Simultaneous Inhibition of SIRT2 Deacetylase and Defatty-Acylase Activities via a PROTAC Strategy

Article abstract of DOI:10.1021/acsmedchemlett.0c00423

As a member of the sirtuin family of enzymes, SIRT2 promotes tumor growth and regulates various biological pathways through lysine deacetylation and defatty-acylation. In the past few years, many SIRT2-selective small molecule inhibitors have been developed, but none have demonstrated simultaneous inhibition of both SIRT2 activities in cells. To further scrutinize the physiological importance and significance of SIRT2 deacetylase and defatty-acylase activities, small molecules that can selectively inhibit both activities of SIRT2 in living cells are needed. Here, we have applied the Proteolysis Targeting Chimera (PROTAC) strategy and synthesized a new SIRT2 inhibitor (TM-P4-Thal) to degrade SIRT2 selectively, which led to simultaneous inhibition of its deacetylase and defatty-acylase activities in living cells. Additionally, this compound exemplifies the advantage of the PROTAC strategy that allows complete eradication of an enzyme and its activity in biological settings.

Full text of DOI:10.1021/acsmedchemlett.0c00423

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Letter
Simultaneous Inhibition of SIRT2 deacetylase and
defatty-acylase activities via a PROTAC strategy
Jun Young Hong, Hui Jing, Ian Robert Price, Ji Cao, Jessica Jingyi Bai, and Hening Lin
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.0c00423 • Publication Date (Web): 21 Sep 2020
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Simultaneous Inhibition of SIRT2 deacetylase and defatty-acylase
activities via a PROTAC strategy
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Jun Young Hong†, Hui Jing†, Ian Robert Price†, Ji Cao†, Jessica Jingyi Bai†, Hening Lin†‡*
† Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY, 14853
‡ Howard Hughes Medical Institute; Department of Chemistry and Chemical Biology Cornell University, Ithaca, NY,
14853
ABSTRACT: As a member of the sirtuin family of enzymes, SIRT2 promotes tumor growth and regulates various biological
pathways through lysine deacetylation and defatty-acylation. In the past few years, many SIRT2-selective small molecule
inhibitors had been developed, but none had demonstrated simultaneous inhibition of both SIRT2 activities in cells. To further
scrutinize the physiological importance and significance of SIRT2 deacetylase and defatty-acylase activities, small molecules
that can selectively inhibit both activities of SIRT2 in living cells are needed. Here, we have applied Proteolysis Targeting
Chimera (PROTAC) strategy and synthesized a new SIRT2 inhibitor (TM-P4-Thal) to degrade SIRT2 selectively, which led to
simultaneous inhibition of its deacetylase and defatty-acylase activities in living cells. Additionally, this compound exemplifies
the advantage of PROTAC strategy that allows complete eradication of an enzyme and its activity in biological settings.
