The signal increases during injection of the analyte at a rate
related to the on-rate, reaching a plateau at the equilibrium level determined by the
K
d
and
the concentrations of fragment and protein. When the injection ends, the signal decreases
back to the original level at a rate related to the off-rate. Hence a distinct advantage of
the method is that it is possible to obtain both equilibrium (
K
d
) and kinetic parameters
(on- and off-rates). Since the magnitude of the signal is directly related to the mass of the
binding ligand, it is also possible to obtain stoichiometries from the signal at saturation
level. The main disadvantage is the necessity to immobilize one of the two components.
Consequently, this is not a true in-solution technique. Immobilization of the target protein
is often protein specific and it could prove difficult to immobilize the protein so that it
remains fully functional. Further, conditions in which the protein remains stable over a
long period must be identified. This assay development phase may require a long time, but
the throughput will be high once everything is set up. If the fragments are immobilized
instead, every fragment must be synthesized with a long, flexible linker attached. The linker
will then serve as an attachment point for the covalent immobilization on the solid support,
usually via a thiol group. Another alternative is to immobilize a previously known high-
affinity ligand which acts as a probe for a defined binding site and then inject a mixture of
the target protein and the fragment(s).
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57
]
If a fragment binds and competes for binding with
the immobilized known ligand, the signal will be lower than when the fragment is absent.
4.3.4 Biochemical Activity or Binding Assay
A biochemical assay can be used directly in the primary screen at very high fragment
concentrations.
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58, 59
]
Modulation of the enzymatic function is detected by monitoring the
rates of either the decreasing substrate or increasing product concentrations. Alternatively,
a binding assay can be used where the displacement of (typically) a fluorophore-tagged
ligand is monitored. For detection of weakly binding fragments, the fluorophore-tagged
ligand should bind fairly weakly but still specifically. Advantages with using a biochemical
assay in the primary fragment screen include direct information on potency, high throughput
and very low protein consumption. The low protein consumption and the possibility of
directly using membrane preparations (lysed and homogenized cells with overexpressed
membrane protein) make it feasible to screen also a wide range of membrane proteins.
