Design and evaluation of 3-(benzylthio)benzamide derivatives as potent and selective SIRT2 inhibitors

Article abstract of DOI:10.1021/acsmedchemlett.5b00075

Inhibitors of sirtuin-2 (SIRT2) deacetylase have been shown to be protective in various models of Huntington's disease (HD) by decreasing polyglutamine aggregation, a hallmark of HD pathology. The present study was directed at optimizing the potency of SI

Full text of DOI:10.1021/acsmedchemlett.5b00075

Letter
pubs.acs.org/acsmedchemlett
Design and Evaluation of 3‑(Benzylthio)benzamide Derivatives as
Potent and Selective SIRT2 Inhibitors
Mohammad A. Khanfar,†,‡ Luisa Quinti,§ Hua Wang,† Johnathan Nobles,§ Aleksey G. Kazantsev,*,§
and Richard B. Silverman*,†
†Department of Chemistry, Department of Molecular Biosciences, Chemistry of Life Processes Institute, Center for Molecular
Innovation and Drug Discovery, Northwestern University, Evanston, Illinois 60208-3113, United States
‡Department of Pharmaceutical Sciences, The University of Jordan, Amman, Jordan
§Department of Neurology, Harvard Medical School and Massachusetts General Hospital, Charlestown, Massachusetts 02129-4404,
United States
S
* Supporting Information
ABSTRACT: Inhibitors of sirtuin-2 (SIRT2) deacetylase have
been shown to be protective in various models of Huntington’s
disease (HD) by decreasing polyglutamine aggregation, a hallmark
of HD pathology. The present study was directed at optimizing the
potency of SIRT2 inhibitors containing the 3-(benzylsulfonamido)-
benzamide scaffold and improving their metabolic stability.
Molecular modeling and docking studies revealed an unfavorable
role of the sulfonamide moiety for SIRT2 binding. This prompted
us to replace the sulfonamide with thioether, sulfoxide, or sulfone
groups. The thioether analogues were the most potent SIRT2 inhibitors with a two- to three-fold increase in potency relative to
their corresponding sulfonamide analogues. The newly synthesized compounds also demonstrated higher SIRT2 selectivity over
SIRT1 and SIRT3. Two thioether-derived compounds (17 and 18) increased α-tubulin acetylation in a dose-dependent manner
in at least one neuronal cell line, and 18 was found to inhibit polyglutamine aggregation in PC12 cells.
KEYWORDS: SIRT2, Huntington’s disease, docking, 3-(benzylthio)benzamide, polyglutamine aggregation
uman sirtuin-2 (SIRT2) belongs to the class III of
Hhistone deacetylases (HDAC), which require nicotina-
mide adenine dinucleotide (NAD+) for their function.1,2
SIRT2 has many known substrates, including α-tubulin,3
histones 3 and 4, transcription factors FOXO1 and FOXO3a,
and others.4 SIRT2 is ubiquitously expressed in all tissues and
highly abundant in the central nervous system (CNS). The
expression of SIRT2 is significantly increased during neuro-
development and remains strikingly high in the adult brain.3
Loss of SIRT2 through small-molecule inhibition or genetic
ablation is beneficial for treatment of a number of neuro-
degenerative diseases. Pharmacological inhibition of SIRT2
increases neuronal survival in animal models of Parkinson’s
disease (PD), which is associated with changes in protein
inclusion body characteristics.5 Moreover, SIRT2 inhibition
mediates protection in neuronal and invertebrate models of
Huntington’s disease (HD), which is also associated with a
reduction in polyglutamine (polyQ) aggregates, a hallmark of
HD pathology.6
The efficacy of the sulfobenzoic acid derivative SIRT2
inhibitor, AK-1 (SIRT2 IC50 = 12.5 μM), in PD and HD
models has been described (Figure 1).5−7 Brain-permeability of
its close structural analogue, the selective SIRT2 inhibitor AK-7
(IC50 = 15.5 μM), has been reported.7 We have shown that
treatment with AK-7 improved motor function, extended
Figure 1. Structures of sulfobenzoic acid derivatives as SIRT2
inhibitors.