KEYWORDS: SIRT2, Sirtuin Inhibitor, PROTAC, Cancer Treatment, Lysine Defatty-acylase, Lysine Deacetylase
Introduction
Many SIRT2-selective inhibitors have been developed
and shown to impede cancer growth. From a high
throughput screen effort, AGK2 was found to be a SIRT2-
selective inhibitor that binds to the “C-pocket” of SIRT2.21
Later, another SIRT2-selective inhibitor, SirReal2, was
synthesized, which decreases migration and invasion of
gastric cancer cells.22-23 NCO-90/140 slowed down cellular
growth of several leukemia cells through induction of
apoptosis and autophagy.24 Compound 53, based on NCO-
90, demonstrated enhanced antiproliferative effect in
breast cancer cells and promoted neurite outgrowth.25
NPD11033 effectively delayed the cell proliferation of
pancreatic cancer cells.26 TM, a mechanism-based SIRT2-
selective inhibitor, portrayed broad anti-cancer effects by
promoting the degradation of c-Myc (Figure 1).17
The mammalian sirtuin family of enzymes remove
various acyl groups from protein lysine residues, using
nicotinamide adenine dinucleotide (NAD+) as
a
cosubstrate.1-4 They are involved in many biological
processes, including metabolism, DNA damage repair, and
cell growth.5-9 Among the seven sirtuins, SIRT2 is the only
one that is primarily localized in the cytosol and can remove
both acetyl and other acyl groups from protein lysine
residues.10-12 SIRT2 was initially reported to deacetylate
protein substrates, including hypoxia-inducible factor 1 α
(HIF1α) and α-tubulin.13-14 Through deacetylation of
various substrates, SIRT2 promotes tumorigenesis. For
example, SIRT2 deacetylates Slug to increase its stability,
and promotes breast cancer progression.15 By deacetylating
lactate dehydrogenase
A (LDH-A), which is often
overexpressed in cancer cells and responsible for lactate
production, SIRT2 promotes pancreatic cancer growth.16 In
many cancer cell lines, the inhibition of SIRT2 leads to
degradation of C-Myc, impeding cancer cell growth in vitro
and tumor growth in mice.17 Besides the deacetylation
activity of SIRT2, SIRT2 can also efficiently remove long-
chain fatty acyl groups from lysine.10, 18 SIRT2 can defatty-
acylate
K-Ras4a
and
promote
K-Ras-mediated
transformation.19 Recently, SIRT2 is also reported to
defatty-acylate RalB, another small GTPase in the Ras
subfamily.20 Through both deacetylation and defatty-
acylation, SIRT2 plays significant roles in cancer growth and
metabolism, which made SIRT2 an attractive target for
cancer treatment.
Figure 1. Examples of previously reported SIRT2 inhibitors
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Scheme 1. Synthesis of TM-P2-Thal and TM-P4-Thal.
Interestingly, recent studies that directly compared these
inhibitors in various assays revealed that AGK2, SirReal2,
and TM inhibit SIRT2 deacetylation activity, but not the
defatty-acylation activity.27 In contrast, a peptide-based
SIRT2 inhibitor, S2DMi-7, could efficiently inhibit the
defatty-acylase activities of SIRT1, SIRT2, and SIRT3.28
Nevertheless, being a peptide, S2DMi-7 is not SIRT2-
selective and may have limited utility for cellular and in vivo
studies. We recently reported that JH-T4 and NH-TM, which
can hinder both activities of SIRT2 (Figure 1).29 However,
these two compounds also inhibit SIRT1 and SIRT3, making
it difficult to study SIRT2’s specific roles in cells.
degrade SIRT2 efficiently in various cancer cells. Through
SIRT2 degradation, we demonstrate that these compounds
inhibit both activities of SIRT2 in living cells and achieve
enhanced antiproliferative effect in cancer cells.
Results and Discussion
PROTAC Design and Synthesis. TM is a thiomyristoyl
lysine-based SIRT2 selective inhibitor. Its thiomyristoyl
lysine is important for SIRT2 inhibition, as this portion
forms a stalled covalent intermediate with NAD+ to inhibit
the activity of SIRT2.17 The C- and N-termini of the
thiomyristoyl lysine serves as the potential modification
sites to attach the linker and thalidomide, the ligand that
recruit the E3 ubiquitin ligase cereblon (CRBN). According
to the previously reported co-crystal structures of SIRT2 in
complex with two inhibitors, BHJH-TM1 and Glucose-TM
(PDB: 4R8M, 6NR0), the thiomyristoyl lysine points inward
towards the enzyme pocket, while both the N- and C-
terminal of the lysine are located outside of the pocket.18, 34
The introduction of a hydroxyl group to the C-terminal
phenyl group of TM, resulting in a new inhibitor, JH-T4,
disrupted the selectivity of TM for SIRT2, as JH-T4 inhibits
SIRT1, SIRT2 and SIRT3.29 From this finding, we
hypothesized that altering the C-terminal phenyl ring for
thalidomide attachment could potentially ruin SIRT2
selectivity. Thus, we decided to attach thalidomide to the N-
terminus of TM (Scheme 1).
