Chemistry Reference
In-Depth Information
ligand for KcsA. These tests indicated that both TSP and the known ligand FPMSMA spe-
cifically bind KcsA and we therefore chose to use TMA as an internal standard. Repeated
application of TMA and FPMSMA, followed by washing with buffer plus 5mM DPC,
demonstrated stability of the immobilized KcsA and so these conditions were used for the
limited library screen. During the screen, the immobilized target showed insignificant loss
of binding capacity for the control compound and only 12% loss after 3 months of storage.
Of the 95 fragments that were screened, 7% showed substantial changes in the NMR spec-
trum that were specific to the target and were considered binders after analysis of spectral
intensities (Figure 6.6B). This is in line with target hit rates obtained for soluble proteins
applied toTINS. Of the potential newhits, two structures had a similar scaffold to the known
binder. The other hits had a variety of scaffolds with a variety of shapes and numbers of
rings.
6.4.3 Development of a High-affinity Inhibitor of Bacterial Membrane
Protein DsbB Using TINS
Very recently we have undertaken a program to develop high-affinity inhibitors to the
bacterial inner membrane protein DsbB in collaboration with Bushweller's group at the
University of Virginia (USA). DsbB is a redox enzyme involved in the production of toxin
in Gram-negative bacteria
[
53
]
and as such is a potentially medically interesting target. The
crystal structure of DsbB bound to its redox partner, DsbA, has been solved
[
54
]
and Bush-
weller's group has solved the solution structure of a disulfide mutant of DPC solubilized
DsbB (in preparation). Since this is very much a research project in progress at the time
of writing, we provide only an overview of the current status here (we will provide a full
report when completed).
For ligand screening, we immobilized both the functional wild-type DsbB (see above)
and OmpA (as a reference) at a solution equivalent of 100 μM. We used the compound
Ubiquinone-5 (Figure 6.7) which binds competitively with the native DsbB ligand and
is in rapid exchange on the NMR time-scale to report on the condition of immobilized
DsbB throughout the screen. Similarly to KcsA, deuterated DPC was included only in
the wash buffer. Using this arrangement, 1270 compounds were screened in mixtures that
averaged a little over five compounds each. Figure 6.7 demonstrates that the immobilized
DsbB remains intact throughout the screen. In the screen we found 93 compounds that
specifically bind DsbB for a hit rate of 7.3%. Follow-up biochemical studies are currently
under way. To date, 41 of the 93 hits have been investigated for enzyme inhibition at 250 μM,
where we find that nearly half are substantially potent (greater than 20% inhibition). The
best nine of these compounds have IC
50
s of 150 μM or better, and a representative curve
is shown in Figure 6.7. We have carried out both competition binding and competition
enzyme inhibition analyses on a limited subset of the hits. Most of the hits are competitive
with ubiquinone binding and this seems to represent the major small-molecule binding
pocket. However, a subset of hits are not competitive with ubiquinone. Docking studies
place the noncompetitive compounds in a secondary pocket which is excitingly, about
7 Å away.
