Novel Lysine-Based Thioureas as Mechanism-Based Inhibitors of Sirtuin 2 (SIRT2) with Anticancer Activity in a Colorectal Cancer Murine Model

Article abstract of DOI:10.1021/acs.jmedchem.9b00191

Sirtuin 2 (SIRT2) is a protein lysine deacylase that has been indicated as a therapeutic target for cancer. To further establish the role of SIRT2 in cancers, it is necessary to develop selective and potent inhibitors. Here, we report the facile synthesis

Full text of DOI:10.1021/acs.jmedchem.9b00191

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Cite This: J. Med. Chem. XXXX, XXX, XXX−XXX
Novel Lysine-Based Thioureas as Mechanism-Based Inhibitors of
Sirtuin 2 (SIRT2) with Anticancer Activity in a Colorectal Cancer
Murine Model
Ali Sohail Farooqi,† Jun Young Hong,† Ji Cao,†,§ Xuan Lu,† Ian Robert Price,† Qingjie Zhao,†,∥
Tatsiana Kosciuk,† Min Yang,† Jessica Jingyi Bai,† and Hening Lin*,‡
†Department of Chemistry and Chemical Biology and ‡Howard Hughes Medical Institute, Department of Chemistry and Chemical
Biology, Cornell University, Ithaca, New York 14853, United States
S
* Supporting Information
ABSTRACT: Sirtuin 2 (SIRT2) is a protein lysine deacylase that has been indicated as a therapeutic target for cancer. To
further establish the role of SIRT2 in cancers, it is necessary to develop selective and potent inhibitors. Here, we report the facile
synthesis of novel lysine-derived thioureas as mechanism-based SIRT2 inhibitors with anticancer activity. Compounds AF8,
AF10, and AF12 selectively inhibited SIRT2 with IC50 values of 0.06, 0.15, and 0.08 μM, respectively. Compounds AF8 and
AF10 demonstrated broad cytotoxicity amongst cancer cell lines, but minimal toxicity in noncancerous cells. AF8 and AF10
inhibited the anchorage-independent growth of human colorectal cancer cell line HCT116 with GI50 values of ∼7 μM.
Furthermore, AF8 potently inhibited tumor growth in a HCT116 xenograft murine model, supporting that SIRT2 is a viable
therapeutic target for colorectal cancer.
the thiomyristoyl lysine compound TM is a SIRT2
mechanism-based inhibitor that displayed cancer cell-selective
toxicity and is active in many human cancer cell lines.10,16 The
sirtuin deacylation mechanism involves nucleophilic attack on
the NAD+ anomeric position by the carbonyl of the acyl group
to release nicotinamide (Scheme 1).17 TM contains a
thiomyristoyl lysine moiety, which can attack the anomeric
position of NAD+ in the presence of SIRT2, forming a stable
covalent intermediate to potently inhibit SIRT2.10
While TM has promising anticancer activity, it is poorly
soluble in water and the synthesis of the thioamide bond is
difficult and results in poor yield. We therefore sought to
develop similar mechanism-based inhibitors that are less
hydrophobic and easier to synthesize. In a previous study by
Hirsch et al., it was shown that a peptidomimetic thiourea-
containing compound could mechanistically inhibit SIRT1 and
lead to the general inhibition of SIRT1−3.18 Modifying TM
with a thiourea moiety could similarly result in mechanism-
based inhibition and facilitate a simplified synthesis route.
Here, we present the development of novel lysine-based
thioureas as potent and selective SIRT2 inhibitors with
INTRODUCTION
■
Sirtuins are a class of nicotinamide adenine dinucleotide
(NAD+)-dependent enzymes with lysine deacetylase activ-
ities.1,2 They are also referred to as class III histone
deacetylases, but can catalyze the deacylation of various
nonhistone substrates.3 In mammals, there are seven sirtuins
(SIRT1−7) that are involved in the regulation of cellular
metabolism, transcription, and differentiation.4,5 Specifically,
SIRT1, SIRT2, and SIRT5 have been implicated in promoting
cancer cell proliferation and tumorigenesis.6−8 While SIRT1
and SIRT2 have also been contradictorily shown to be tumor
suppressors,7,9 the therapeutic potential of targeting SIRT2 has
been demonstrated by isoform specific inhibition in breast
cancer cell lines.10 Recently, SIRT2 was shown to promote
tumor growth by regulating oncogenes c-Myc and KRas.6,11
SIRT2 also promotes basal-like breast cancer phenotype and
tumor growth through the deacetylation and stabilization of
transcription factor Slug.12 As such, SIRT2 regulates multiple
oncogenic pathways and is a promising therapeutic target for
cancer. However, there is a need for more studies in animal
models of cancer to convincingly demonstrate the therapeutic
potential of SIRT2 inhibition.
Several SIRT2-selective inhibitors, such as AGK2, tenovin-6,
Received: January 30, 2019
TM, and SirReal2, have been reported.10,13−15 Among these,
© XXXX American Chemical Society
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Scheme 1. Inhibition of Sirtuin Lysine Deacylation by Thiourea Compounds
a
Scheme 2. Synthesis of Thiourea Compounds with Varying Chain Lengths
a
Reactions and conditions: (a) (i) dichloromethane (DCM), N-methylmorpholine, isobutyl chloroformate, 1 h; (ii) 3-aminophenol, 16 h. (b) (i)
DCM, N-methylmorpholine, isobutyl chloroformate, 1 h; (ii) aniline, 16 h. (c) (i) DCM/trifluoroacetic acid, 1 h; (ii) tetrahydrofuran,
triethylamine (TEA), 1,1’-thiocarbonyldiimidazole, 16 h. (d) dimethylformamide, TEA, corresponding primary alkylamine, 16 h.
anticancer activity in various cancer cell lines and a colorectal
cancer xenograft murine model.
inhibitors varied in chain length from one to 12 carbons and
the five AF6−AF12 inhibitors varied from six to 12 carbons.
AF8, AF10, and AF12 are Potent and Selective SIRT2
Inhibitors in Vitro. We then measured the ability of these
compounds to inhibit SIRT1, SIRT2, and SIRT3. Because
SIRT1, SIRT2, and SIRT3 are similar structurally and
comprise the class I sirtuins,21 it was important to test for
selectivity among these three isoforms. A liquid chromatog-
raphy (LC)-based assay with acetyl H3K9 peptide2 and
purified sirtuins was used to test the compounds for SIRT1−3
inhibition. The previously reported TM compound was also
assayed and used as a reference compound. Nicotinamide, a
nonspecific sirtuin inhibitor, was used as a positive control for
SIRT3 inhibition and the SIRT1 selective inhibitor EX527
used as a positive control for SIRT1 inhibition.22−24
Nicotinamide weakly and nonspecifically inhibited SIRT3
(IC50 76 30 μM) and SIRT2 (IC50 70 5 μM) while EX527
more potently inhibited SIRT1 (IC50 0.085 0.01 μM) over
RESULTS AND DISCUSSION
■
Design and Synthesis of New SIRT2 Inhibitors. Two
different classes of thiourea inhibitors with varying alkyl chain
lengths were synthesized through a divergent synthesis route
(Scheme 2). The C class of inhibitors C1−12 contained a 3′
hydroxyl group on the C-terminal while the AF class of
inhibitors AF6−12 did not. Modeling the inhibitors into the
known SIRT2 structure suggested the 3′ hydroxyl group of the
C inhibitors could facilitate additional hydrogen bonding
interactions with the backbone of residues Val266 or Gln267
(Figure 1).19 The C inhibitors could therefore have improved
potency and solubility as compared to the AF inhibitors.
SIRT2 contains a hydrophobic pocket for acyl group
binding,15,20 and this pocket accommodates the thiomyristoyl
group present in TM. Toward the goal of reducing the
hydrophobicity of the inhibitors, we decided to synthesize
thiourea compounds with various alkyl chain lengths.
SIRT2 (IC50 1.80
0.03 μM), consistent with reported
literature values.22,25
As shown in Table 1, none of the synthesized inhibitors
demonstrated significant inhibition of SIRT3 with IC50 values
of 50 μM or greater. Except for inhibitors C1 and C3, all
inhibitors had micromolar (C5) or submicromolar IC50 values
for SIRT2. The lack of SIRT2 deacetylase inhibition for
compounds C1 and C3 is likely because of the shorter side
chains of one and three carbons, respectively, which led to
Commercially available Cbz-L-lysine(Boc)-OH was con-
verted to the corresponding isothiocyanate intermediates 2a
and 2b in two steps with 54% and 83% yield, respectively. The
isothiocyanate compounds were then coupled with varying
alkyl chain primary amines to form the resultant thiourea
compounds, with up to 94% yield. In total, the six C1−C12
B
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AF10, AF12, and TM showed no inhibition of SIRT1. The
inhibition of SIRT3 was the weakest and only AF8 showed
slight inhibition of SIRT3. The selectivity for SIRT2 over
SIRT1 correlated with the alkyl chain length of the inhibitors,
as demonstrated by the 47, 137, 180, >1000, and >1000-fold
selectivity of AF6, AF7, AF8, AF10, and AF12, respectively.
This trend suggests that longer alkyl chains can exploit isoform
specific binding for selective SIRT2 inhibition, despite the fact
that SIRT1−3 have similar hydrophobic acyl pockets.26 AF8
inhibition of SIRT2 (IC50 0.061
0.03 μM) was the most
potent amongst the thiourea inhibitors and comparable to TM
(IC50 0.034 0.01 μM).