Given the two different physiological catalytic activities of
SIRT2, one interesting question is whether small molecules
that can inhibit both activities of SIRT2 would produce
different biological effects. To address this question, new
inhibitors that can inhibit both the deacetylase and defatty-
acylase activities of SIRT2 in live cells, but are specific for
SIRT2 are needed.
Proteolysis Targeting Chimera (PROTAC) uses a hetero-
dimeric compound consisting of a ligand interacting with
the protein-of-interest and another ligand recruiting E3
ligase to degrade the protein of interest.30-32 This strategy
gained attention for its ability to enhance the effects of small
molecule inhibitors. Traditionally, most inhibitors bind to
an enzyme to inhibit its activity. PROTAC compounds,
instead, catalytically remove the target proteins and thus
not only inhibit the catalytic activity but also eliminate non-
enzymatic functions of target proteins through
degradation.30-31 Here we set out to test another possibility
that could make the PROTAC strategy more effective: when
an inhibitor of SIRT2 could only inhibit the deacetylation
activity of SIRT2, we could enable it to inhibit all the
catalytic activities of SIRT2 using the PROTAC strategy. In
other words, applying PROTAC strategy to specifically
degrade SIRT2 would inhibit both catalytic activities of
SIRT2, deacetylation and defatty-acylation, which currently
existing small molecule inhibitors could not achieve.
PROTAC utilizing SirReal2 as the ligand was synthesized,
but was not tested to show the advantage of the PROTAC
strategy.33 Here we introduce two new PROTAC
compounds, TM-P2-Thal and TM-P4-Thal, which can
TM contains the carboxybenzoyl group (Cbz) on the N-
terminal of the lysine backbone. We replaced this Cbz group
with thalidomide via a 2- or 4-repeating units of the
polyethylene glycol (PEG) linker (Scheme 1). The PEG linker
was chosen because of its aqueous solubility.35 In addition,
being flexible on its own, the PEG linker could allow the
thalidomide-CRBN complex to reach SIRT2’s ubiquitination
site easier. PEG linkers with two different lengths were used
because the linker may affect the ubiquitination efficiency.
If the linker is too short, SIRT2 and CRBN could clash with
each other and not be able to form the PROTAC complex.
However, if the linker is too long, CRBN and SIRT2 could be
too far apart and decrease ubiquitination.
The synthesis of TM-P2-Thal or TM-P4-Thal required a
total of 10 steps (Scheme 1). PEG linkers with two different
lengths were attached to thalidomide. tert-Butyloxycarbonyl
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(Boc)-L-lysine was used to synthesize the TM analog with a
free N-terminus. Then, in the last step, thalidomide with the
PEG linkers was coupled to the TM analog with a free N-
terminus using isobutyl chloroformate to produce TM-P2-
Thal and TM-P4-Thal as the final products (Scheme 1).
with and without pre-incubation. Even without pre-
incubation, these three compounds could inhibit SIRT2
deacetylase efficiently. However, without pre-incubation,
they could not inhibit SIRT2 defatty-acylase activity even at
83 µM. With pre-incubation, the IC50 for TM, TM-P2-Thal,
and TM-P4-Thal were 0.37, 0.17 and 0.11 µM, respectively.
This IC50 difference between with and without pre-
incubation may explain why TM could not inhibit SIRT2
defatty-acylase activity in cells from previous studies.29
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Table 1. In Vitro IC50 values of TM-P2-Thal and TM-P4-
Thal.
