Chemistry Reference
In-Depth Information
4.6.1 Expansion of Primary Fragment Hit - A-FABP
Fatty acid binding proteins (FABPs) constitute a family of homologous proteins, all of w
h
ich
reversibly bind long-chain fatty acids, bile acids and retinoic acid with high affinity.
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]
Members of this protein family have MWs of 14-15 kDa and a prominent feature is a large
and well-defined binding pocket that can accommodate these lipophilic ligands. Although
the exact physiological role of the FABPs is still not clearly understood, they are thought
to be involved in the st
o
rage, transport and targeting of fatty acids to appropriate loca-
tions within the cells.
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]
A-FABP, for example, is known to interact physically with
hormone-sensitive lipase,
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]
possibly to enable A-FABP to bind and transport the product
of the enzymatic reaction. In 1996, Hotamisligil
et al
. found that mice with a disruption
in the gene encoding A-FABP develop dietary obesity comparable to wild-type animals
but, unlike control mice, do not develop insulin resistance or diabetes.
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]
This was further
supported by data in a genetically obese mouse model.
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]
Based on these results, it was
hypothesized that blocking of A-FABP may represent a route to prevent obesity-induced
insulin resistance.
In the A-FABP fragment screening campaign,
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24
]
transverse relaxation-filtered
1
H1D
NMR was employed as the binding assay, a fluorescence polarization assay utilizing a
fatty acid analogue with a fluorescent group was used to determine potency and X-ray
crystallography was used to obtain structural information. The fragment library that was
screened contained 531 diverse fragments divided into 57 cocktails. The relaxation filter
spectra for one cocktail containing 10 fragments are shown in Figure 4.4, together with the
identified hit BVT.1960. Atotal of 52 primary fragment hits were detected. The hit criterion
used was
>
80% reduction in signal intensity in the presence of A-FABP as compared with
the reference spectrum when applying a spinlock time of 400 ms. The primary hits were
confirmed by repeating the relaxation filter experiment with two spinlock times (100 and
400 ms) on the individual compounds This allowed a classification of the hits into weaker
(14) and stronger (38) binders. Most of the initial hits (43 out of 52, 83%) comprised a
CO
2
,SO
3
or PO
3
group, indicating that they are fatty acid mimics. This feature was further
accentuated in the strong binder category, where 95% (36 out of 38) of the hits comprised
aCO
2
,SO
3
or PO
3
group. It was possible to obtain X-ray crystallographic structures of
A-FABP in complex with several of the primary fragment hits.
The left panel of Figure 4.5 shows a region of the crystal structure of A-FABP in com-
plex with the primary screening hit BVT.1960. As in pre
v
iously published structures of
ligand-target complexes between FAPB and fatty acids,
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115 117
]
an interaction between the
carboxylate group of the NMR hit and a conserved tyrosine side-chain (Tyr128) is clearly
observed. In addition, a novel interaction, not found in complexes between FABPs and
fatty acids, was found for a distal hydroxyl group, which forms a hydrogen bond to the
side-chain ofAsp76. More than 30 structures of ligand-target complexes of humanA-FABP
were determined and an outstanding feature of these structures is how the ligands cluster
in one region of the binding pocket (see the right panel of Figure 4.5). Noteworthy is that
there are a large number of conserved water molecules in the binding pocket and only a
few of these are displaced by ligands.
A-FABP belongs to a large family of fatty acid binding proteins and selectivity against
particularly the heart and muscle isoform (H-FABP) is considered important. Mice with
a disruption of the gene encoding H-FABP have been reported to suffer from stress