survival, reduced brain atrophy, and was associated with marked
reduction of aggregated mutant huntingtin in two genetic
mouse models of HD.8
Recently, we developed and characterized several 3-
(benzylsulfonamido)benzamide derivatives as potent and
Received: February 15, 2015
Accepted: March 26, 2015
© XXXX American Chemical Society
A
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selective SIRT2 inhibitors.9 Many of the synthesized com-
pounds increased α-tubulin acetylation and inhibited polyQ
aggregation in several neuronal cell lines.9 However, the
reported compounds suffered from rapid metabolic oxidative
demethylation at the sulfonamide nitrogen to the correspond-
ing inactive metabolites (Figure 1).9
The inactivity of the demethylated analogues can be
attributed to the hydrophilic properties of the sulfonamide
group and, more decisively, to the formation of an acidic
moiety, which can be partially ionized at physiological pH. For
example, the calculated pKa of demethylated analogue 2 using
MarvinSketch is 6.7, which means that 83.4% (as calculated by
the Henderson−Hasselbalch equation) of this compound is
negatively charged and involved in electrostatically enforced
hydrogen bonding interactions with water molecules, resulting
in a high desolvation energy. Furthermore, analysis of the
SIRT2 active site using the Discovery Studio suite revealed that
the surface is highly hydrophobic (Figure 2A) and lacks any
Figure 3. Highest ranked docked pose of demethylated analogue 2
within the active site of SIRT2 (pdb code 3ZGO). Side chains of the
amino acids are only visualized for clarity.
bonding interaction is the cyano nitrogen with the imidazole
group of His111. The sulfonamide group is surrounded by
three hydrophobic amino acid residues; Phe96, Ile118, and
Phe119. The 4-cyanophenyl moiety undergoes a hydrophobic
interaction with Leu107 and Leu112 and has a sandwich-type
π-stack with Tyr104. The chlorophenyl group is imbedded in
the hydrophobic pocket of Ile169 and Ile232 and has a T-
shaped π-stack with Phe119.
Similar docking results were obtained using a recent SIRT2
crystal structure with a cocrystallized inhibitor bound (pdb
code 4RMH). The highest ranked docked pose shares
interactions with amino acid residues similar to those identified
with apo-SIRT2; however, the docked pose is inserted deeper
within the active site inside the Phe143, Phe190, and Leu206
pocket, and the cyano group is hydrogen bonded to the amidic
nitrogens of Phe243 and Phe235 (Figure S1 in Supporting
Information).
These results add further confirmation to the unfavorable
and insignificant role of the sulfonamide; however, N-
methylation of the sulfonamide nitrogen can partially mask
this effect and enhance ligand binding in a hydrophobic pocket.
In an attempt to overcome the metabolic inactivation and
unfavorable role of the sulfonamide moiety, it was replaced by
sulfone, sulfoxide, and thioether moieties, as shown in Scheme
1.
Commercially available 3-mercaptobenzoic acid and 3-benzyl
bromide derivatives were coupled to yield 3-(benzylthio)-
benzoic acid derivatives 3a−3c, which were subjected to
oxidation with excess H2O2 in acetic acid to generate the
corresponding sulfones (4a−4c) or with one equivalent of
H2O2 to form the sulfoxide intermediates (5a and 5b).
Subsequently, the thioether, sulfoxide, and sulfone derivatives
were coupled with aniline, pyridine, or pyridazine amines using
EDCI and DMAP to form the final compounds (6−22); the
sulfoxide chirality was not resolved.
Figure 2. Surface of SIRT2 (pdb code 3ZGO) active site visualized by
Discovery Studio suite. (A) Active site visualized by hydrophobicity;
hydrophobic and hydrophilic amino acids are colored by brown and
blue colors, respectively. (B) Active site visualized by ionizability;
negatively and positively ionizable are colored in red and blue,
respectively.
basic amino acids, except Arg97, which protrudes outside of the
active site and is less likely to be involved in any ionic
interaction with a bound ligand (Figure 2B). These results
mean that the highly hydrophilic and possibly negatively
charged sulfonamide moiety is not favorable, as it is not
neutralized upon binding by an ionic interaction with a
positively charged residue or incorporated in strong hydrogen
bonding interactions inside the active site of SIRT2 to
compensate for the high desolvation penalty of the sulfonamide
moiety.
To further understand the binding interaction and
orientation of 3-(benzylsulfonamido)benzamide derivatives
within the SIRT2 active site, 2 was docked, in ionized and
unionized states, in the active site of apo-SIRT2 (pdb code
3ZGO) using the LigandFit docking engine implemented in the
Discovery Studio package.10,11 The SIRT2 active site was
defined as described in the literature.12,13 Out of 100 poses
generated by LigandFit, none of those with 2 in the ionized
state was involved in an ionic interaction with Arg97. Moreover,
in some of the docked poses the sulfonamide group formed one
hydrogen bonding interaction, while it can form up to five
hydrogen bonding interactions with water molecules in
solution. Figure 3 shows the highest-ranked binding pose of
2 in the unionized form within the binding site of SIRT2.