TM-P2-
Thal
TM-P4-
Thal
TM-P2-Thal and TM-P4-Thal degrade SIRT2
selectively in cells. We next set out to check the effects of
TM, TM-P2-Thal, and TM-P4-Thal on SIRT2 protein levels in
two breast cancer cell lines, MCF7 and BT549. After 48
hours of treatment at various concentrations, TM-P2-Thal
and TM-P4-Thal degraded SIRT2 selectively without
changing the levels of SIRT1 and SIRT3 (Figure 2A). As
expected, TM did not decrease the level of SIRT2 in the two
cell lines. In MCF7 cells, 5 µM of the PROTAC compounds
degraded SIRT2 more effectively than 25 µM, which is likely
due to the Hook effect of the PROTAC strategy.36 High
concentrations of PROTAC primarily form binary complex
with either the E3 ligase or the protein target, and thereby
preventing the ternary complex formation that is for the
efficient degradation of target protein. Similarly, SIRT2
selective degradations were observed in MDA-MB-231 and
MDA-MB-468 cell lines (Supplementary Figure 1A and 1B)
in the presence of TM-P2-Thal and TM-P4-Thal.
9
IC50 (µM)
TM
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SIRT1
SIRT2
37 ± 3.5
41 ± 5.1
>83
0.069 ±
0.032
0.078 ±
0.018
0.093 ±
0.012
Deacetylase
(with
pre-incubation)
SIRT2
0.12 ±
0.020
0.093 ±
0.025
0.15 ±
0.036
Deacetylase
(without
pre-incubation)
SIRT2
Defatty-
acylase
(with
0.17 ±
0.044
0.11 ±
0.063
0.37 ±
0.031
pre-incubation)
SIRT2
Defatty-
acylase
(without
>83
>83
>83
In MCF7 cells, we then further tested SIRT2-selective
degradation with lower than 5 µM of TM-P4-Thal for 48
hours. Even at 0.5 µM, SIRT2 level was significantly
decreased compared to that of a vehicle-treated sample.
Furthermore, the decreased levels of SIRT2 at both 0.5 and
10 µM were similar, suggesting the high efficiency of TM-
P4-Thal (Figure 2B). Again, at 50 µM of TM-P4-Thal, SIRT2
level did not change much likely due to the hook effect.36
pre-incubation)
SIRT3
SIRT5
SIRT6
>83
>83
>83
>83
>83
>83
>83
>83
>83
TM-P2-Thal and TM-P4-Thal maintain SIRT2
selectivity. In vitro IC50 values of TM-P2-Thal and TM-P4-
Thal on the sirtuin family members, SIRT1, SIRT2, SIRT3,
SIRT5 and SIRT6, were measured to see if the selectivity
toward SIRT2 had been preserved. Each compound at
different concentrations was added to the sirtuin enzymatic
reaction mixture to measure its inhibitory activities.17, 27
Because the tested compounds inhibit sirtuin activities
through forming a stalled covalent intermediate with NAD+,
we have pre-incubated these compounds with NAD+ and the
corresponding sirtuin enzyme, prior to the addition of the
substrate peptide. With pre-incubation, the IC50 of TM-P2-
Thal and TM-P4-Thal for SIRT2 deacetylase were 0.069 and
0.078 µM, respectively. Meanwhile, the IC50 of TM-P2-Thal
and TM-P4-Thal for SIRT1 deacetylase were 37 and 41 µM,
respectively. The selectivity for SIRT2 over SIRT1 (IC50
SIRT1/IC50 SIRT2) was about 500 times for both TM-P2-
Thal and TM-P4-Thal. In addition, both compounds did not
show inhibitory activity on SIRT3, SIRT5, or SIRT6 (Table
1). In addition, Compound 8, thalidomide with PEG4 linker,
could not inhibit any of the sirtuin activities
(Supplementary Table 1). Overall, even with the
modifications on the N-terminus, both TM-P2-Thal and TM-
P4-Thal retained SIRT2 selectivity.
