Chemistry Reference
In-Depth Information
with and without the target biomolecule. The equilibrium concentration profiles of all
library ligands when generated in both the presence and absence of the target protein (fol-
lowing disruption of the ligand-target complexes) are compared and the detection of ligand
enrichment in the targeted DCL is the basis of identifying the 'best binders'. Only recently
has the original promise to integrate DCC synthesis and screening occurred, as repor-
ted by Poulsen
[
15
]
and followed by Schofield and colleagues
[
28
]
using mass spectrometry
and Congreve
et al
.
[
14
]
using X-ray crystallography. These direct screening approaches
represent a major leap forward in addressing the inconvenient and somewhat impractical
indirect analysis that has come to be associated with drug discovery applications of DCC.
X-ray crystallography is without doubt unrivalled with regard to the resolution of structural
information yielded; however, it is a relatively slow turn-around experiment with a high
consumption of protein and so from a primary screening viewpoint is somewhat restricted.
By describing some recently published examples we will demonstrate that mass spectro-
metry allows direct screening of DCLs, requiring only a minimal protein sample, and is
capable of generating a rapid result that yields the mass of any ligands noncovalently bound
to the target biomolecule. Importantly, when using ESI-FTMS this mass determination is
of high accuracy and hence with a knowledge of the fragments and reversible chemistry
employed this analysis permits the unique identification of the bound ligand and is there-
fore an excellent primary screen to evaluate DCLs for drug discovery applications. An
exception to this unique identification will occur only if the mass determined for the bound
ligand(s) can be attributed to isobaric DCL constituents; these are structural isomers that
share the same molecular formula and hence share an identical molecular mass and isotope
distribution fingerprint. The contribution of these ambiguous DCL constituents to a 'hit'
identified in the MS screen can be differentiated if required to evaluate the identity of the
fragments in question, for example by tandem MS experiments or alternatively 'knockout'
MS experiments, both ready extensions of the basic experiment. Mass spectrometry has no
requirement for modification or labelling of ligand or target to permit the analysis. Through-
out this discussion, we will aim to highlight the key practical considerations involved to
deliver a successful DCL screening outcome using mass spectrometry.
Poulsen was the first to describe the application of mass spectrometry to the direct
screening of a DCLwherein a biomolecule was the target.
[
15
]
This study applied ESI-FTMS
to the screening of a DCL against the Zn(II) metalloenzyme target of carbonic anhydrase II
(CAII). Recent evidence implicates CAinhibition as a validated target and/or biomarker for
a range of disease states.
[
55
]
The classical CArecognition moiety is an aromatic sulfonamide
(ArSO
2
NH
2
); the sulfonamide group (deprotonated asArSO
2
NH
−
) serves as a zinc binding
function in the active site of CAs (Figure 7.11). When this privileged CA anchor fragment
is derivatized with 'tail' fragments, CA inhibitors with optimized target affinity, selectivity
and other pharmaceutical properties may be generated (Figure 7.11).
[
55, 56
]
In this ESI-FTMS screening proof-of-concept experiment, a DCL was generated using
the hydrazone exchange reaction (described earlier in this chapter) from two hydrazide frag-
ments (
1
and
2
) and five aldehyde fragments (
A
-
E
) (Figure 7.12). Hydrazide
1
was designed
as the CA anchor fragment and hence necessitated dual functionality: an ArSO
2
NH
2
moi-
ety for reliable CA affinity and a hydrazide moiety to take part in hydrazone exchange.
Hydrazide
2
lacked the sulfonamidemoiety of
1
, but was still able to participate in hydrazone
exchange and thus functioned as a control compound through provision of hydrazone
compounds expected to have
no
affinity for the target CA. Fragment aldehydes
A
-
E
, the