AF8 was confirmed to work through mechanism-based
inhibition of SIRT2. AF8 was incubated with an excess of
SIRT2 and NAD+ and the reaction mixture analyzed by LC−
mass spectrometry (MS). As shown in Figure 2, the covalent
AF8-ADP-ribose intermediate formed by the release of
nicotinamide was detected. When AF8 was removed from
the reaction mixture, the intermediate mass was not detected.
This result confirmed that AF8, and likely the other thiourea
AF inhibitors, could covalently react with NAD+. This is
consistent with the MS detection of the stalled S-alkylamidate
intermediate of a previously reported, nonspecific lysine-based
thiourea sirtuin inhibitor upon incubation with SIRT1.18
AF8 and AF10 Demonstrated Cytotoxicity Against
Human Colon, Lung, Breast, and Pancreatic Cancer Cell
Lines. The thiourea modification and shorter alkyl chains of
compounds AF10 and AF8 were predicted to improve
solubility as TM had a calculated cLogP value of 8.8, while
the thiourea analogues AF10 and AF8 had calculated cLogP
values of 7.04 and 6.13, respectively (Table 1). Thus, the lower
cLogP values suggest that AF8 and AF10 may have improved
solubility while still maintaining reasonable selectivity and
potency for SIRT2. Because of this, AF8 and AF10 were tested
for possible anticancer activity amongst various cell lines.
As shown in Table 2, toxicity was observed in all cancer lines
upon treatment with AF8, AF10, and TM. None of the
inhibitors were toxic to noncancerous HME1 epithelial cells. In
a normal colon cell line, CCD 841 CoN, the GI50 values of
AF8 and AF10 were also higher in general than those in the
cancer cell lines tested. These observations suggested that,
while there is variation, cancer cells could rely more on SIRT2-
driven pathways for survival and growth. AF10 was the most
potent inhibitor and was approximately two-fold more potent
than TM in MCF7, MDA-MB-231, A549, SW948, and
HCT116 cells (Table 2). AF8 was comparable to TM in
most cell lines except the pancreatic BxPC-3 cell line, which
showed little inhibition by AF8. Both AF8 and AF10 were
twice as potent in the colorectal HCT116 cell line as compared
to TM (Table 2). AF8 demonstrated minimal inhibition of
noncancerous colon cell line CCD 841 CoN, suggesting that
AF8 could be used to selectively target cancerous colon cells.
Consistent with its increased potency, AF10 showed higher
toxicity in CCD 841 CoN cells, which indicated it may have a
lower therapeutic index (Table 2).
Figure 1. C class inhibitors modeled into the peptide-binding site of
SIRT2 (PDB code 4X3O) show additional possible hydrogen bonds
as compared to the AF inhibitors. For both classes of inhibitors,
hydrogen bonds are predicted to form between the lysine carbonyl
and the Gly236 backbone nitrogen, between the N-terminus lysine
backbone nitrogen and the Glu237 backbone carbonyl, and between
the C-terminus backbone nitrogen and the Gln267 backbone
carbonyl. The 3′ hydroxyl of the C class inhibitors (circled in
green) could make additional hydrogen bonds with the Gln267
backbone nitrogen or the Val266 backbone carbonyl or nitrogen
(circled in pink).
decreased interactions in the sirtuin hydrophobic pocket. A
trend of increased chain length and increased SIRT2 inhibition
was observed, suggesting that a longer alkyl chain length
facilitates increased binding in the hydrophobic pocket.
Compounds C8−C12 were potent for SIRT2 with IC50
values of 0.12 μM or lower, but also inhibited SIRT1 with IC50
values of 1.0, 0.45, and 1.0 μM for C8, C10, and C12,
respectively. The decreased selectivity is likely because the
additional hydrogen bonding capability of C8−C12 increases
the SIRT1 inhibition more than the SIRT2 inhibition. The
resultant 8 to 17-fold selectivity for SIRT2 over SIRT1 was not
adequate to ensure SIRT2-specific inhibition for biological
testing. As a result, the nonselective inhibitors C8−C12 were
not used in further biological activity assays.
AF8, AF10, and TM Inhibited the Anchorage-
Independent Growth of HCT116 Cells. The ability of
cancer cells to survive and proliferate without adhesion to an
extracellular matrix is one of the hallmarks of oncogenic
transformation.27,28 To examine anchorage-independent
growth, HCT116 cells were suspended in a soft agar matrix
and incubated until colony growth was observed.29 Slightly less
overall growth was observed in the TM treated group, likely
AF6, AF7, AF8, AF10, and AF12 inhibited SIRT2
deacetylase activity with IC50 values of 0.64, 0.16, 0.061,
0.15, and 0.081 μM, respectively (Table 1). AF8 slightly
inhibited SIRT1 deacetylase activity (IC50 11 5 μM) while
C
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Table 1. In Vitro IC50 Values of Inhibitors for SIRT1-3 and Relative Selectivity for SIRT2 Over SIRT1
a
b
compound
C1
C3
C5
C8
C10
C12
AF6
AF7
R1
chain length
SIRT1 IC50 (μM)
SIRT2 IC50 (μM)
SIRT3 IC50 (μM)
SIRT2/SIRT1 selectivity
cLogP
OH
OH
OH
OH
OH
OH
H
H
H
H
H
n = 0
n = 2
n = 4
n = 7
n = 9
n = 11
n = 5
n = 6
n = 7
n = 9
n = 11
NA
>100
>100
>100
>100
1.0 0.3
>100
>100
>100
>100
>100
>100
68 19
83 11
51 0.4
>200
NA
NA
34
8
12
2.65
3.52
4.42
5.79
6.7
34
2
1.0 0.6
0.45 0.1
1.0 0.6
30 11
0.12 0.001
0.036 0.0001
0.060 0.0005
0.64 0.02
0.16 0.006
0.061 0.030
0.15 0.06
0.081 0.030
0.034 0.010
17
47
7.6
5.22
5.68
6.13
7.04
7.95
8.80
22
11
2
5
137
180
>1000
>1000
>1000
AF8
AF10
AF12
TM
Nicotinamide
EX527
>200
>200
>200
>200
>100
76 30
NA
NA
NA
NA
NA
70
5
0.085 0.01
1.80 0.03
a
In vitro IC50 values were calculated in duplicate using acetylated H3K9 peptide and LC. Statistical analysis was done on GraphPad Prism, and
b
reported values include the SEM. cLogP values computed using DataWarriors software.
Figure 2. Detecting the covalent intermediate formed between AF8 and NAD+ in the presence of SIRT2 by LC−MS. When AF8 was removed
from the reaction mixture, the intermediate mass was not detected as shown through the 1067.5−1068.5 m/z ion trace.
Table 2. GI50 Values of SIRT2 Inhibitors Across a Variety of Cell Lines
cell line
MCF7
MDA-MB-468
MDA-MB-231
BxPC-3
NCI-H23
A549
SW948
type
TM GI50 (μM)
AF8 GI50 (μM)
AF10 GI50 (μM)
breast cancer
breast cancer
breast cancer
pancreatic cancer
lung cancer
37.0 4.5
15.7 2.5
42.8 6.5
13.3 3.5
16.4 1.8
17.3 6.0
19.2 3.5
131.6 54.0
42.6 18.5
26.1 6.4
27.2 8.1
72.4 25.5
35.8 14.2
20.8 6.9
30.0 8.9
58.9 26.5
136.8 53.5
n.i.
18.4 6.0
13.4 1.7
14.8 4.1
24.3 5.0
15.6 3.5
9.0 3.9
12.8 3.5
54.4 12.6
47.4 7.0
n.i.
lung cancer
colorectal cancer
colorectal cancer
colon cells
HCT116
CCD 841 CoN
HME1
a
n.d.
n.i.
b
epithelial cells
a
b
n.d.: not determined. n.i.: no inhibition.
because of a smaller number of cells seeded per well.
Treatment with AF8 and AF10 potently inhibited colony
formation with GI50 values of 7.4 and 7.0 μM, respectively,
while TM had a weaker effect with a GI50 value of 16.7 μM
(Figure 3). The potency of the compounds in the soft agar
colony formation assay shows that they can inhibit anchorage-
independent growth better than normal 2D growth (cytotox-
icity assay described in the previous section), suggesting that
D
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Figure 3. AF8, AF10, and TM inhibited the anchorage-independent growth of HCT116 cells in a soft agar colony formation assay. At a
concentration of 10 μM and greater, AF8 and AF10 completely inhibited the growth of HCT116 cells. The GI50 values for AF8 and AF10 were 7.4
and 7.0 μM, respectively, whereas the GI50 for TM was 16.7 μM.
SIRT2 inhibitors preferentially target the transformed
phenotype of cancer cells. This is also consistent with the
fact that the inhibitors exhibit less toxicity toward non-
cancerous cells in the 2D growth assay shown in Table 2.
AF8 and AF10 Inhibit SIRT2 but Not SIRT1 in Cells.
We next sought to confirm that AF8 and AF10 could
selectively inhibit SIRT2 deacetylase activity in cells.
Compounds C3, C5, and C10 were also tested to determine
if our in vitro sirtuin inhibition assay correlated with SIRT2
inhibition in cells. We monitored α-tubulin acetylation as a
readout for SIRT2 inhibition as α-tubulin is a well-known
SIRT2 deacetylation substrate.30 HCT116 cells were incubated
with dimethylsulfoxide (DMSO) alone (control) or varying
concentrations of inhibitor for 6 h before α-tubulin acetylation
was detected by immunofluorescence. As shown in Figure
4A,C, acetyl α-tubulin levels of HCT116 cells increased upon
treatment with TM at 25 and 50 μM, suggesting the inhibition
of SIRT2 activity. AF8 and AF10 treatment at 25 and 50 μM
also resulted in extensive α-tubulin hyperacetylation as
compared to DMSO. Thus, AF8 and AF10 can also inhibit
SIRT2 activity in cells.