Next, we examined the time course of TM-P4-Thal-
induced SIRT2 degradation in MCF7 cells. As the incubation
time increased, 10 µM of TM-P4-Thal lowered SIRT2 even
further. Thus, SIRT2 was degraded by TM-P4-Thal in a time-
dependent manner (Figure 2C). However, with 25 µM of
TM-P4-Thal, SIRT2 level decreased slightly at 24 hour and
no significant SIRT2 degradation was observed in later time
points (Supplementary Figure 1C). The decreased
degradation at 25 µM of TM-P4-Thal was again due to the
hook effect. We also treated cells with 10 and 25 µM of TM-
P2-Thal in different time points. Similar result was
observed, but the overall efficiency in the degradation of
SIRT2 was lower compared to that with TM-P4-Thal
(Supplementary Figure 1C). Thus, we mainly used TM-P4-
Thal for the rest of the study. Furthermore, we treated
MCF7 cells with Compound 8, thalidomide with a PEG4
linker, and detected SIRT2 level through immunoblotting.
As expected, without the TM moiety, Compound 8 could not
degrade SIRT2 (Supplementary Figure 2A). Also, we
synthesized TM-P4-Thal-CH3 with a methyl group on
thalidomide, which disrupts the overall binding to CRBN
and hinder PROTAC degradation.37 Like Compound 8, TM-
P4-Thal-CH3 could not degrade SIRT2 in cells
(Supplementary Figure 2B).
We have further tested TM, TM-P2-Thal and TM-P4-Thal
against SIRT2 deacetylase and defatty-acylase activities
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Figure 2. TM-P2-Thal and TM-P4-Thal degrade SIRT2 selectively in cells. (A) Immunoblots for SIRT1, SIRT2 and SIRT3
after treating with indicated concentrations of inhibitors for 48 hours in MCF7 and BT-549 cells. (B) Immunoblot for SIRT2
after MCF7 cells were treated with indicated concentrations of TM-P4-Thal for 48 hours. (C) Immunoblot for SIRT2 after
MCF7 cells were treated with 10 µM of TM-P4-Thal for the indicated incubation times.
Figure 3. TM-P4-Thal inhibits both SIRT2 deacetylase and defatty-acylase activities in cells. (A) Immunofluorescence
images of acetyl α-tubulin (K40) in MCF7 cells treated with DMSO (control), TM-P4-Thal (5, 10 µM) and TM (5, 10 µM) for 12
hours. (B) Normalized Ac-α-tubulin fluorescence intensity level of 5 µM TM-P4-Thal and TM for 12 hours, using ImageJ
software for quantification. (C) Immunoblot with pan-Ras antibody to show the stable overexpression of Flag-tagged K-
Ras4a-G12V in HEK 293T cells. (D) Immunoblots for SIRT1, SIRT2, and SIRT3 after treating HEK 293T cells with 1 µM of TM-
P4-Thal or 10 µM of TM in the presence of 50 µM of Alk-14 for 24 hours. (E) Alk14 labeling and in-gel fluorescence to detect
lysine fatty acylation of K-Ras4a in HEK 293T cells. Cells were treated with 1 µM of TM-P4-Thal or 10 µM of TM in the presence
of 50 µM of Alk-14. (F) Quantified relative lysine fatty acylation level of K-Ras4a from Figure 3E, using ImageJ for
quantification.
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To validate that SIRT2 degradation occurs through CRBN
and ubiquitin-proteasome system, we tried to rescue the
degradation through co-treatment of proteasome
inhibitor, MG132. MCF7 cells were pre-treated with MG132
(5 µM) for 2 hours, followed by co-treatment of TM-P4-Thal
(5 µM) and MG132 (5 µM) for additional 16 hours. While the
treatment with TM-P4-Thal decreased SIRT2 level, the co-
treatment with TM-P4-Thal and MG132 did not decrease
SIRT2 level, suggesting that TM-P4-Thal promotes
DMSO control, suggesting efficient inhibition of SIRT2
defatty-acylation by TM-P4-Thal. In contrast, TM did not
significantly increase the fluorescence level of K-Ras4a
(Figure 3E, F). This suggests that while TM could not inhibit
SIRT2 defatty-acylation, its PROTAC-version induced
degradation of SIRT2 efficiently and inhibited the defatty-
acylation activity in live cells.
