Bivalent SIRT1 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2016.11.082

In the current study, bivalent compounds 1–17 constructed by covalently linking the ?-amino group of lysine in a tripeptidic scaffold to a functionality via a linker were prepared and examined for their inhibitory potencies against SIRT1, a prototypical member of the β-nicotinamide adenine dinucleotide (β-NAD⁺)-dependent sirtuin family of protein N^ε-acyl-lysine deacylases. A few of them were found to be stronger SIRT1 inhibitors than the N^?-acetyl-lysine-containing monovalent counterparts 18 and 19. As exemplified with compounds 6 and 18, a bivalent SIRT1 inhibitor could exhibit a greater degree of inhibitory selectivity among SIRT1/2/3 than the corresponding monovalent counterpart. This study has laid a foundation for the future development of superior bivalent inhibitors against the (patho)physiologically and therapeutically important sirtuin family of deacylase enzymes.

Full text of DOI:10.1016/j.bmcl.2016.11.082

Accepted Manuscript
Bivalent SIRT1 inhibitors
Juan Wang, Wenwen Zang, Jiajia Liu, Weiping Zheng
PII:
S0960-894X(16)31255-0
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.11.082
BMCL 24477
Reference:
Bioorganic & Medicinal Chemistry Letters
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Received Date:
Revised Date:
Accepted Date:
17 July 2016
22 November 2016
26 November 2016
Please cite this article as: Wang, J., Zang, W., Liu, J., Zheng, W., Bivalent SIRT1 inhibitors, Bioorganic & Medicinal
Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.11.082
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Bivalent SIRT1 inhibitors
Juan Wang§, Wenwen Zang§, Jiajia Liu§, Weiping Zheng*
School of Pharmacy, Jiangsu University, 301 Xuefu Road, Zhenjiang 212013, Jiangsu
Province, P. R. China
*Corresponding author. Tel: +86-151-8912-9171; Fax: +86-511-8879-5939; E-mail:
wzheng@ujs.edu.cn
§These authors contributed equally to this work.
1

Abstract
In the current study, bivalent compounds 1-17 constructed by covalently linking the
-amino group of Nε-acetyl-lysine in a tripeptidic scaffold to a functionality via a linker were
prepared and examined for their inhibitory potencies against SIRT1, a prototypical member of
the -nicotinamide adenine dinucleotide (-NAD+)-dependent sirtuin family of protein
Nε-acyl-lysine deacylases. A few of them were found to be stronger SIRT1 inhibitors than the
N-acetyl-lysine-containing monovalent counterparts 18 and 19. As exemplified with
compounds 6 and 18, a bivalent SIRT1 inhibitor could exhibit a greater degree of inhibitory
selectivity among SIRT1/2/3 than the corresponding monovalent counterpart. This study has
laid a foundation for the future development of superior bivalent inhibitors against the
(patho)physiologically and therapeutically important sirtuin family of deacylase enzymes.
Key words: bivalent, SIRT1, sirtuin, inhibitor.
2

Abbreviations:
1. -NAD+, -nicotinamide adenine dinucleotide
2. NAM, nicotinamide
3. 2’-O-AADPR, 2’-O-acyl-ADP-ribose
4. RP-HPLC, reversed-phase high performance liquid chromatography
5. HRMS, high-resolution mass spectrometry
6. EDC-MeI, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide methiodide
7. DIC, N,N’-diisopropylcarbodiimide
8. HBTU, 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium hexafluorophosphate
9. HOBt, N-hydroxybenzotriazole
10. NMM, N-methylmorpholine
11. EDC-HCl, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
12. TFA, trifluoroacetic acid
13. IC50, the inhibitor concentration at which an enzymatic reaction velocity is reduced by
50%
14. DIPEA, N,N-diisopropylethylamine
3