SIRT2 inhibition in cells as measured by α-tubulin
acetylation was consistent with calculated in vitro SIRT2
inhibition results. As shown in Figure 4B,D, treatment with 25
or 50 μM of C3 did not increase the acetylation of α-tubulin,
corroborating the lack of in vitro SIRT2 inhibition (IC50 > 100
μM). C5 slightly increased α-tubulin acetylation at 25 μM and
more robustly increased acetylation at 50 μM, consistent with
moderate in vitro SIRT2 inhibition (IC50 1.0 μM). C10
increased α-tubulin acetylation at both 25 and 50 μM,
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Figure 5. AF8 and AF10 did not noticeably increase p53 acetylation.
HCT116 cells were co-incubated with 400 nM TSA and 25 μM of
TM, AF8, AF10, C3, C5, C10, and EX527 for 6 h and p53 K382
acetylation levels analyzed by western blot. Known SIRT1 inhibitor
EX527 dramatically increased acetyl p53 levels, while compounds C5
and C10 moderately increased acetyl p53 levels. A dose-dependent
increase of acetyl p53 was observed upon treatment with C10, but not
AF8 and AF10.
Figure 4. AF8, AF10, TM, C5, and C10 inhibit SIRT2 activity in
HCT116 cells as shown through an increase in α-tubulin acetylation.
HCT116 cells were incubated with 25 or 50 μM of inhibitor for 6 h at
37 °C and α-tubulin acetylation detected by immunofluorescence.
(A) At 25 μM, TM, AF8, and AF10 increased α-tubulin acetylation in
HCT116 cells. (B) At 25 μM, C3 did not increase α-tubulin
acetylation, C5 slightly increased α-tubulin acetylation, and C10 more
strongly increased α-tubulin acetylation. (C) At 50 μM, TM, AF8,
and AF10 also increased α-tubulin acetylation. (D) At 50 μM, C3 did
not increase α-tubulin acetylation, but C5 and C10 increased α-
tubulin acetylation.
in a dose-dependent manner, indicating a lack of SIRT1
inhibition. A very slight increase in acetyl p53 was observed
with AF10, but there was no correlation between AF10
concentration and acetyl p53 level, indicating a lack of
meaningful SIRT1 inhibition (Figure 5). This result suggests
that the in vitro selectivity of AF8 and AF10 (203 and >1000-
fold selectivity, respectively), is sufficient to ensure SIRT2
specific inhibition in cells.
consistent with more potent in vitro SIRT2 inhibition (IC50
0.036 μM) (Figure 4B,D). These observations helped to
further validate the results from the in vitro sirtuin inhibition
assay.
AF8 and AF10 Significantly Reduced Tumor Growth
in a HCT116 Xenograft Mice Model of Colorectal
Cancer. Immunodeficient NOD scid gamma mice (NSG
mice) were grafted with HCT116 cells to form tumors and
then treated with varying doses of AF8, AF10, or vehicle by
intraperitoneal injection over the course of 11 days before the
mice were sacrificed and the tumors weighed. As shown in
Figure 6A, AF8 at 25 mg/kg slightly decreased tumor growth
by volume and weight but did not result in a statistically
significant difference. AF8 at 100 mg/kg significantly (p <
0.0001) inhibited tumor growth by 80% as measured by the
average tumor volume on the last day of treatment. This was
consistent with the significant (p = 0.0001) difference in
average tumor weight that demonstrated an inhibition of 78%.
However, AF10 was less potent in this xenograft model. At 100
mg/kg, AF10-inhibited tumor growth significantly (p =
0.0114), but only by 37% as measured by the final tumor
volume (Figure 6B). This was similar to the significant (p =
0.0152) tumor inhibition of 49% as measured by final tumor
weight. No significant weight loss was observed in either the
AF8 or AF10 treatment groups.
We next tested whether AF8 and AF10 inhibit SIRT1
deacetylase activity in HCT116 cells by monitoring the
acetylation of p53, a well-known SIRT1 deacetylation
substrate.31 We also tested compounds C3, C5, and C10 to
determine if our in vitro SIRT1 inhibition results were
consistent with SIRT1 inhibition in cells. HCT116 cells were
treated with Trichostatin A, an inhibitor of the zinc-dependent
HDACs, and with or without 25 μM of our sirtuin inhibitors.
The SIRT1-selective inhibitor EX527 increased p53 acetyla-
tion, consistent with previous reports (Figure 5).25,32 TM,
AF8, and AF10 did not increase the levels of acetyl p53,
indicating that they inhibit SIRT2 but not SIRT1 in cells.
Compound C3 also did not increase the acetylation of p53,
consistent with the lack of in vitro SIRT1 inhibition (IC50
>
100 μM). Inhibitors C5 and C10 moderately increased the
acetylation of p53, consistent with the lack of significant SIRT2
selectivity for the compounds (34 and 12-fold respective
SIRT2/SIRT1 selectivity). To more closely examine the
inhibition of SIRT1 in cells, HCT116 cells were cotreated
with trichostatin A and 10−50 μM of AF8, AF10, and C10.
Compound C10 demonstrated a dose-dependent increase in
acetyl p53 levels, consistent with in vitro SIRT1 inhibition
(IC50 0.45 μM) and lack of significant SIRT2/SIRT1
selectivity (Figure 5). AF8 did not increase acetyl p53 levels
HCT116 xenograft tumor data suggest that thiourea SIRT2
inhibitors present a possible new therapeutic approach for
treating colorectal cancers. Targeted therapies for colorectal
cancer are generally more limited to antibodies targeting
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Figure 6. AF8 and AF10 significantly inhibit tumor growth by volume and weight in a HCT116 xenograft murine model of colorectal cancer. (A)
Tumor volume, tumor weight, and mouse body weight for mice treated with vehicle (n = 4), AF8 25 mg/kg (n = 3), AF8 100 mg/kg (n = 4). (B)
Tumor volume, tumor weight, and mouse body weight for mice treated with vehicle (n = 6) or AF10 100 mg/kg (n = 8). Statistical results were
analyzed with an unpaired, two-tailed t-test. * indicates P value < 0.05 and ** indicates P value < 0.01.
angiogenesis through VEGF/VEGFR and growth through
EGFR.33,34 Such therapies have met moderate success because
of the high-mutation load found in colorectal cancer, and are
frequently less effective against colorectal cancers with
constitutively active KRas mutations.35−37 The potent
inhibition of HCT116 (KRASG13D) xenograft mice tumor
growth by AF8 suggests that targeting SIRT2 activity can be a
viable therapeutic strategy in colorectal cancers containing a
KRAS mutation. Given the broad toxicity of AF8 and AF10 in
other cancer cell lines, it is also possible that SIRT2 inhibitors
can be useful for treating other type of cancers.
anticancer effect of SIRT2 inhibition in a mouse xenograft
model of tumors. The results further support that SIRT2
inhibition is a promising cancer treatment strategy and that
developing new and improved SIRT2 inhibitors is warranted, a
task that will be facilitated by the facile synthesis procedure of
the thiourea-type of SIRT2 inhibitors.
EXPERIMENTAL SECTION
■
General. All reagents were purchased from commercial sources
and used without further purification. Reactions were checked for
completion by LC−MS and compounds purified through flash
chromatography (SiliaFlash Silica Gel, P60). The Cornell University
NMR Facility was used to obtain NMR spectra. 1H spectra (500
MHz, chloroform-d) and 13C NMR spectra (126 MHz, chloroform-d)
were obtained on a Bruker 500 MHz with cryoprobe. High-resolution
MS was obtained using either an ESI-Orbitrap mass spectrometer or
DART-Orbitrap mass spectrometer. The purity of synthesized
compounds was confirmed to be ≥95% purity by qNMR.
CONCLUSIONS
■
We have developed thiourea mechanism-based inhibitors for
SIRT2. Among them, AF8−AF12 are highly selective and
potent at inhibiting SIRT2 in vitro. Compared with the
previous thioamide-type inhibitor TM, these thiourea com-
pounds are much easier to synthesize, which allowed us to
quickly prepare many compounds. In addition, AF8 is less
hydrophobic than TM and thus is more desirable as a
therapeutic candidate.
LC−MS System. Analysis of reaction mixtures and purified
compounds was carried out using a Shimadzu HPLC LC20-AD
system connected to a Thermo Scientific LCQ Fleet Ion Trap mass
spectrometer, Rugged Ion Max Source (electrospray ionization), and
reverse phase Phenomenex Kinetex EVO C18 column (30 × 2.1 mm,
5 μM). Samples were diluted in water/acetonitrile (1:1) and analyzed
using water with 0.1% acetic acid (solvent A) and acetonitrile with
0.1% acetic acid (solvent B) at a constant flow rate of 0.2 mL/min,
recording UV absorbance at 215 and 260 nm and positive mode
detection of ions.
General Procedure for the Synthesis of 1a−b as Shown through
the Preparation of Benzyl tert-Butyl (6-((3-Hydroxyphenyl)amino)-
6-oxohexane-1,5-diyl)(S)-dicarbamate (1a). Cbz-L-Lys(Boc)-OH (3
g, 7.89 mmol, 1 equiv) was dissolved in dichloromethane (48 mL).