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a
TM-P4-Thal shows improved cytotoxicity in breast
cancer cells at lower concentrations. After
demonstrating that TM-P4-Thal can inhibit both activities
of SIRT2, we evaluated its effect on the proliferation of
MCF7 and MDA-MB-231 cells (Figure 4). In both breast
cancer cell lines, we observed selective SIRT2 degradation
after treatment of TM-P4-Thal (Figure 2 and Supplementary
Figure 1). In MCF7 cells, after 72 hours incubation, TM-P4-
Thal showed much stronger cytotoxicity than TM at lower
concentrations. However, towards higher concentrations,
the difference between TM and TM-P4-Thal became
smaller. Similar trend was observed in MDA-MB-231.
However, in MDA-MB-231 cells, at higher concentrations,
cytotoxicity of TM exceeded that of TM-P4-Thal (Figure 4).
This is likely because of inefficient SIRT2 degradation at
high concentrations of TM-P4-Thal due to the hook effect.36
proteasome-dependent
degradation
of
SIRT2
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(Supplementary Figure 3).
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To further validate SIRT2 degradation by TM-P4-Thal, we
performed Tandem Mass Tag (TMT) global proteomic with
HEK 293T cells treated with 5 μM of TM-P4-Thal for 16
hours. Consistent with the immunoblots, the SIRT2
abundance ratio between TM-P4-Thal and DMSO treated
cells was about 0.36, suggesting substantial degradation of
SIRT2. Meanwhile, abundance ratios of other sirtuins,
including SIRT1, 3, 5 and 6, did not alter significantly after
TM-P4-Thal treatment. Overall, the TMT global proteomic
result further confirms selective SIRT2 degradation by TM-
P4-Thal (Supplementary Figure 4, Supplementary Table 3).
TM-P4-Thal efficiently inhibits both SIRT2
deacetylation and defatty-acylation in cells. To assess
the inhibition of SIRT2 deacetylation by TM-P4-Thal in cells,
acetylation of α-tubulin, a previously reported SIRT2
There were some discrepancy between the SIRT2 level
and the cytotoxicity. For instance, 1 µM of TM-P4-Thal
induced SIRT2 degradation but did not exert significant
cytotoxicity in the cellular proliferation assay. SIRT2 may
need to be completely inhibited/degraded for a longer
period to see a stronger impact. Also, there could be an
undiscovered reparative biological mechanism for the
deceased protein level. Further studies are needed to
understand this difference.
deacetylation
target,
was
examined
by
immunofluorescence after treating MCF7 cells with 5 and
10 µM of TM-P4-Thal for 12 hours.14 If SIRT2 deacetylation
is inhibited, the level of acetylation would be increased by
the treatments. TM-P4-Thal or TM at 5 and 10 µM
concentrations significantly increased the acetylation of α-
tubulin (Figure 3A). Furthermore, quantification of the
fluorescence signals indicated that at 5 µM, TM-P4-Thal
increased approximately 2.9-fold while TM increased about
2.6-fold compared to the vehicle control (Figure 3B),
suggesting both compounds inhibit SIRT2 deacetylase with
similar efficiency.
To test the cellular permeabilities, after treatment of TM-
P4-Thal and TM (50 µM) for 6 hours, MCF7 cells were
washed with cold PBS three times, and the small molecules
were extracted using methanol. The extracted samples
were analyzed using liquid chromatography-mass
spectrometry (LC-MS). Because these compounds with poor
aqueous solubility are not suitable for traditional PAMPA
assays, we have tried to measure the cellular permeability
in this way. Overall, TM-P4-Thal showed less cell
permeability, which could explain the lower cytotoxicity of
TM-P4-Thal than that of TM at high concentrations
(Supplementary Table 2). Thalidomide did not affect
cellular proliferation, suggesting that the cytotoxicity of TM-
P4-Thal did not originate from the thalidomide moiety
(Figure 4).