The sirtuins refer to a family of intracellular enzymes able to catalyze the protein
N-acyl-lysine deacylation in a -nicotinamide adenine dinucleotide (-NAD+)-dependent
manner (Figure 1).1,2 Sirtuins are evolutionarily conserved ancient enzymes and are found in
both prokaryotes and eukaryotes.3 In eukaryotes, while the original native sirtuin substrates
identified were histone proteins, a large number of non-histone proteins have also been
identified later on as native sirtuin substrates.4 These non-histone sirtuin substrates are not
only found in nucleus where histone proteins also reside, but also found in cytoplasm and
mitochondrion. Moreover, all the currently know eukaryotic sirtuins also reside in these three
intracellular compartments.5 These observations are consistent with the increasingly
demonstrated importance of the sirtuin-catalyzed deacylation reaction in regulating not only
nuclear events (e.g. transcription, DNA damage repair) but also cellular events occurring in
cytoplasm and mitochondrion (primarily metabolism).6-8 The sirtuin-catalyzed deacylation
reaction has also been regarded as a therapeutic target for human diseases including cancer,
metabolic diseases, and neurodegeneration.9-11 Therefore, the chemical modulators (inhibitors
and activators) for the sirtuin-catalyzed deacylation reaction have been actively pursued
during past few years,1,9,12-14 with a hope of developing novel therapeutic agents for the
above-mentioned diseases. A potent, selective, metabolically stable, and cell permeable
chemical modulator for the sirtuin-catalyzed deacylation reaction can also be employed to
further explore the therapeutic potentials of this enzymatic reaction. To facilitate the
exploration and exploitation of this enzymatic reaction, developing its effective chemical
modulators with the afore-mentioned calibers seems to represent a bottleneck step.
In terms of the development of sirtuin inhibitors, chemical library screening and
4

Figure 1. The sirtuin-catalyzed -NAD+-dependent N-acyl-lysine deacylation reaction. -NAD+, -nicotinamide adenine dinucleotide; NAM,
nicotinamide; 2’-O-AADPR, 2’-O-acyl-ADP-ribose.
catalytic mechanism-based design have been two primary approaches employed, with the identification of a few fairly potent and/or selective
inhibitors.15-23 As an effort in developing a novel avenue for sirtuin inhibitor design, in the current study, we performed a preliminary study on
5

bivalent compounds as for their sirtuin inhibitory efficacy. We found that this design was able
to furnish strong bivalent inhibitors against the deacetylation reaction catalyzed by the
prototypical sirtuin SIRT1, which were also stronger than their monovalent counterparts. We
further found that a bivalent SIRT1 inhibitor could also exhibit a greater inhibitory selectivity
among SIRT1/2/3 than the corresponding monovalent counterpart.
Our design of the bivalent sirtuin inhibitors was inspired by the observed presence of an
open space in sirtuin 3-dimensional structures that extends from the side chain of the bound
substrate’s N-acetyl-lysine residue (depicted in Figure 2A).24,25 We reasoned that, if a
functionality (R) is covalently anchored onto a N-acetyl-lysine substrate via a linker, as
depicted in Figure 2B, the resulting bivalent compound could be a stronger sirtuin inhibitor
than a N-acetyl-lysine-containing inhibitor (a monovalent counterpart) if the linker and R
could also be accommodated at a sirtuin active site. This potency enhancement would be due
to a decreased entropic penalty associated with the binding of a bivalent compound to a
sirtuin active site, as compared with the binding of its monovalent counterpart. Also from the
depiction in Figure 2A, the binding of the linker and R of a bivalent compound could also
prevent -NAD+ from binding to the same sirtuin active site, due to a steric clash with the
nicotinamide moiety of -NAD+. Therefore, such a bivalent compound would not be
deacylated by a sirtuin. It should be noted that, except for the different conformations of the
cyclopentane ring of carba-NAD+ (depicted in Figure 2A) and the N-ribose of -NAD+
bound in a Sir2Tm/-NAD+/N-acetyl-lysine substrate Michaelis complex, the binding modes
of carba-NAD+ and -NAD+ are similar, especially concerning the nicotinamide and adenine
moieties of these two molecules.26
6

Figure 2. The design of bivalent compounds 1-17. (A) A 3-dimensional rendering of the
X-ray crystallographically determined structure of the ternary complex of the yeast sirtuin
Hst2 with carba-NAD+ and a K16 N-acetylated peptide substrate derived from the histone H4
protein (Protein Data Bank code: 1SZC) (adapted from ref. 25 and used with permission),
illustrating the presence of an open space extending from the side chain of the bound
substrate’s N-acetyl-lysine residue. (B, C) The chemical structures of compounds 1-17
designed in the current study. NAM, nicotinamide; Cmpd, Compound.
7