To this solution, N-methylmorpholine (1.39 mL, 12.6 mmol, 1.6
equiv) and isobutyl chloroformate (1.43 mL, 11.0 mmol, 1.4 equiv)
were sequentially added and the reaction mixture stirred for 1 h at
room temperature (RT). Then, 3-aminophenol (1.022 g, 9.45 mmol,
1.2 equiv) (or aniline for 1b) was added and the reaction mixture
stirred for 16 h at RT. The reaction was checked for completion by
AF8 and AF10 showed cytotoxicity across a variety of
breast, lung, pancreatic, and colorectal cancer cell lines, but
showed generally less toxicity in normal cells which suggested
that cancer cells could have an increased dependence on
SIRT2 activity. AF8 and AF10 also inhibited SIRT2, but not
SIRT1 in cells. AF8 at 100 mg/kg inhibited tumor growth by
80% in a HCT116 xenograft mouse model. Although in cell
culture AF10 is more potent than AF8, in the mouse xenograft
model, AF8 is better than AF10. This is likely because other
properties, such as solubility (AF8-predicted cLogS is −6.5,
AF10-predicted cLogS is −7.0), are more important for in vivo
efficacy, which should be useful for the future development of
new SIRT2 inhibitors as anticancer therapeutic candidates.
This represents one of the few studies examining the
G
DOI: 10.1021/acs.jmedchem.9b00191
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Journal of Medicinal Chemistry
Article
thin-layer chromatography (hexanes/ethyl acetate 1:1) and LC−MS.
The reaction mixture was further diluted in dichloromethane (75 mL)
and sequentially washed with water (3 × 75 mL) and saturated brine
(1 × 75 mL). The organic layer was collected, dried over Na2SO4, and
concentrated by rotary evaporation to yield a viscous, yellow oil. The
crude product was purified by silica gel column chromatography
(hexanes/ethyl acetate 1:1) to yield 1a as a pale yellow solid in 80%
yield (2.98 g). 1H NMR (500 MHz, chloroform-d): δ 8.74 (d, J = 31.9
Hz, 1H), 7.45 (s, 1H), 7.41−7.30 (m, 5H), 7.20−7.08 (m, 1H), 6.87
(s, 1H), 6.64 (dd, J = 8.1, 2.3 Hz, 1H), 5.93 (d, J = 23.4 Hz, 1H),
5.18−5.02 (m, 2H), 4.75 (s, 1H), 4.37 (d, J = 8.4 Hz, 1H), 3.08 (d, J
= 7.0 Hz, 2H), 1.90 (d, J = 13.6 Hz, 1H), 1.82−1.64 (m, 1H), 1.56−
1.32 (m, 13H). 13C NMR (126 MHz, chloroform-d): δ 170.9, 157.0,
156.9, 156.6, 138.5, 135.9, 129.9, 128.6, 128.3, 128.2, 112.0, 111.6,
107.4, 79.7, 67.4, 55.9, 39.7, 31.6, 29.5, 28.4, 22.5. HRMS [DART-
Orbitrap] m/z: calcd for C24H34N3O4 ([M − CO2 + H]+), 428.2544;
observed, 428.2567.
0.98−0.72 (m, 3H). 13C NMR (126 MHz, chloroform-d): δ 181.3,
170.2, 156.7, 137.5, 136.0, 129.0, 128.6, 128.3, 128.0, 124.7, 120.1,
67.3, 55.3, 43.8, 31.8, 31.4, 29.7, 28.9, 28.2, 26.6, 22.5 (d, J = 3.3 Hz),
14.0. HRMS [DART-Orbitrap] m/z: calcd for C27H39N4O3S ([M +
H]+), 499.2737; observed, 499.2766.
Benzyl (S)-(6-(3-Heptylthioureido)-1-oxo-1-(phenylamino)-
hexan-2-yl)carbamate (AF7). White solid, 85% yield. 1H NMR
(500 MHz, chloroform-d): δ 8.51 (s, 1H), 7.54 (d, J = 8.0 Hz, 2H),
7.41−7.27 (m, 7H), 7.17−7.08 (m, 1H), 6.08 (s, 2H), 5.71 (d, J = 8.0
Hz, 1H), 5.13 (d, J = 5.2 Hz, 2H), 4.39 (d, J = 8.0 Hz, 1H), 3.43 (d, J
= 71.3 Hz, 3H), 1.96 (dt, J = 14.1, 7.5 Hz, 1H), 1.76 (dtd, J = 13.9,
8.6, 5.8 Hz, 1H), 1.72−1.60 (m, 2H), 1.60−1.37 (m, 4H), 1.37−1.12
(m, 9H), 0.94−0.82 (m, 3H). 13C NMR (126 MHz, chloroform-d): δ
181.1, 170.2, 156.7, 137.5, 136.0, 129.0, 128.6, 128.3, 128.0, 124.6,
120.1, 67.3, 55.2, 43.8, 31.8, 31.7, 28.9, 28.3−28.0 (m), 26.9, 22.6,
22.5, 14.1 (d, J = 3.0 Hz). HRMS [DART-Orbitrap] m/z: calcd for
C28H41N4O3S ([M + H]+), 513.2894; observed, 513.2930.
General Procedure for C1−C12 and AF6−AF12 Synthesis as
Shown through the Preparation of Benzyl (S)-(6-(3-octylthiour-
eido)-1-oxo-1-(phenylamino)hexan-2-yl)carbamate (AF8). Com-
pound 2b (250 mg, 0.63 mmol, 1 equiv) (or 2a for C1−C12) was
dissolved in dimethylformamide (4.2 mL). To this mixture,
octylamine (0.26 mL, 1.57 mmol, 2.5 equiv) and triethylamine
(0.17 mL, 1.25 mmol, 2 equiv) were sequentially added and the
reaction was stirred for 16 h at RT. The reaction mixture was diluted
in dichloromethane (50 mL) and washed with water (3 × 25 mL) and
saturated brine (1 × 25 mL) before being dried over Na2SO4 and
concentrated by rotary evaporation. The crude mixture was purified
by flash chromatography (hexanes: ethyl acetate 2:1) to afford AF8 as
a white, crystalline solid in 74% yield (247 mg). 1H NMR (500 MHz,
chloroform-d): δ 8.51 (s, 1H), 7.54 (d, J = 8.0 Hz, 2H), 7.43−7.24
(m, 7H), 7.17−7.08 (m, 1H), 6.08 (s, 2H), 5.72 (d, J = 8.1 Hz, 1H),
5.22−5.02 (m, 2H), 4.39 (d, J = 8.5 Hz, 1H), 3.35 (s, 2H), 2.03−1.85
(m, J = 6.8 Hz, 1H), 1.76 (dtd, J = 13.9, 8.6, 5.7 Hz, 1H), 1.66 (h, J =
6.5 Hz, 2H), 1.51 (dq, J = 36.6, 7.0 Hz, 4H), 1.37−1.12 (m, 11H),
0.90 (t, J = 7.0 Hz, 3H). 13C NMR (126 MHz, chloroform-d): δ
181.0, 170.2, 156.7, 137.5, 136.0, 129.0, 128.6, 128.3, 128.0, 124.6,
120.1, 67.3, 55.2, 43.8, 31.8, 29.24, 29.17, 29.0, 28.2, 26.9, 22.6, 22.5,
14.1. HRMS [DART-Orbitrap] m/z: calcd for C29H43N4O3S ([M +
H]+), 527.3050; observed, 527.3078.
Benzyl (S)-(6-(3-Decylthioureido)-1-oxo-1-(phenylamino)hexan-
2-yl)carbamate (AF10). White solid, 70% yield. 1H NMR (500 MHz,
chloroform-d): δ 8.51 (s, 1H), 7.54 (d, J = 7.9 Hz, 2H), 7.42−7.24
(m, 7H), 7.18−7.08 (m, 1H), 6.08 (s, 2H), 5.72 (d, J = 8.1 Hz, 1H),
5.22−5.04 (m, 2H), 4.39 (d, J = 7.9 Hz, 1H), 3.42 (d, J = 76.0 Hz,
4H), 1.95 (dt, J = 14.2, 7.5 Hz, 1H), 1.84−1.71 (m, 1H), 1.55 (dddq,
J = 44.3, 37.4, 14.7, 7.4, 6.9 Hz, 5H), 1.36−1.14 (m, 16H), 0.90 (t, J =
6.9 Hz, 3H). 13C NMR (126 MHz, chloroform-d): δ 181.0, 170.2,
156.7, 137.5, 136.0, 129.0, 128.6, 128.3, 128.0, 124.6, 120.1, 67.3,
55.2, 44.3, 43.8, 31.9, 31.8, 29.5 (d, J = 2.4 Hz), 29.3 (d, J = 1.9 Hz),
29.0, 28.2, 26.9, 22.7, 22.5, 14.1. HRMS [DART-Orbitrap] m/z: calcd
for C31H47N4O3S ([M + H]+), 555.3363; observed, 555.3394.
Benzyl (S)-(6-(3-Dodecylthioureido)-1-oxo-1-(phenylamino)-
hexan-2-yl)carbamate (AF12). White solid, 57% yield. 1H NMR
(500 MHz, chloroform-d): δ 8.48 (s, 1H), 7.54 (d, J = 8.0 Hz, 2H),
7.43−7.24 (m, 7H), 7.17−7.08 (m, 1H), 6.07 (s, 2H), 5.69 (d, J = 8.1
Hz, 1H), 5.14 (d, J = 4.5 Hz, 2H), 4.38 (d, J = 7.9 Hz, 1H), 3.72−
3.21 (m, 2H), 2.04−1.88 (m, 1H), 1.85−1.72 (m, 1H), 1.67 (h, J =
6.7 Hz, 1H), 1.60−1.39 (m, 4H), 1.37−1.13 (m, 20H), 0.94−0.86
(m, 3H). 13C NMR (126 MHz, chloroform-d): δ 181.1, 170.1, 156.7,
137.5, 136.0, 129.0, 128.6, 128.3, 128.0, 124.6, 120.1, 67.4, 55.2, 43.8,
31.9, 31.7, 29.7, 29.64, 29.59, 29.53, 29.4, 29.3, 29.0, 28.2, 28.0, 26.9,
22.7, 22.4, 19.1, 14.1. HRMS [DART-Orbitrap] m/z: calcd for
C33H51N4O3S ([M + H]+), 583.3676; observed, 583.3704.
