C. B. Stevens et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1703–1706
1705
arise from the increased sample volume. These results indicate that
BPT and OTT do not compete with CR to bind Ab. As such, the de-
creased levels of bound CR–Ab complex observed in the presence
of these tetrols (e.g., as shown in Figs. 1 and 2) are confirmed to re-
sult from inhibition of Ab aggregation, rather than displacement of
bound CR dye. With the addition of 10 equiv of MTT, the CR–Ab con-
centration decreased by an average of 9%. This decrease is slightly
greater than the 2.6% change expected for increased sample vol-
ume, and likely indicates a small degree of competition between
MTT and CR to bind Ab. However, this small decrease in CR–Ab can-
not explain the MTT results in Figure 1: although MTT may displace
a small percentage of bound CR molecules, it also significantly
inhibits Ab aggregation.
Collectively, the results described herein demonstrate that the
geometry around the linker phenyl ring significantly affects inhib-
itory efficacy in the terphenyltetrols OTT, MTT and PTT, perhaps
because it impacts inhibitor binding to Ab assemblies. Surprisingly,
the biphenyltetrol BPT, which lacks the linker phenyl ring, is the
most effective inhibitor of Ab aggregation. Although this result
may seem to contradict the linker requirements proposed by Rein-
ke and Gestwicki,26 BPT is more conformationally restricted than
the inhibitors included in their study, such that the characteristics
they associated with inhibitory efficacy may not apply in this case.
Our finding that BPT inhibits Ab aggregation by 50% when present
at stoichiometric levels renders it a promising architecture for fur-
ther study. Future work will focus on designing and evaluating re-
lated inhibitor molecules and identifying the Ab species they are
targeting.
Figure 2. Dose–response plot for determination of the IC50 for biphenyltetrol BPT
monitored by Congo red spectral-shift assay. Peptide samples with and without
inhibitor were incubated with agitation at 37 °C and 1500 rpm prior to addition of
Congo red. Once Ab aggregation was confirmed to reach equilibrium levels, CR–Ab
concentrations for samples containing various amounts of BPT were compared to
those for inhibitor-free control samples (1X = 25 lM). All measurements for a
particular IC50 determination were made in duplicate. A logistic sigmoidal fit was
used to determine the IC50 for each experiment. IC50 values in Table 1 represent
averages from two runs.
Table 1
Acknowledgments
IC50 values for terphenyl and biphenyl tetrols
inhibiting Ab40 aggregation.
The project described was supported by Grants from the Na-
tional Center for Research Resources (P20 RR016461) and the Na-
tional Institute of General Medical Sciences (P20 GM103499)
from the National Institutes of Health. Additional support was pro-
vided by the Winthrop University Department of Chemistry, Phys-
ics, and Geology.
a
Inhibitor
IC50 (equiv, X)
BPT
OTT
MTT
1.0 0.3
2.7 0.3
3.7 0.4
a
Each IC50 value is an average determined
from multiple experiments. 1X = 25 M.
l
Supplementary data
approximately the same as those evident at time zero; for PTT, the
maximal CR–Ab concentration is near one-half that of the control
(Fig. 1).
Supplementary data associated with this article can be found, in
To quantify inhibitory efficacy more precisely, we determined
IC50 values for the most successful terphenyltetrols, OTT and MTT.
For comparison, we also examined the biphenyltetrol BPT, to probe
the effects of removing the linker phenyl ring. A representative
dose–response plot for BPT is shown in Figure 2. For each IC50 deter-
mination, the equilibrium CR–Ab levels present at various inhibitor
concentrations were measured in duplicate and compared to those
of control (inhibitor-free) samples. A logistic sigmoidal fit was used
to determine the IC50 characteristic of each run. Runs were per-
formed in duplicate to determine the average IC50 values listed in
Table 1. Of the two most promising terphenyltetrols, OTT exhibits
greater efficacy than MTT (IC50 ꢂ2.7X vs 3.7X); both of these out-
perform PTT, which has an IC50 on the order of 10X, as shown in Fig-
ure 1. Interestingly, the biphenyltetrol lacking the linker phenyl
ring is the most successful inhibitor, with an IC50 near 1.0X.
To confirm that the decreased CR–Ab levels observed in the pres-
ence of these tetrols are, in fact, due to inhibition of Ab aggregation,
competitive binding studies were performed. For these measure-
ments, 10 equiv of inhibitor were added only after Ab40 aggrega-
tion had reached equilibrium, and CR–Ab concentrations were
compared for inhibitor-free versus inhibitor-containing samples.
For BPT and OTT, inhibitor addition did not affect the concentration
of bound complex beyond the slight (2.6%) decrease calculated to
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