As shown in Figures 2B and 2C, we used the depicted tripeptidic scaffold to construct
bivalent compounds 1-17 by covalently linking the side chain -amino group of its central
residue to a distal functionality via a linker. Compounds 1-17 were designed to primarily
assess their inhibitory power against SIRT1, a prototypical sirtuin family member. For this set
of seventeen compounds, we used simple aliphatic chains with different lengths (numbers of
methylene unit) as their linkers; as for the R groups, we used aromatic instead of aliphatic
groups due to the considerations on structural bulkiness and ease of manifestation of different
electrosteric properties. With aromatic groups, unique -electron-mediated interactions at
sirtuin active site can also be introduced. Given the demonstrated capability of SIRT1 to also
robustly catalyze the lysine N-defatty acylation, e.g. the catalytic removal of the slim
myristoyl group from N-myristoyl-lysine,27,28 the use of the bulkier aromatic R groups would
also help to prevent the SIRT1-catalyzed deacylation of compounds 1-17. It should be noted
that the tripeptidic scaffold in compounds 1-17 is the one that has also been used in our
laboratory over the past few years to evaluate the performance of a sirtuin inhibitory warhead
embedded in the scaffold.1,14
Compounds 1-17 were synthesized according to Schemes 1 and 2. The overall synthetic
strategy is to prepare the conjugate of R and the linker with a terminal carboxyl for the
subsequent condensation with the tripeptidic starting material 1a under peptide coupling
reaction condition. For compounds 1-16, the linker-R conjugate was prepared by an alkylation
reaction between commercially available R-SH and a bromoalkanoic acid. For compound 17,
the linker-R conjugate was prepared by a more extensive series of synthetic manipulation, as
depicted in Scheme 2. The crude 1-17 from respective reaction mixtures were purified by
8

Scheme 1. The synthesis of compounds 1-16. HBTU,
2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium hexafluorophosphate; HOBt,
N-hydroxybenzotriazole; NMM, N-methylmorpholine. The tripeptidic compound 1a was
purchased from China Peptides Co., Ltd. through a custom synthesis order; its purity was
≥98% based on RP-HPLC analysis and its exact mass was confirmed by ESI-MS analysis.
Scheme 2. The synthesis of compound 17. EDC-HCl,
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; TFA, trifluoroacetic acid;
DIPEA, N,N-diisopropylethylamine; HBTU/HOBt/NMM, as defined in Scheme 1. The
tripeptidic compound 1a came from the same source with the same quality as that used for the
synthesis described in Scheme 1.
semi-preparative reversed-phase high performance liquid chromatography (RP-HPLC). The
purified 1-17 were each shown to be >95% pure based on analytic RP-HPLC analysis, and
9

their exact masses were confirmed by high-resolution mass spectrometry (HRMS) analysis
(Table 1).
Table 1. HRMS analysis of compounds 1-19a
Compound
Ionic Formula
Calculated m/z Observed m/z
[C35H53N8O7S2]+
761.3473
692.3800
771.3970
757.3136
817.4099
813.3762
763.3630
791.4345
784.4426
784.4426
748.4426
774.4331
775.4171
805.4099
819.4256
819.4658
849.3746
718.2396
753.2791
761.3469
692.3806
771.3972
757.3111
817.4092
813.3788
763.3635
791.4340
784.4420
784.4411
748.4412
774.4323
775.4168
805.4084
819.4226
819.4652
849.3732
718.2390
753.2788
1
[C33H54N7O7S]+
[C36H55N10O7S]+
[C33H50N8O7S2Na]+
[C39H61N8O7S2]+
[C37H58N8O7S2Na]+
[C35H55N8O7S2]+
[C35H59N12O7S]+
[C40H62N7O7S]+
[C40H62N7O7S]+
[C37H62N7O7S]+
[C37H60N9O7S]+
[C37H59N8O8S]+
[C38H61N8O7S2]+
[C39H63N8O7S2]+
[C37H63N12O7S]+
[C37H57N10O9S2]+
[C26H46IN7O7Na]+
[C28H50IN8O8]+
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
aAll the compounds were measured with the positive ion mode of electro-
spray ionization.
10