Benzyl tert-Butyl (6-oxo-6-(Phenylamino)hexane-1,5-diyl)(S)-di-
1
carbamate (1b). White, powdery solid. 94% yield. H NMR (500
MHz, chloroform-d): δ 8.46 (d, J = 47.9 Hz, 1H), 7.53 (d, J = 7.9 Hz,
2H), 7.44−7.29 (m, 7H), 7.17−7.08 (m, 1H), 5.71 (d, J = 28.3 Hz,
1H), 5.13 (d, J = 3.2 Hz, 2H), 4.69 (s, 1H), 4.33 (s, 1H), 3.11 (s,
2H), 2.07−1.88 (m, 1H), 1.75 (dtd, J = 14.1, 8.6, 5.4 Hz, 1H), 1.44
(s, 14H). 13C NMR (126 MHz, chloroform-d): δ 170.2, 156.7, 156.4,
137.7, 136.1, 128.9 (d, J = 1.2 Hz), 128.6, 128.3 (d, J = 1.4 Hz), 128.1
(d, J = 2.7 Hz), 124.4, 120.0 (d, J = 2.4 Hz), 79.4, 67.3, 55.6, 39.5,
31.6, 29.5, 28.4, 22.5, 19.1. HRMS [DART-Orbitrap] m/z: calcd for
C24H34N3O3 ([M − CO2 + H]+), 412.2595; observed, 412.2618.
General Procedure for 2a−b Synthesis as Shown through the
Preparation of Benzyl (S)-(1-((3-Hydroxyphenyl)amino)-6-isothio-
cyanato-1-oxohexan-2-yl)carbamate (2a). Compound 1a (1 g, 2.12
mmol, 1 equiv) was dissolved in dichloromethane (14 mL) and
trifluoroacetic acid (7 mL). Reaction was stirred for 1 h at RT before
being concentrated by rotary evaporation to remove the trifluoro-
acetic acid. The crude mixture was dissolved in tetrahydrofuran (21
mL) before 1,1′-thiocarbonyl diimidazole (454 mg, 2.54 mmol, 1.2
equiv) and triethylamine (TEA, 0.73 mL, 5.3 mmol, 2.5 equiv) were
sequentially added. The reaction was stirred for 16 h at RT and then
further diluted in ethyl acetate (100 mL) and washed with water (3 ×
60 mL) and saturated brine (1 × 60 mL). The organic layer was
collected, dried over Na2SO4, and concentrated by rotary evaporation.
The crude product was purified by flash chromatography (hexanes/
ethyl acetate 2:1) to afford 2a as a white solid in 68% yield (594 mg).
1H NMR (500 MHz, chloroform-d): δ 8.55 (s, 1H), 7.46 (s, 1H),
7.35 (d, J = 3.0 Hz, 5H), 7.14 (t, J = 8.1 Hz, 1H), 6.79 (d, J = 7.9 Hz,
1H), 6.64 (dd, J = 8.2, 2.3 Hz, 1H), 5.65 (d, J = 7.2 Hz, 1H), 5.15 (q,
J = 12.2 Hz, 2H), 4.37 (d, J = 8.2 Hz, 1H), 3.56−3.35 (m, J = 6.9, 6.4
Hz, 2H), 2.01−1.84 (m, 1H), 1.70 (tdd, J = 22.8, 11.4, 6.1 Hz, 2H),
1.61−1.35 (m, 2H). 13C NMR (126 MHz, chloroform-d): δ 170.5,
156.8, 138.2, 135.7, 130.2, 130.0, 128.6, 128.4, 128.1, 112.2, 111.6,
107.4, 67.7, 55.7, 44.7, 31.4, 29.4, 22.8. HRMS [DART-Orbitrap] m/
z: calcd for C21H24N3O4S ([M + H]+), 414.1482; observed, 414.1507.
Benzyl (S)-(6-Isothiocyanato-1-oxo-1-(phenylamino)hexan-2-
1
yl)carbamate (2b). White solid, 86% yield. H NMR (500 MHz,
chloroform-d): δ 8.37 (s, 1H), 7.49 (d, J = 7.9 Hz, 2H), 7.42−7.25
(m, 7H), 7.13 (t, J = 7.4 Hz, 1H), 5.60 (d, J = 8.8 Hz, 1H), 5.14 (q, J
= 12.2 Hz, 2H), 4.38 (q, J = 7.9, 7.3 Hz, 1H), 3.62−3.40 (m, J = 7.0,
6.2 Hz, 2H), 1.98 (ddt, J = 13.7, 10.1, 6.1 Hz, 1H), 1.87−1.63 (m,
4H), 1.55 (tdd, J = 16.2, 11.6, 7.0 Hz, 2H). 13C NMR (126 MHz,
chloroform-d): δ 169.8, 156.7, 137.4, 135.9, 130.4, 129.0, 128.6,
128.4, 128.1, 124.7, 120.1, 67.5, 55.4, 44.8, 31.5, 29.5, 22.7. HRMS
[DART-Orbitrap] m/z: calcd for C21H24N3O3S ([M + H]+),
398.1533; observed, 398.1556.
Benzyl (S)-(6-(3-Hexylthioureido)-1-oxo-1-(phenylamino)hexan-
1
2-yl)carbamate (AF6). White solid, 80% yield. H NMR (500 MHz,
Benzyl (S)-(1-((3-Hydroxyphenyl)amino)-6-(3-methylthioureido)-
1
chloroform-d): δ 8.56 (s, 1H), 7.54 (d, J = 7.9 Hz, 2H), 7.41−7.23
(m, 7H), 7.15−7.08 (m, 1H), 6.09 (s, 2H), 5.76 (d, J = 7.8 Hz, 1H),
5.20−5.03 (m, 2H), 4.39 (t, J = 7.5 Hz, 1H), 3.42 (d, J = 68.8 Hz,
4H), 1.94 (dt, J = 14.5, 7.6 Hz, 1H), 1.83−1.71 (m, 1H), 1.64 (d, J =
7.6 Hz, 2H), 1.50 (dq, J = 38.3, 6.9 Hz, 4H), 1.37−1.14 (m, 7H),
1-oxohexan-2-yl)carbamate (C1). White solid, 48% yield. H NMR
(500 MHz, DMSO-d6): δ 9.87 (s, 1H), 9.37 (s, 1H), 7.53 (d, J = 7.9
Hz, 1H), 7.47−7.28 (m, 5H), 7.23−7.14 (m, 1H), 7.07 (t, J = 8.0 Hz,
1H), 6.99−6.93 (m, 1H), 6.45 (dd, J = 8.0, 2.3 Hz, 1H), 5.04 (s, 2H),
4.11 (td, J = 8.5, 5.2 Hz, 1H), 3.33 (s, 7H), 2.79 (s, 3H), 1.73−1.55
H
DOI: 10.1021/acs.jmedchem.9b00191
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(m, 2H), 1.55−1.21 (m, 3H). 13C NMR (126 MHz, DMSO-d6): δ
171.4, 158.0, 156.5, 140.4, 137.5, 129.8, 129.4, 128.8, 128.7, 128.3,
128.2, 110.8, 110.4, 106.8, 65.9, 55.9, 32.1, 29.0, 23.6. HRMS [ESI-
Orbitrap] m/z: calcd for C22H29N4O4S ([M + H]+), 445.1904;
observed, 445.1908.
Sirtuin Purification. Human SIRT1-3 were all expressed and
purified as previously reported.10
Enzyme IC50 Assay. Inhibitors were stored at −20 °C as stock
solutions in DMSO (20−50 mM) and further serial dilutions were
prepared on the day of the assay. Inhibitors were tested in duplicate at
final concentrations ranging from 0.002 to 417 μM with DMSO as
control. At 0 °C, sirtuins were diluted in the reaction buffer (20 mM
Tris, 1 mM NAD+, 1 mM dithiothreitol, pH 8.0) to the final
concentration (0.05 μM SIRT1, 0.2 μM SIRT2, 0.05 μM SIRT3).
Inhibitors were added to the appropriate SIRT1−3 reaction mixture
and preincubated at 37 °C for 15 min. Acetyl H3K9 peptide (10 μM
final concentration) was added to start the reaction, and the reaction
mixture further incubated at 37 °C until 10−18% of the peptide was
deacetylated (3 min for SIRT1, 6 min for SIRT2, and 15 min for
SIRT3). The reaction was quenched by the addition of aqueous acid
(0.2 M HCl, 6 M acetic acid) at an equal volume, followed by
vigorous vortexing. Samples were then centrifuged at 17 000g for 2
min at RT to pellet the enzyme. The supernatant was then analyzed
by analytical HPLC on a Shimadzu LC, Phenomenex Kinetex EVO
C18 column (100 × 4.60 mm, 5 μM, 100 Å), UV absorbance
measurement at 215 and 326 nm, 0.1% trifluoroacetic acid in water
(solvent A), 0.1% trifluoroacetic acid in acetonitrile (solvent B), and
0.5 mL/min flow rate. The peak areas of deacetylated to acetylated
H3K9 peptide were used to quantify deacetylase activity. Deacetylase
activity was then normalized to the DMSO control, and log(inhibitor
Benzyl (S)-(1-((3-Hydroxyphenyl)amino)-1-oxo-6-(3-
propylthioureido)hexan-2-yl)carbamate (C3). White solid, 41%
1
yield. H NMR (500 MHz, chloroform-d): δ 8.82 (s, 1H), 7.47 (s,
2H), 7.40 (t, J = 2.2 Hz, 1H), 7.33 (s, 4H), 7.13 (t, J = 8.1 Hz, 1H),
6.93 (d, J = 8.0 Hz, 1H), 6.65 (dd, J = 8.1, 2.3 Hz, 1H), 6.12 (d, J =
50.5 Hz, 1H), 5.89 (d, J = 8.0 Hz, 1H), 5.22−5.00 (m, 2H), 4.42 (d, J
= 7.7 Hz, 1H), 3.36 (d, J = 19.1 Hz, 3H), 1.86 (s, 1H), 1.70 (s, 5H),
1.56 (h, J = 7.3 Hz, 4H), 1.47−1.22 (m, 1H), 0.92 (t, J = 7.4 Hz, 3H).
