small molecules in this study (9a-m vs 5a-m, Table 3). In the
current study, we found that 9g and 9h were the best two
inhibitors against SIRT2 with IC50 values of 20 μM and 35 μM,
respectively. Notably, both of them (9g and 9h) show 10-fold
higher SIRT2 inhibition than that of other sirtuins. Therefore, this
novel scaffold of L-S-(3-carboxamidopropyl) cysteine (L-CAPC)
could be exploited for developing more potent and highly
selective sirtuin inhibitors. Due to the limited cell permeability of
peptide-based sirtuin inhibitors, L-CAPC small molecules as
sirtuin inhibitors are under further investigation in our laboratory.
To further evaluate the inhibitory potency and selectivity of
peptides 9f-h, we measured their IC50 values using an HPLC
assay (Table 4). Interestingly, the IC50 values of peptides 9f-h
were all higher against SIRT1/3 catalyzed-deacetylation reaction,
SIRT5 catalyzed-desucinylation reaction and SIRT6 catalyzed-
demyristoylation reaction. Exceptionally, an IC50 value of 20 μM
was obtained when 9g was subjected to an inhibition assay
against the SIRT2-catalyzed deacetylation reaction while an IC50
value of 35 μM was obtained when 9h was used under the same
conditions. Considering that L-ACAH is a known weaker
SIRT1/2/3 inhibitory warhead than thioacetyl group, it’s
reasonable that peptides 9f-h with the warhead of L-CAPC like
L-ACAH demonstrated lower SIRT2 inhibitory potency than that
of TM with thioacetyl as a warhead (Table 4)40, 50. Notably, L-
CAPC (9g or 9h) show better selectivity toward SIRT2 compared
with our previously reported sirtuin inhibitor of BHJH-TM1, a
thiomyristoyl lysine peptide (Table 4)51. On the other hand, the L-
ACAH (7) with “inverted amide”, which contains the
carboxamide NH2-dodecylated analog with the similar sequence
as peptides 9 all derived from histone H3K9, has shown an
inhibitory potency against SIRT6 with IC50 of 20.9 M40 while L-
CAPC (9g or 9h) containing the carboxamide NH2- decylated or
tridecylated or analogs only show the inhibitions against SIRT2
(Table 4). The replacing the methylene at 4-position of L-ACAH
with the atom of sulphur may increase the flexibility of the
carboxamide NH2-long chain fatty acylated side chain and thus
more readily orientate and target SIRT2 (Fig. 3). However, too
long or short for the length of hydrophobic acyl group is not quite
appropriate for SIRT2 inhibition in L-CAPC (9g vs 9f or 9h,
Table 4). Therefore, peptide 9g was come from the coupling of
decyl amine (Fig. 3), which showed the best inhibitory potency
with an IC50 value of 20 μM against SIRT2 and the best
inhibitory selectivity with over 10-fold higher than that of other
tested sirtuins (SIRT1, SIRT3, SIRT5 and SIRT6). Additionally,
we have performed the mechanism analysis by doing the kinetics
study to find out whether L-CAPC peptides 9 are mechanism-
based inhibitors. As shown in Figure S4 in supporting
information, the apparent Km value for H3K9AcWW (SIRT2
substrate) increased with increasing concentrations of 9g (0μM,
5μM and 10μM). Furthermore, plots of 1/ V versus 1/[S] revealed
a series of lines that intersected at the 1/V axis at each inhibitor
concentration (V stands for initial velocities, [S] stands for
substrate concentration, Figure S5 in supporting information).
The features of kinetics and the double reciprocal plot indeed
demonstrated that 9g is a competitive inhibitor for SIRT2
(Figures S4 and S5).
Acknowledgments
We thank the following for the financial support to this
work: National Natural Science Foundation of China (No.
21662010), the Innovation Team of Natural Science Foundation
of Department of Educationof Guizhou Province (No.
QJHRCTDZ [2015] 57), the Cooperative Project of Department
of Science and Technology of Guizhou Province (No.
QKHLZ[2015]7350), the Talent Team Project of Department of
Science and Technology of Guizhou Province (No.
QKHPTRC[2016]5613\5677).
Supplementary Material
Supplementary data associated with this article can be found,
in the online version, at http://
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Due to the concern about the possible toxicity associated with
thioamide-based
sirtuin
inhibitors30-34
.
L-2-amino-7-
carboxamidoheptanoic acid (L-ACAH) as sirtuin inhibitors have
been developed for several years39, 40. However, the starting
material of L--aminosuberic acid (L-Asu) as an unnatural amino
acid is not readily available. An alternative strategy here has been
proposed and employed to develop novel sirtuin mechanism-
based inhibitors, which involves in simply replacing the
methylene at 4-position of L-ACAH with an atom of sulphur to
give a novel scaffold of L-S-(3-carboxamidopropyl) cysteine (L-
CAPC) constructed by starting from L-cysteine, a nature amino
acid (Fig. 2). According to recent discoveries of sirtuin novel
enzymatic activities for preferentially removing some acyl
groups, like malonyl44, succinyl44, glutaryl45, crotonyl46,
myristoyl47 and so on5, 48, we have designed and synthesised L-
CAPC small molecules 5a-m and L-CAPC peptides 9a-m.
Similar to the results from the strategies of thioamide or L-
ACAH15, 21, peptides show better inhibition compared to that of
4