When compounds 1-4 were initially prepared and screened for their SIRT1 inhibitory
potency, we found that the R groups in compounds 1 and 4 seemed to confer stronger SIRT1
inhibition than those in compounds 2 and 3 (Table 2). Therefore, the two R groups in 1 and 4
were selected to construct compounds 5 and 6 whose linker chain was more extended than
that in compounds 1 and 4. We would like to see if stronger SIRT1 inhibition could be
realized by this linker chain extension. Indeed, while 5 was found to be slightly stronger than
1, 6 exhibited a much stronger SIRT1 inhibition than 4. More importantly, 6 was also found
to be modestly stronger than 5 (Table 2). Encouraged by this finding, we then prepared 7
whose R group was the same as that in 6, yet linker chain was two methylene units less than
that in 6. We found that 7 was a weaker SIRT1 inhibitor than 6 (Table 2), suggesting that
linker chain shortening from that in 6 had a deleterious effect on SIRT1 inhibition. We
subsequently prepared 8-13 that had the same linker chain as that in 6, yet different R groups
from that in 6. We found that, while 9 and 10 were slightly weaker SIRT1 inhibitors than 6,
all the remaining compounds (i.e. 8 and 11-13), especially 8, were much weaker SIRT1
inhibitors than 6 (Table 2), suggesting that the binding pocket for R group is mostly
hydrophobic in nature yet is able to confer a specific interaction environment for R group. We
further explored the effect of linker chain elongation from that in 6 on SIRT1 inhibitory
potency. For this, we prepared and assessed 14 and 15 that had the same R group as that in 6,
yet had linker chains one and two methylene units, respectively, more than that in 6. We
found that, while 14 was a weaker SIRT1 inhibitor than 6, 15 exhibited a ~2.3-fold
enhancement in SIRT1 inhibitory potency as compared with 6 (Table 2). Prompted by this
finding, we further prepared and assessed 16 (same linker yet different R as compared with 15)
11

and 17 (same R yet different linker as compared with 15). Both compounds were found to be
much weaker SIRT1 inhibitors than 15 (Table 2), suggesting that the binding regions for both
R and the linker of a bivalent compound are hydrophobic in nature. Of note, the hydrophilic R
in 16 is the one also found in 8, and the linker in 17 can be regarded as the hydrophilic
counterpart of the hydrophobic linker in 15 with roughly same length. It is worth noting that
compounds 1-17 were all found not to be deacylated under our assay condition.
Table 2. The SIRT1 inhibition of compounds 1-19a
Compound
IC50(M)
57.7
Compound IC50(μM)
18
171
1
2
3
4
5
6
7
11
12
13
14
15
16
17
187.3
133.1
30.0
149.5
77.5
15.4
12.4
27.0
5.3
45.8
116
>900
14.6
14.6
39.3
8
9
18
19
>200
10
aSee “Supplementary Material” for SIRT1 inhibition
assay details.
Since a bivalent ligand is known to exhibit stronger target binding affinity than its
monovalent counterpart, due to the decreased entropic penalty for the binding of a bivalent
ligand than its monovalent counterpart to the same target,29 we also prepared and assessed the
SIRT1 inhibitory potency of the two compounds shown in Figure 3. Our use of the iodoacetyl
compounds 18 and 19 as the monovalent controls to be evaluated along with compounds 1-17
12

were not only based on a structural intuition that 18 and 19 are R-less, but also based on our
fortuitous finding that these two compounds did not behave as irreversible SIRT1 inhibitors
nor behave as SIRT1 substrates under our assay condition; they behaved as simple reversible
SIRT1 inhibitors.
Figure 3. The chemical structures of the two
monovalent compounds used in the current study.
As shown in Table 2, 18 and 19 were found to be weaker SIRT1 inhibitors than most
compounds among 1-17, especially compounds 5, 6, 9, 10, and 15. Therefore, the screen of
compounds 1-17 for their SIRT1 inhibitory potency generated a few compounds that were
found to be stronger than their monovalent counterparts 18 and 19.
Compounds 18 and 19 were prepared according to Scheme 3. The iodoacetylation of a
precursor amine was performed under the amide bond formation condition with
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide methiodide (EDC-MeI) or
N,N’-diisopropylcarbodiimide (DIC) as the coupling reagent. The crude 18 and 19 from
respective reaction mixtures were also purified by semi-preparative RP-HPLC. The purified
18 and 19 were also each shown to be >95% pure based on analytic RP-HPLC analysis, and
their exact masses were also confirmed by HRMS analysis (Table 1).
13

Scheme 3. The synthesis of compounds 18 and 19. EDC-MeI,
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide methiodide; DIC,
N,N’-diisopropylcarbodiimide; TFA, as defined in Scheme 2. The tripeptidic compound 1a
also came from the same source with the same quality as that used for the synthesis described
in Scheme 1.
14

