Chemistry Reference
In-Depth Information
screened against a single target, with the results obtained by mass spectrometry correl-
ating well with those obtained by conventional means.
[
50
]
Methods for simultaneously
screening multiple binders require that the interaction between the target and any com-
ponent in the screen be independent of the interactions with other components. This
requirement can be met by using a sufficient excess of the target in each screen.
[
49
]
Thus, when screening ligand libraries with unknown binding affinities, it is also pos-
sible to include a small amount of compound with a known binding constant (
K
d
)to
act as an internal calibrant for determining the
K
d
s for those library ligands for which
noncovalent complexes with the target are observed.
[
49
]
Mass spectrometry has very high
sensitivity and a wide dynamic range and previous results have shown that using these
techniques it is possible to detect and determine
K
d
s for noncovalent complexes in the
range 10 nM-1 mM.
[
49
]
An important consideration in the success of direct observation of specific noncova-
lent complexes of native protein with small molecules using ESI MS is the nature of the
sample itself, which is aqueous, near physiological pH and with an ionic strength cap-
able of maintaining the native target conformation and specific noncovalent interactions
thereof. These considerations will be discussed fully later. Under such conditions, the pro-
tein remains fully folded with many of the acidic and basic side-chains either involved
in strong hydrogen bonding interactions (that help maintain the native state protein struc-
ture) or otherwise inhibited from charging by their position in the protein structure or
p
K
a
value (Figure 7.6d). The fully folded protein typically shows a very narrow
m
/
z
distribution with just a few charge states observed and with a maximum in the distri-
bution at much higher
m
/
z
than for the denatured protein. This is shown for carbonic
anhydrase in Figure 7.6e, where only three charge states are observed (
10)
compared with the more extensive charge state distribution under denaturing conditions
(Figure 7.6b). The mass spectrum can similarly be deconvoluted as already described and
will result in the same protein molecular weight as determined under denaturing conditions
(Figure 7.6f). If an active site inhibitor for carbonic anhydrase is added, the noncovalent
complex of carbonic anhydrase
+
8,
+
9 and
+
inhibitor is then observed in the ESI mass spectrum
together with some unbound protein (Figure 7.6g). The mass difference between the peaks
for the unbound protein and the protein-inhibitor complex (
m
/
z
) can be multiplied by the
charge state to give directly the molecular weight of the binding inhibitor, i.e.
MW
inhibitor
=
m
/
z
+
z
.
In general, the speed, simplicity and sensitivity of mass spectrometry-based screening
of ligands against targets make it an excellent choice for a primary screen. This is espe-
cially true for
in situ
medicinal chemistry applications where the identity of the binding
species is not initially known, except that any binding species must be composed of the
building block fragments employed in the
in situ
experiment. The mass spectral detection
of protein-ligand complexes readily allows the determination of the mass of the binding
ligand, which can be used to ascertain the building block fragments comprising the ligand.
On this basis, the integration of
in situ
medicinal chemistry synthetic approaches (both DCC
and Click) with a mass spectrometry-based screen should fulfil the screening requirements
and provide a very effective means for identifying the combination of fragments that bind
to a given target under biologically relevant conditions. Ligands identified by mass spec-
trometric screening may then be verified by more traditional biological activity secondary
screening tests.
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