Clearly, the only moiety that participates in a hydrogen
Substituent selection was based on our previous work;9 those
that showed high SIRT2 inhibitory activities were substituted at
the para positions (R1 and R2 in Scheme 1) of the two phenyl
rings. Therefore, chloro, methylthio, and methoxyl groups were
added at the R1 position, while cyano, halo, methylsulfinyl, and
methylsulfonyl groups were added at the R2 position. The
potency and selectivity of the synthesized compounds were
evaluated by applying robust, sensitive, and quantitative
biochemical sirtuin deacetylation assays (SIRT1, 2, 3), as
previously described.5,9 Compounds were screened at a single
10 μM dose in triplicate in the primary SIRT2 assay and
counter-screened in SIRT1 and SIRT3 assays. AK-1 was
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a
Scheme 1. Synthetic Routes of Sulfone, Sulfoxide, and Thioether Derivatives
a
Reagents and conditions: (a) (i) KOH, CH3CH2OH, H2O, reflux, 2 h; (ii) 20% aq. NaOH, reflux, 1 h; (b) excess H2O2 (30%), 65 °C, 1 h; (c) Ar-
NH2, EDCI, DMAP, CH2Cl2; (d) H2O2 (30%, 1 equiv), 70 °C, 1 h.
included as a reference compound with each assay. Hits
demonstrating higher or equal potency for SIRT2 inhibition
compared to that of AK-1 were selected for dose−response
studies, which were performed in multiple doses in the primary
SIRT2 assay, including AK-1 for direct comparison.
These compounds also were subjected to SIRT1 and SIRT3
assays in multiple doses to determine sirtuin selectivity. The
dose−response assays identified three analogues (9, 16, 18) as
being more potent SIRT2 inhibitors than compound 1 (Table 1
and Figure S2 in the Supporting Information), and six
analogues (9, 15−19) as being more potent than AK-1.
These inhibitors showed a high degree of selectivity, having
weak SIRT1 and SIRT3 inhibition when tested at a
concentration of 10 μM. The thioether analogues were the
most potent and selective SIRT2 inhibitors, verifying the
nonessential role of the sulfonamide moiety for SIRT2 binding.
Furthermore, although the thioether moiety is known to
undergo metabolic oxidation, it may not be detrimental because
several sulfoxide and sulfone analogues were still active as
SIRT2 inhibitors (e.g., 9, 15, 19). Other sulfoxide and sulfone
analogues were less active (or inactive) than SIRT2 inhibitors.
This might be a result of the high desolvation binding energy of
analogues 10−14 and 20−22 within a highly hydrophobic
active site or the absence of a hydrogen bond acceptor at the R2
position for analogues 6−8 and 14. However, despite the
metabolic oxidation potential, thioether (sulfoxide and sulfone)
analogues are an improvement over the sulfonamides.
The dose−response curves of the tested compounds
exhibited Hill slope values between −1 and −2, excellent
correlation coefficients (Table S1 in Supporting Information),
and low standard errors, which strongly suggest the authenticity
(i.e., nonpromiscuity) of the SIRT2 inhibitors.14
To assess SIRT2 inhibition activity in live cells, we measured
an increase in α-tubulin K40 acetylation, a known substrate for
SIRT2 deacetylase, in neuronal rat ST14A15 and mouse
Neuro2a cell lines. In this bioactivity test, extracts from cells
treated with compounds for 6 h at a range of concentrations
were resolved on SDS-PAGE and subjected to Western blot
analysis using a primary antibody specific to acetylated lysine-
40 of α-tubulin. Active SIRT2 inhibitors (9, 16−18) were
tested, and two of them, 17 and 18, increased α-tubulin
acetylation in a dose-dependent manner in at least one cell line.
Figure 4A,B shows the bioactivity of SIRT2 inhibitors 18 and
17, respectively, after 6 h treatment in ST14A and Neuro2a
Table 1. SIRT2 Activity and Selectivity of Sulfone, Sulfoxide,
and Thioether Derivatives
% of SIRT2
inhibition at
10 μM
% SIRT1
inhibition at
10 μM
% SIRT3
inhibition at
10 μM
cmpd
no.
IC50
(μM)
AK-1
1
12.5
6.1
10
10
5
8
67
0
6
7
0
8
2
9
40
31
36
17
23
0
4.7
0
0
10
11
12
13
14
15
16
17
18
19
20
21
22
50
81
60
83
47
24
18
21
6.7
2.9
8.4
2.4
7.1
3
0
8
0
10
3
8
0
12
11
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Figure 4. SIRT2 inhibitor effects on acetylation of α-tubulin K40. (A) Treatment of ST14A striatal cells and Neuro2a cells with 18 for 6 h resulted in
an increase of α-tubulin acetylation as detected by immunoblotting; GAPDH is shown as a loading control. (B) Treatment of ST14A striatal cells
with 17 for 6 h resulted in a weak increase of α-tubulin acetylation as detected by immunoblotting; GAPDH is shown as a loading control. (C)
Compound dose-dependent increase of α-tubulin acetylation in ST14A striatal cells, normalized to GAPDH and quantified from Western blots in
panels A and B.
cells at concentrations ranging from 0.5 to 50 μM. Compound
18 increased acetylation of α-tubulin in both neuronal cell lines
(Figure 4A) and was found to be the most bioactive in this
assay in a manner corresponding to its highest SIRT2 inhibition
activity in vitro. Compound 17 showed weak activity against
ST14A; however, it was inactive against Neuro2a cells. A
comparison of 17 and 18 in ST14A cells is shown in Figure 4C.