In order to shed light on the mechanism of SIRT1 inhibition by the bivalent compounds,
we next determined the SIRT1 inhibition kinetics for 6. It should be noted that, even though
15 is the strongest bivalent SIRT1 inhibitor among 1-17, due to its lower aqueous solubility
than the second strongest compound (i.e. 6), we therefore picked the more manageable 6 for
the SIRT1 inhibition kinetics determination. As indicated in Figure 4, 6 was found to be
competitive versus the N-acetyl-lysine substrate and non-competitive versus -NAD+. This
finding would be consistent with an ordered sequential kinetic mechanism for the
SIRT1-catalyzed deacetylation reaction, in which the N-acetyl-lysine substrate binding
precedes the binding of -NAD+.30 This finding also suggested that the bivalent SIRT1
inhibitors seemed indeed to be able to occupy the open space observed in sirtuin structural
analysis and thus prevent the binding of the N-acetyl-lysine substrate (to free sirtuin) and
-NAD+ (to its binding site created on sirtuin following the binding of the N-acetyl-lysine
substrate to free sirtuin).
(A)
(B)
Figure 4. SIRT1 inhibition kinetics of compound 6. (A) Competitive inhibition of 6 versus
the SIRT1 substrate H2N-HK-(N-acetyl-lysine)-LM-COOH (AcK). Kis and the apparent Km
for AcK were determined to be 6.4 ± 3.9 M and 200.4 ± 15.2 M, respectively. (B)
Non-competitive inhibition of 6 versus -NAD+. Kii and the apparent Km for -NAD+ were
determined to be 31.1 ± 5.4 M and 168.9 ± 46.7 M, respectively. See “Supplementary
Material” for assay details.
15

Since a bivalent ligand is also known to be able to exhibit an enhanced binding
selectivity than its monovalent counterpart toward a desired target versus other homologous
targets, due to the possible existence of different distances between the two binding pockets
engaged by a bivalent ligand among different targets,29 we also performed a comparative
assessment of the bivalent compound 6 and its monovalent counterpart 18 for their inhibitory
potencies against SIRT2 and SIRT3, two sirtuins closely related to SIRT1 in terms of the
substrate N-acyl head specificity.1,27,28,31 As shown in Table 3, while 18 was found to be
essentially a pan-SIRT1/2/3 inhibitor, 6 exhibited an appreciable degree of inhibitory
selectivity toward SIRT1 versus SIRT2 and SIRT3.
Table 3. The comparative assessment of SIRT1/2/3 inhibition for compounds
6 and 18a
IC50(M)
Compound
SIRT1/ SIRT1/
SIRT2b SIRT3c
SIRT1
SIRT2
>200
SIRT3
>1,000
12.4 ± 2.0
>16
>81
6
39.3 ± 18.0 22.2 ± 7.4 54.2 ± 16.3
0.56
1.38
18
aSee “Supplementary Material” for SIRT1/2/3 inhibition assay details. bThe
inhibitory selectivity of SIRT1 versus SIRT2. cThe inhibitory selectivity of
SIRT1 versus SIRT3.
To summarize, in the current study, we preliminarily explored the feasibility of
developing bivalent-type sirtuin inhibitors with SIRT1 as the model sirtuin. As compared with
the monovalent counterpart, several bivalent compounds among 1-17 were found to be
stronger SIRT1 inhibitors. Moreover, as demonstrated with compounds 6 and 18, as well as
human SIRT1/2/3, a bivalent compound could also exhibit a higher degree of inhibitory
selectivity among different sirtuins than its monovalent counterpart. Our findings suggested
that, when trying to develop potent and selective sirtuin inhibitors, bivalent compounds could
16

be also considered as another class of inhibitors to pursue.
Acknowledgements
We would like to thank the following for the financial support to this work: the National
Natural Science Foundation of China (grant no. 21272094), the Jiangsu provincial
specially appointed professorship, the Jiangsu provincial “innovation and venture talents”
award plan, and Jiangsu University.
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19

Graphical Abstract
Bivalent SIRT1 inhibitors
Juan Wang§, Wenwen Zang§, Jiajia Liu§, Weiping Zheng*
A study on seventeen bivalent compounds (1-17) and their monovalent counterparts (18 and
19) with SIRT1/2/3 supported the feasibility of developing bivalent-type sirtuin inhibitors
able to furnish a stronger and more selective sirtuin inhibition
IC50(M)
SIRT2
>200
than their monovalent counterparts.
Compound
SIRT1
6
12.4 ± 2.0
18
39.3 ± 18.0 22.2 ± 7.4 54
20