13C NMR (126 MHz, chloroform-d): δ 181.2, 171.0, 156.9, 138.3,
135.8, 130.0, 128.6, 128.4, 128.1, 112.3, 112.0, 107.6, 67.5, 55.6, 43.6,
31.9, 29.7, 28.3, 22.6, 22.3, 11.4. HRMS [ESI-Orbitrap] m/z: calcd for
C24H33N4O4S ([M + H]+), 473.2217; observed, 473.2222.
Benzyl (S)-(1-((3-Hydroxyphenyl)amino)-1-oxo-6-(3-
pentylthioureido)hexan-2-yl)carbamate (C5). White solid, 94%
yield. 1H NMR (500 MHz, DMSO-d6): δ 9.87 (s, 1H), 9.37 (s,
1H), 7.53 (d, J = 7.9 Hz, 1H), 7.37 (d, J = 5.4 Hz, 4H), 7.35−7.25
(m, 2H), 7.19 (q, J = 2.8, 2.2 Hz, 1H), 7.07 (t, J = 8.1 Hz, 1H), 6.99−
6.93 (m, 1H), 6.45 (dd, J = 8.1, 2.3 Hz, 1H), 5.04 (s, 2H), 4.11 (td, J
= 8.6, 5.3 Hz, 1H), 3.33 (s, 10H), 1.63 (ddd, J = 18.2, 9.4, 4.4 Hz,
1H), 1.45 (p, J = 7.2 Hz, 3H), 1.41−1.16 (m, 5H), 0.87 (t, J = 7.1 Hz,
3H). 13C NMR (126 MHz, DMSO-d6): δ 171.4, 158.0, 156.5, 140.4,
137.5, 129.8, 128.8, 128.3, 128.2, 110.8, 110.4, 106.8, 65.9, 55.9, 32.1,
29.1, 28.9, 23.6, 22.4, 14.4. HRMS [ESI-Orbitrap] m/z: calcd for
C26H37N4O4S ([M + H]+), 501.2530; observed, 501.2532.
concentration) versus normalized deacetylase activity fitted by
)
nonlinear regression to Y = 100/(1 + 10(X − log IC ) on GraphPad
50
Prism version 6.01.
Cell Cytotoxicity Assay. To a flat bottom 96-well plate, 1000−
3000 cells were seeded into each well and incubated for 20 h at 37 °C
(1000 cells for HCT-116, 2000 cells for MDA-MB-231, MDA-MB-
468, NCI-H23, A549, SW948 and 3000 cells for MCF7, BxPC-3,
CCD 841 CoN, and HME1). Inhibitors in varying final concen-
trations (1−100 μM) were added to each well and the cells incubated
for 72 h at 37 °C. Then, 100 μL of media was removed from each well
and 20 μL of CellTiter-Blue (Promega) was added to each well. The
96-well plate was then incubated for 4 h at 37 °C to measure cell
viability. The fluorescence of each well (560 nm excitation/590 nm
emission) was measured using a Fluoroskan Ascent FL microplate
fluorometer. The fluorescence was normalized to DMSO-treated cells,
and GraphPad Prism software used to plot the cell viability curves and
calculate the GI50 value for each cell line.
Benzyl (S)-(1-((3-Hydroxyphenyl)amino)-6-(3-octylthioureido)-1-
1
oxohexan-2-yl)carbamate (C8). White solid, 79% yield. H NMR
(500 MHz, chloroform-d): δ 9.07 (s, 1H), 8.18 (s, 1H), 7.39 (s, 1H),
7.29 (p, J = 6.8, 5.7 Hz, 5H), 7.05 (t, J = 8.0 Hz, 1H), 6.89 (d, J = 8.0
Hz, 1H), 6.71−6.55 (m, 1H), 6.55−6.19 (m, 2H), 6.14 (d, J = 7.9 Hz,
1H), 5.21−4.87 (m, 2H), 4.39 (q, J = 7.3 Hz, 1H), 3.71−2.98 (m,
4H), 2.58 (s, 1H), 1.78 (d, J = 12.0 Hz, 1H), 1.71−1.57 (m, 1H),
1.48 (dt, J = 19.7, 8.5 Hz, 3H), 1.39−1.04 (m, 14H), 0.88 (t, J = 7.0
Hz, 3H). 13C NMR (126 MHz, chloroform-d): δ 180.9, 171.4, 156.9,
138.3, 135.8, 129.9, 128.6, 128.3, 128.0, 112.3, 112.0, 107.8, 67.4,
55.7, 44.2 (d, J = 89.0 Hz), 32.0, 31.8, 29.7, 29.3, 29.2, 29.1, 28.3,
26.9, 22.7, 22.6, 14.1. HRMS [ESI-Orbitrap] m/z: calcd for
C29H43N4O4S ([M + H]+), 543.3000; observed, 543.3004.
Soft Agar Assay. HCT116 cells in a 10 cm dish (70−80%
confluent) were rinsed with phosphate-buffered saline (PBS),
trypsinized for 1 min, resuspended in media (McCoy’s medium
+10% calf serum), and counted by hemocytometry. To make the solid
base layer, a stock 3% agar solution in water was heated to 42 °C,
diluted to 0.6% in media, and mixed with inhibitors to final
concentrations of 2.5−50 μM. To each well in a six-well plate, 2
mL of the base layer was added and allowed to solidify for 1 h at room
temperature. To make the growth layer, the stock 3% agar solution
was diluted to 0.3% in media and inhibitors were added to final
concentrations of 2.5−50 μM. Then, HCT116 cells were added to the
growth layer to a concentration of 500 cells/mL. In triplicate, 2 mL of
the growth layer-containing inhibitor and HCT116 cells was added to
each well and incubated at 37 °C. After five days, another 2 mL of
growth layer-containing inhibitor was added to each well. After 9−11
days, 200 μL of nitro blue tetrazolium chloride in water (2 mg/mL)
was added to each well and incubated overnight at 37 °C. The plates
were imaged using a ChemiDoc MP Imaging System and colonies
were counted with ImageJ software.
Xenograft Mice Study. All animals used in this study were
handled in accordance with federal and institutional guidelines, under
a protocol approved by the Cornell University Institutional Animal
Care and Use Committee. Immunodeficient female and male NSG
mice were purchased from The Jackson Laboratory. When mice were
approximately four to eight weeks old, 1 × 106 HCT116 cells were
injected on both sides of the abdomen. The cells were allowed to
grow for 3−5 days until the tumors were just large enough to be
visible and measured. Inhibitors were dissolved in solution (80% PBS,
Benzyl (S)-(6-(3-Decylthioureido)-1-((3-Hydroxyphenyl)amino)-
1-oxohexan-2-yl)carbamate (C10). White solid, 80% yield. 1H
NMR (500 MHz, chloroform-d): δ 9.04 (s, 1H), 8.09 (s, 1H), 7.39 (s,
1H), 7.29 (s, 5H), 7.06 (t, J = 8.1 Hz, 1H), 6.90 (d, J = 8.0 Hz, 1H),
6.68−6.54 (m, 1H), 6.29 (d, J = 69.3 Hz, 2H), 6.10 (d, J = 7.9 Hz,
1H), 5.21−4.88 (m, 2H), 4.40 (q, J = 7.4 Hz, 1H), 3.60−3.01 (m,
4H), 2.42 (s, 1H), 1.78 (s, 1H), 1.71−1.57 (m, 1H), 1.49 (dt, J =
21.7, 7.2 Hz, 4H), 1.26 (d, J = 5.6 Hz, 15H), 0.89 (t, J = 7.0 Hz, 3H).
13C NMR (126 MHz, chloroform-d): δ 180.9, 171.4, 156.9, 138.3,
135.8, 130.0, 128.6, 128.3, 128.0, 112.3, 112.0, 107.8, 67.4, 55.7, 44.2
(d, J = 82.2 Hz), 32.0, 31.9, 29.6, 29.4, 29.3, 29.1, 28.4, 27.0, 22.8,
22.7, 14.1. HRMS [ESI-Orbitrap] m/z: calcd for C31H47N4O4S ([M +
H]+), 571.3313; observed, 571.3311.
Benzyl (S)-(6-(3-dodecylthioureido)-1-((3-Hydroxyphenyl)-
amino)-1-oxohexan-2yl)carbamate (C12). White solid, 82% yield.