Previous work demonstrated that SIRT2 can efficiently
remove long chain fatty acyl groups from K182, 184, and
185 of K-Ras4a. SIRT2 knockdown consequently increased
K-Ras4a lysine fatty acylation.19 Furthermore, we also
found that TM could not inhibit SIRT2’s activity on K-Ras4a
lysine fatty acylation.29 Because TM-P4-Thal degrades
SIRT2, we expected that TM-P4-Thal could effectively
inhibit not only the deacetylase but also the defatty-acylase
activity of SIRT2.
To test this, we generated HEK 293T stably expressing
Flag-tagged K-Ras4a-G12V (Figure 3C). In these cells, 1 µM
of TM-P4-Thal treatment for 48 hours decreased SIRT2
level. Meanwhile, both SIRT1 and SIRT3 levels remained
unchanged upon treatment (Figure 3D). Next, to visualize K-
Ras4a fatty acylation levels, the cells were treated with 1 µM
of TM-P4-Thal or 10 µM of TM for 42 hours and then
incubated with Alkyne-14 (Alk-14) for additional 6 hours to
label fatty acylated proteins. After immunoprecipitating the
Flag-tagged K-Ras4a-G12V, attaching a fluorescent dye
through click chemistry and treating with NH2OH to remove
cysteine fatty acylation, we detected the lysine fatty
acylation level by in-gel fluorescence (Figure 3E, F). TM-P4-
Thal increased fluorescence significantly compared to the
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significance of SIRT2’s defatty-acylation activity.
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ASSOCIATED CONTENT
Supporting Information
Materials and methods, Supplementary Figure 1,2,3 and
Supplementary Table 1, 2. The Supporting Information is
available free of charge via the internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Email: hl379@cornell.edu.
Author Contributions
J.Y.H., and H.L. wrote the manuscript. All the authors have
given approval to the final version of the manuscript.
Funding Sources
The work is support in part by an NIH grant R01DK107868.
ACKNOWLEDGMENT
Figure 4. TM-P4-Thal shows improved cellular cytotoxicity
in breast cancer cell lines at lower concentrations. Cell
viability of MCF7 and MDA-MB-231 cells after treatment of TM,
TM-P4-Thal, and thalidomide for 72 hours.
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. This work had use of the
Cornell University NMR facility, which is supported, in part, by
the NSF through MRI award CHE-1531632. Proteomic data
was acquired by the Proteomic and Metabolomics Facility of
Cornell University, and was supported by HHMI
Transformative Technology 2019 program.
The stronger cytotoxicity of TM-P4-Thal may come from
the inhibition of SIRT2 defatty-acylase activity. As shown
earlier, at the same concentrations (5 and 10 µM), TM and
TM-P4-Thal inhibited SIRT2 deacetylase similarly (Figure
3A and 3B), while only TM-P4-Thal could inhibit SIRT2
defatty-acylase. As such, the inhibition of SIRT2 defatty-
acylase activity may have led to the stronger cytotoxicity of
TM-P4-Thal, suggesting that inhibiting the defatty-acylation
activity of SIRT2 could enhance the anticancer activity of
SIRT2 inhibitors. We note that it remains possible that the
stronger cytotoxicity of TM-P4-Thal may also come from the
inhibition of the non-enzymatic function of SIRT2. However,
given no such non-enzymatic function has been reported for
SIRT2, we think the stronger cytotoxicity of TM-P4-Thal is
likely due to the inhibition of SIRT2 defatty-acylase activity.
ABBREVIATIONS
PROTAC, Proteolysis-Targeting Chimera; NAD+, Nicotinamide
Adenine Dinucleotide; HIF1α, Hypoxia-Inducible Factor 1 α;
LDH-A, Lactate Dehydrogenase A; PROTAC, Proteolysis
Targeting Chimera; CRBN, Cereblon; PEG, Polyethylene Glycol;
UPS, Ubiquitin-Proteasome System; TMT, Tandem Mass Tag;
tert-Butyloxycarbonyl, Boc; Carboxybenzoyl, Cbz
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