Chemistry Reference
In-Depth Information
(b)
Figure 6.3
(
Continued
)
.
and target spectra for all well-resolved peaks. This cut-off was chosen for practical reasons
based on the fact that the difference was sufficiently large to overcome artifacts related to
spectral noise, minor lineshape differences between the two samples and spectral crowding,
and therefore allowed reliable detection of a hit. This last fact is particularly important since
we wish to automate the data analysis process. Since screening on these targets has only
been carried out using TINS, it is not possible to compare directly the observed hit rates
with other methods, including high-concentration screening (i.e. screens based on inhibiting
and enzymatic activity). Where Hajduk
et al.
reported essentially a 0% hit rate for the pH
domain of AKT.
[
16
]
we in fact do detect some compounds binding, but our 'hit rate' is about
0.2%, some 10-fold lower than the lowest rate obtained for a target that is expected to be
'druggable'. In their work, Hajduk
et al
. reported hit rates of up to 1% for SAR by NMR.
Interestingly, the 3% hit rate for TINS was found when screening a soluble 'NTPase' in
the NDP-bound form. The hit rate for the apo-protein was about 9%. The low hit rate
found when the nucleotide binding pocket is occupied is expected and suggests that the
high hit rates that we observe are not due to artifacts, but rather to reliable sensitivity to
binding events. This idea is further supported by follow-up biochemical studies that we have
now performed for two targets with enzymatic activity. Considering a soluble enzymatic
target for which we found a hit rate of 9.5%, approximately 50% of the TINS hits showed
significant inhibitory activity at 500 μM, and we would expect this number to increase even
further if tested at the 1-2mM typically used in high-concentration screening. A similar
pattern has been observed for membrane proteins (see below).