1H NMR (500 MHz, chloroform-d): δ 9.01 (s, 1H), 8.00 (d, J = 8.0
Hz, 1H), 7.39 (s, 1H), 7.30 (s, 5H), 7.08 (t, J = 8.1 Hz, 1H), 6.91 (d,
J = 8.0 Hz, 1H), 6.62 (dd, J = 8.0, 2.2 Hz, 1H), 6.54−6.10 (m, 2H),
6.06 (d, J = 7.9 Hz, 1H), 5.20−4.92 (m, 2H), 4.40 (q, J = 7.4 Hz,
1H), 3.66−3.01 (m, 4H), 2.20 (s, 1H), 1.98−1.72 (m, 1H), 1.66 (d, J
= 11.1 Hz, 1H), 1.50 (dt, J = 16.3, 7.7 Hz, 4H), 1.42−1.06 (m, 20H),
0.90 (t, J = 6.9 Hz, 3H). 13C NMR (126 MHz, chloroform-d): δ
181.0, 171.3, 156.9, 138.3, 135.8, 130.0, 128.6, 128.3, 128.0, 112.3,
112.0, 107.7, 67.5, 55.7, 44.2 (d, J = 103.5 Hz), 32.0, 31.9, 29.69,
29.67, 29.64, 29.59, 29.4 (d, J = 1.4 Hz), 29.1, 28.3, 27.0, 22.7, 14.2.
HRMS [ESI-Orbitrap] m/z: calcd for C33H51N4O4S ([M + H]+),
599.3626; observed, 599.3625.
I
DOI: 10.1021/acs.jmedchem.9b00191
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
10% DMSO, 10% Kolliphor EL) to a concentration of 10 mg/mL
before injection into mice. AF8 at 25 mg/kg (n = 3) and 100 mg/kg
(n = 4), or vehicle (n = 4), was intraperitoneally injected every day
over the course of 12 days. AF10 at 100 mg/kg (n = 8) or vehicle (n =
6) was also intraperitoneally injected every day over the course of 12
days. Tumor volume was measured daily and mice were sacrificed on
the last day and the tumors extracted and weighed.
Immunofluorescence Microscopy. 400 000 HCT116 cells were
seeded per dish (MatTek 35 mm, no. 1.5 coverslip, 14 mm glass
diameter, poly-D-lysine coated) and incubated for 24 h at 37 °C. Each
well was then treated with DMSO or inhibitor at a final concentration
of 50 μM and incubated for 6 h at 37 °C. Cells were fixed in ice cold
methanol for 10 min and rinsed 3× in detergent containing buffer
(Tris-buffered saline or TBS, 0.1% TX-100). Cells were blocked in
blocking buffer (TBS, 0.1% TX-100, 5% BSA) for 1 h at RT and then
incubated in primary acetyl α-tubulin antibody (Millipore MABT868
1:100 dilution) overnight at 4 °C. Cells were then washed 3× in
detergent containing buffer and incubated with secondary antibody
(Cy3-conjugated goat α-mouse, Thermo Fisher A10521, 1:500
dilution) for 1 h at RT. Cells were rinsed 5× in detergent containing
buffer and mounted overnight at RT (DAPI Fluoromount-G,
SouthernBiotech). Cells were imaged using a Zeiss LSM880 inverse
laser scanning confocal microscope.
P53 Acetylation Assay and Western Blotting. 400 000
HCT116 cells were seeded per well in a six-well plate and incubated
for 24 h at 37 °C. Subsequently, each well was cotreated with 400 nM
TSA and respective concentration of AF8, AF10, TM, EX527, C3,
C5, or C10 for 6 h at 37 °C before cells were collected and lysed in
buffer [4% sodium dodecyl sulfate (SDS), 150 mM NaCl, 50 mM
triethanolamine, Universal Nuclease, pH 7.4]. The protein concen-
trations of cell lysates were calculated using the BCA assay. To
evaluate AcP53 (K382) levels, 50 μg of lysate was run on a 12% SDS-
polyacrylamide gel electrophoresis gel. To check protein loading, 8 μg
of lysate was also loaded on the same gel. The protein gel was
transferred to a polyvinylidene difluoride membrane, blocked in buffer
(TBS, 5% BSA, 0.1% Tween-20) for 1 h at RT, and incubated
overnight at 4 °C with AcP53 (K382) antibody (CST 2525S), or with
β-actin antibody (SCBT sc-47778) for 1 h at RT. Membranes were
rinsed with TBST (5 ×, 7 min each) and incubated with antirabbit or
antimouse secondary antibody (CST 7074S and CST 7076S) for 1 h
at RT. Membranes were rinsed again with TBST (5×, 7 min each)
before being incubated with ECL substrate and imaged on a Typhoon
FLA 7000.
ORCID
Ali Sohail Farooqi: 0000-0001-7763-6152
Hening Lin: 0000-0002-0255-2701
Present Addresses
§Zhejiang Province Key Laboratory of Anti-Cancer Drug
Research, College of Pharmaceutical Sciences, Zhejiang
University, Hangzhou, China. 310058.
∥Shanghai Institute of Materia Medica, Chinese Academy of
Sciences, No. 501, Haike Rd., Pudong New District, Shanghai
201203, China.
Author Contributions
A.S.F., J.Y.H., and J.C. contributed equally to this work. The
manuscript was written through contributions of all authors.
All authors have given approval to the final version of the
manuscript.
Funding
The work was supported by funds from HHMI and Cornell
University. This work made use of the Cornell University
NMR Facility, which is supported, in part, by the NSF through
MRI award CHE-1531632. Imaging data was acquired through
the Cornell University Biotechnology Resource Center, with
NYSTEM (CO29155) and NIH (S10OD018516) funding for
the shared Zeiss LSM880 confocal/multiphoton microscope.
Notes
The authors declare the following competing financial
interest(s): Cornell University has patent issued or pending
on SIRT2 inhibitors.
ACKNOWLEDGMENTS
A.S.F. thanks Dr. Ying-Ling Chiang for his guidance in organic
synthesis.
■
ABBREVIATIONS
■
BSA, bovine serum albumin; FBS, fetal bovine serum; LC−
MS, liquid chromatography−mass spectrometry; HPLC, high
performance liquid chromatography; PBS, phosphate buffered
saline; DMSO, dimethyl sulfoxide; NAD+, nicotinamide
adenine dinucleotide; TEA, triethylamine; TSA, Trichostatin
A; TBS, Tris-buffered saline; NEAA, nonessential amino acids
Cell Culture. Cell lines were cultured in Dulbecco’s modified
Eagle medium with 10% fetal bovine serum (FBS; MCF-7, 293T,
MDA-MB-231, MDA-MB-468, HME1, CCD 841 CoN), RPMI with
10% FBS (A549, SW948, NCIH23, BxPC-3), McCoy’s media with
10% calf serum (HCT116). Cells were incubated at 37 °C with 5%
CO2.
REFERENCES
■
(1) Houtkooper, R. H.; Pirinen, E.; Auwerx, J. Sirtuins as regulators
of metabolism and healthspan. Nat. Rev. Mol. Cell Biol. 2012, 13, 225.
(2) Imai, S.-i.; Armstrong, C. M.; Kaeberlein, M.; Guarente, L.
Transcriptional silencing and longevity protein Sir2 is an NAD-
dependent histone deacetylase. Nature 2000, 403, 795−800.
(3) Bheda, P.; Jing, H.; Wolberger, C.; Lin, H. The substrate
specificity of sirtuins. Annu. Rev. Biochem. 2016, 85, 405−429.
(4) Chang, H.-C.; Guarente, L. SIRT1 and other sirtuins in
metabolism. Trends Endocrinol. Metab. 2014, 25, 138−145.
(5) Rodriguez, R. M.; Fernandez, A. F.; Fraga, M. F. Role of sirtuins
in stem cell differentiation. Genes Cancer 2013, 4, 105−111.
(6) Liu, P. Y.; Xu, N.; Malyukova, A.; Scarlett, C. J.; Sun, Y. T.;
Zhang, X. D.; Ling, D.; Su, S.-P.; Nelson, C.; Chang, D. K.; Koach, J.;
Tee, A. E.; Haber, M.; Norris, M. D.; Toon, C.; Rooman, I.; Xue, C.;
Cheung, B. B.; Kumar, S.; Marshall, G. M.; Biankin, A. V.; Liu, T. The
histone deacetylase SIRT2 stabilizes Myc oncoproteins. Cell Death
Differ. 2013, 20, 503−514.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jmed-
chem.9b00191.
■
S
In vitro inhibition assays of SIRT1-3 with compounds
AF8−AF12, cell cytotoxicity assay of various cell lines
1
comparing compounds AF8, AF10, and TM, and H
and 13C NMR spectra of all synthesized intermediates
and final compounds (PDF)
qNMR of synthesized compounds (PDF)
Molecular formula strings (CSV)
Modeling of AF and C inhibitors into SIRT2 (PDB)
(7) Song, N.-Y.; Surh, Y.-J. Janus-faced role of SIRT1 in
tumorigenesis. Ann. N.Y. Acad. Sci. 2012, 1271, 10−19.
(8) Xiangyun, Y.; Xiaomin, N.; linping, G.; Yunhua, X.; Ziming, L.;
Yongfeng, Y.; Zhiwei, C.; Shun, L. Desuccinylation of pyruvate kinase
M2 by SIRT5 contributes to antioxidant response and tumor growth.
Oncotarget 2017, 8, 6984−6993.
AUTHOR INFORMATION
Corresponding Author
*E-mail: hl379@cornell.edu.
■
J
DOI: 10.1021/acs.jmedchem.9b00191
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(9) Kim, H.-S.; Vassilopoulos, A.; Wang, R.-H.; Lahusen, T.; Xiao,
Z.; Xu, X.; Li, C.; Veenstra, T. D.; Li, B.; Yu, H.; Ji, J.; Wang, X. W.;
Park, S.-H.; Cha, Y. I.; Gius, D.; Deng, C.-X. SIRT2 maintains
genome integrity and suppresses tumorigenesis through regulating
APC/C activity. Cancer cell 2011, 20, 487−499.