Active SIRT2 inhibitor 18 was tested in a polyQ aggregation
assay using rat neuronal PC12 cells expressing a short fragment
of mutant huntingtin in an inducible fashion.16,17 The effect of
18 on polyQ aggregation was examined visually by fluorescence
microscopy at doses of 25, 10, and 5 μM (Figure 5). Expression
of the mutant huntingtin fragment was induced in PC12 cells
simultaneously to compound treatment with 2.5 μM
concentration of inducer muristerone A for 24 h, which
resulted in the formation of polyQ aggregates, visible in cells as
large fluorescent inclusion bodies (Figure 5A). Inhibition of
aggregation in this cell model can be detected as a reduction in
the number and size of fluorescent polyQ inclusions. Analogue
18 was bioactive in this assay and significantly reduced the
number and size of fluorescent polyQ inclusions (Figure 5B). A
higher concentration (25 μM) of analogue 18 was required to
shift the acetylation level in neuronal cell lines compared to the
aggregation assay (5 μM). α-Tubulin is a very abundant
protein, and a high concentration is required to observe a
biological response.
In conclusion, starting with sulfonamide-derived compounds,
we have been able to alter their structures to enhance potency
and metabolic stability. We rationalized the insignificant role of
the sulfonamide moiety in SIRT2 binding, which can be
replaced by a thioether group. Several sulfone, sulfoxide, and
thioether analogues were synthesized and tested for SIRT2
inhibitory activity and selectivity. The thioether analogues were
the most potent and selective with a three-fold increase in
potency. Two compounds (17 and 18) increased α-tubulin
acetylation in a dose-dependent manner in at least one
neuronal cell line. Additionally, active SIRT2 inhibitors were
tested in a tertiary aggregation assay, and compound 18 was
found to inhibit polyQ aggregation in PC12 cells. The results
from this study are essential for further improvements of
selective SIRT2 inhibitors.
ASSOCIATED CONTENT
■
S
* Supporting Information
Analytical data of all compounds synthesized in this study and
the experimental procedures. This material is available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Authors
Figure 5. Effects of SIRT2 inhibitors on polyQ aggregation in PC12
cells. (A−D) Detection of aggregate inhibition using a microscopic
epifluorescent assay. (A) Aggregation of extended polyQ peptides
containing HD103Q-EGFP in PC12 cells induced with 2.5 μM
muristerone for 24 h, which results in the formation of punctuated
fluorescent inclusions (indicated by arrow). (B) Treatment with
aggregation inhibitor 18 at 5 μM results in the reduction of fluorescent
inclusion number and size. (C−D) Phase contrast cell images of panels
A and B demonstrate equal cell density, i.e., lack of cytotoxicity.
*(R.B.S.) Phone: 1-847-491-5653. Fax: 1-847-491-7713. E-
mail: agman@chem.northwestern.edu.
*(A.G.K.) Phone: 1-617-726-1274. Fax: 617-724-1480. E-mail:
akazantsev@mgh.harvard.edu.
Funding
The authors are grateful to the National Institutes of Health
(Grant U01 NS066912) for financial support of this research.
D
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Rigamonti, D.; Cattaneo, E. ST14A cells have properties of a medium-
size spiny neuron. Exp. Neurol. 2001, 167, 215−226.
Notes
The authors declare no competing financial interest.
(16) Kazantsev, A.; Preisinger, E.; Dranovsky, A.; Goldgaber, D.;
Housman, D. Insoluble detergent-resistant aggregates form between
pathological and nonpathological lengths of polyglutamine in
mammalian cells. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 11404−11409.
(17) Apostol, B. L.; Kazantsev, A.; Raffioni, S.; Illes, K.; Pallos, J.;
Bodai, L.; Slepko, N.; Bear, J. E.; Gertler, F. B.; Hersch, S.; Housman,
D. E.; Marsh, J. L.; Thompson, L. M. A cell-based assay for aggregation
inhibitors as therapeutics of polyglutamine-repeat disease and
validation in Drosophila. Proc. Natl. Acad. Sci. U.S.A. 2003, 100,
5950−5955.
ABBREVIATIONS
■
CNS, central nervous system; HD, Huntington’s disease;
HDAC, histone deacetylase; Parkinson’s disease, PD; PolyQ,
polyglutamine; SIRT2, sirtuin-2
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