(25) Solomon, J. M.; Pasupuleti, R.; Xu, L.; McDonagh, T.; Curtis,
R.; DiStefano, P. S.; Huber, L. J. Inhibition of SIRT1 catalytic activity
increases p53 acetylation but does not alter cell survival following
DNA damage. Mol. Cell. Biol. 2006, 26, 28−38.
(26) Teng, Y.-B.; Jing, H.; Aramsangtienchai, P.; He, B.; Khan, S.;
Hu, J.; Lin, H.; Hao, Q. Efficient demyristoylase activity of SIRT2
revealed by kinetic and structural studies. Sci. Rep. 2015, 5, 8529.
(27) Paoli, P.; Giannoni, E.; Chiarugi, P. Anoikis molecular pathways
and its role in cancer progression. Biochim. Biophys. Acta 2013, 1833,
3481−3498.
(28) Hanahan, D.; Weinberg, R. A. The hallmarks of cancer. Cell
2000, 100, 57−70.
(29) Patel, S.; Pine, S.; Rameshwar, P. Noble Agar Assay for Self-
Renewal. Protoc. Exch. 2013, DOI: 10.1038/protex.2013.021.
(30) North, B. J.; Marshall, B. L.; Borra, M. T.; Denu, J. M.; Verdin,
E. The human Sir2 ortholog, SIRT2, is an NAD+-dependent tubulin
deacetylase. Mol. Cell 2003, 11, 437−444.
́
(10) Jing, H.; Hu, J.; He, B.; Negron Abril, Y. L.; Stupinski, J.;
Weiser, K.; Carbonaro, M.; Chiang, Y.-L.; Southard, T.; Giannakakou,
P.; Weiss, R. S.; Lin, H. A SIRT2-selective inhibitor promotes c-Myc
oncoprotein degradation and exhibits broad anticancer activity.
Cancer Cell 2016, 29, 297−310.
(11) Jing, H.; Zhang, X.; Wisner, S. A.; Chen, X.; Spiegelman, N. A.;
Linder, M. E.; Lin, H. SIRT2 and lysine fatty acylation regulate the
transforming activity of K-Ras4a. Elife. 2017, 11, e32436.
(12) Zhou, W.; Ni, T. K.; Wronski, A.; Glass, B.; Skibinski, A.; Beck,
A.; Kuperwasser, C. The SIRT2 deacetylase stabilizes Slug to control
malignancy of basal-like breast cancer. Cell Rep. 2016, 17, 1302−1317.
(13) Outeiro, T. F.; Kontopoulos, E.; Altmann, S. M.; Kufareva, I.;
Strathearn, K. E.; Amore, A. M.; Volk, C. B.; Maxwell, M. M.; Rochet,
J.-C.; McLean, P. J.; Young, A. B.; Abagyan, R.; Feany, M. B.; Hyman,
B. T.; Kazantsev, A. G. Sirtuin 2 Inhibitors Rescue -Synuclein-
Mediated Toxicity in Models of Parkinson’s Disease. Science 2007,
317, 516.
(31) Vaziri, H.; Dessain, S. K.; Eaton, E. N.; Imai, S.-I.; Frye, R. A.;
Pandita, T. K.; Guarente, L.; Weinberg, R. A. hSIR2SIRT1 Functions
as an NAD-Dependent p53 Deacetylase. Cell 2001, 107, 149−159.
(32) Gertz, M.; Fischer, F.; Nguyen, G. T. T.; Lakshminarasimhan,
M.; Schutkowski, M.; Weyand, M.; Steegborn, C. Ex-527 inhibits
sirtuins by exploiting their unique NAD+-dependent deacetylation
mechanism. Proc. Natl. Acad. Sci. U.S.A. 2013, 110, No. E2772.
(33) Moriarity, A.; O’Sullivan, J.; Kennedy, J.; Mehigan, B.;
McCormick, P. Current targeted therapies in the treatment of
advanced colorectal cancer: a review. Ther. Adv. Med. Oncol. 2016, 8,
276−293.
(14) Lain, S.; Hollick, J. J.; Campbell, J.; Staples, O. D.; Higgins, M.;
Aoubala, M.; McCarthy, A.; Appleyard, V.; Murray, K. E.; Baker, L.;
Thompson, A.; Mathers, J.; Holland, S. J.; Stark, M. J. R.; Pass, G.;
Woods, J.; Lane, D. P.; Westwood, N. J. Discovery, in vivo activity,
and mechanism of action of a small-molecule p53 activator. Cancer
Cell 2008, 13, 454−463.
(34) El Zouhairi, M.; Charabaty, A.; Pishvaian, M. J. Molecularly
targeted therapy for metastatic colon cancer: proven treatments and
promising new agents. Gastrointest. Cancer Res. 2011, 4, 15−21.
(15) Rumpf, T.; Schiedel, M.; Karaman, B.; Roessler, C.; North, B.
́
J.; Lehotzky, A.; Olah, J.; Ladwein, K. I.; Schmidtkunz, K.; Gajer, M.;
́
Pannek, M.; Steegborn, C.; Sinclair, D. A.; Gerhardt, S.; Ovadi, J.;
̀
(35) Lievre, A.; Bachet, J.-B.; Boige, V.; Cayre, A.; Le Corre, D.; Buc,
Schutkowski, M.; Sippl, W.; Einsle, O.; Jung, M. Selective Sirt2
inhibition by ligand-induced rearrangement of the active site. Nat.
Commun. 2015, 6, 6263.
́
́
E.; Ychou, M.; Bouche, O.; Landi, B.; Louvet, C.; Andre, T.; Bibeau,
F.; Diebold, M.-D.; Rougier, P.; Ducreux, M.; Tomasic, G.; Emile, J.-
F.; Penault-Llorca, F.; Laurent-Puig, P. KRAS Mutations As an
Independent Prognostic Factor in Patients With Advanced Colorectal
Cancer Treated With Cetuximab. J. Clin. Oncol. 2008, 26, 374−379.
(36) Hsu, H.-C.; Thiam, T. K.; Lu, Y.-J.; Yeh, C. Y.; Tsai, W. S.; You,
J. F.; Hung, H. Y.; Tsai, C.-N.; Hsu, A.; Chen, H.-C.; Chen, S.-J.;
Yang, T.-S. Mutations of KRAS/NRAS/BRAF predict cetuximab
resistance in metastatic colorectal cancer patients. Oncotarget 2016, 7,
22257−22270.
(16) Spiegelman, N. A.; Price, I. R.; Jing, H.; Wang, M.; Yang, M.;
Cao, J.; Hong, J. Y.; Zhang, X.; Aramsangtienchai, P.; Sadhukhan, S.;
Lin, H. Direct comparison of SIRT2 inhibitors: potency, specificity,
activity-dependent inhibition, and on-target anticancer activities.
ChemMedChem 2018, 13, 1890−1894.
(17) Cen, Y.; Sauve, A. A. Transition state of ADP-Ribosylation of
acetyllysine catalyzed by Archaeoglobus fulgidus Sir2 determined by
kinetic isotope effects and computational approaches. J. Am. Chem.
Soc. 2010, 132, 12286−12298.
(18) Hirsch, B. M.; Hao, Y.; Li, X.; Wesdemiotis, C.; Wang, Z.;
Zheng, W. A mechanism-based potent sirtuin inhibitor containing Nε-
thiocarbamoyl-lysine (TuAcK). Bioorg. Med. Chem. Lett. 2011, 21,
4753−4757.
(37) Raponi, M.; Winkler, H.; Dracopoli, N. C. KRAS mutations
predict response to EGFR inhibitors. Curr. Opin. Pharmacol. 2008, 8,
413−418.
(19) Wang, Y.; Fung, Y. M. E.; Zhang, W.; He, B.; Chung, M. W. H.;
Jin, J.; Hu, J.; Lin, H.; Hao, Q. Deacylation Mechanism by SIRT2
Revealed in the 1′-SH-2′-O-Myristoyl Intermediate Structure. Cell
Chem. Biol. 2017, 24, 339−345.
(20) Madsen, A. S.; Andersen, C.; Daoud, M.; Anderson, K. A.;
Laursen, J. S.; Chakladar, S.; Huynh, F. K.; Colaco̧ , A. R.; Backos, D.
S.; Fristrup, P.; Hirschey, M. D.; Olsen, C. A. Investigating the
sensitivity of NAD+-dependent sirtuin deacylation activities to
NADH. J. Biol. Chem. 2016, 291, 7128−7141.
(21) Michan, S.; Sinclair, D. Sirtuins in mammals: insights into their
biological function. Biochem. J. 2007, 404, 1−13.
(22) Guan, X.; Lin, P.; Knoll, E.; Chakrabarti, R. Mechanism of
inhibition of the human sirtuin enzyme SIRT3 by nicotinamide:
computational and experimental studies. PLoS One 2014, 9,
No. e107729.
(23) Avalos, J. L.; Bever, K. M.; Wolberger, C. Mechanism of sirtuin
inhibition by nicotinamide: altering the NAD+ cosubstrate specificity
of a Sir2 enzyme. Mol. Cell 2005, 17, 855−868.
(24) Kim, B. S.; Lee, C.-H.; Chang, G.-E.; Cheong, E.; Shin, I. A
potent and selective small molecule inhibitor of sirtuin 1 promotes
differentiation of pluripotent P19 cells into functional neurons. Sci.
Rep. 2016, 6, 34324.
K
DOI: 10.1021/acs.jmedchem.9b00191
J. Med. Chem. XXXX, XXX, XXX−XXX