Discovery of Dihydro-1,4-Benzoxazine Carboxamides as Potent and Highly Selective Inhibitors of Sirtuin-1

Article abstract of DOI:10.1021/acs.jmedchem.1c00017

Sirtuins are signaling hubs orchestrating the cellular response to various stressors with roles in all major civilization diseases. Sirtuins remove acyl groups from lysine residues of proteins, thereby controlling their activity, turnover, and localization. The seven human sirtuins, SirT1-7, are closely related in structure, hindering the development of specific inhibitors. Screening 170,000 compounds, we identify and optimize SirT1-specific benzoxazine inhibitors, Sosbo, which rival the efficiency and surpass the selectivity of selisistat (EX527). The compounds inhibit the deacetylation of p53 in cultured cells, demonstrating their ability to permeate biological membranes. Kinetic analysis of inhibition and docking studies reveal that the inhibitors bind to a complex of SirT1 and nicotinamide adenine dinucleotide, similar to selisistat. These new SirT1 inhibitors are valuable alternatives to selisistat in biochemical and cell biological studies. Their greater selectivity may allow the development of better targeted drugs to combat SirT1 activity in diseases such as cancer, Huntington's chorea, or anorexia.

Full text of DOI:10.1021/acs.jmedchem.1c00017

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Discovery of Dihydro-1,4-Benzoxazine Carboxamides as Potent and
Highly Selective Inhibitors of Sirtuin‑1
Martin Spinck,∥ Matthias Bischoff,∥ Philipp Lampe, Franz-Josef Meyer-Almes, Sonja Sievers,
and Heinz Neumann*
Cite This: J. Med. Chem. 2021, 64, 5838−5849
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ABSTRACT: Sirtuins are signaling hubs orchestrating the cellular
response to various stressors with roles in all major civilization
diseases. Sirtuins remove acyl groups from lysine residues of
proteins, thereby controlling their activity, turnover, and local-
ization. The seven human sirtuins, SirT1−7, are closely related in
structure, hindering the development of specific inhibitors.
Screening 170,000 compounds, we identify and optimize SirT1-
specific benzoxazine inhibitors, Sosbo, which rival the efficiency and
surpass the selectivity of selisistat (EX527). The compounds inhibit
the deacetylation of p53 in cultured cells, demonstrating their
ability to permeate biological membranes. Kinetic analysis of
inhibition and docking studies reveal that the inhibitors bind to a
complex of SirT1 and nicotinamide adenine dinucleotide, similar to
selisistat. These new SirT1 inhibitors are valuable alternatives to
selisistat in biochemical and cell biological studies. Their greater selectivity may allow the development of better targeted drugs to
combat SirT1 activity in diseases such as cancer, Huntington’s chorea, or anorexia.
intramolecular rearrangement and is eventually hydrolyzed,
releasing the deacetylated peptide and OAADPR.17 The
cleavage product NAM can rebind the enzyme and regenerate
NAD+ creating a negative feedback mechanism.18
Sirtuin-activating compounds promise to improve healthy
aging2 because increased levels of sirtuin activity have been
correlated with an increase in the lifespan of various
organisms.19−21 Inhibitors of SirT2 are being explored for
their benefits in the treatment of neurodegenerative disorders,
whereas SirT1 inhibitors may find applications in the treatment
of cancer, Huntington’s disease, or anorexia.22,23
The best in class inhibitor for SirT1, selisistat, is currently
tested in clinical trials for the treatment of Huntington’s
chorea.24 Selisistat inhibits SirT1 with two-digit nanomolar
IC50 and is approximately 100-fold less potent against closely
related sirtuins SirT2 and SirT3.22
Nevertheless, selisistat is routinely used in cells at micro-
molar concentration to inhibit SirT1, a level sufficient to affect
other sirtuins.25,26 Alternative scaffolds for the design of even
INTRODUCTION
■
Sirtuins are nicotinamide adenine dinucleotide (NAD+)-
dependent lysine deacylases conserved in all three domains
of life. Their biological importance is illustrated by the
multitude of physiological processes they contribute to.
Accordingly, there is a role for a sirtuin in nearly every
prominent disease, including neurodegeneration, diabetes,
arteriosclerosis, cancer, and aging.1 Drugs to modulate sirtuin
activity, both positively and negatively, are highly sought after.2
Particularly, SirT1, whose activity has been positively
correlated with longevity,3 has received much attention in
the past two decades. SirT1 substrates encompass, i.a.,
histones4 and transcription factors such as PGC1-α,5,6
FOXOs,7,8 and p53.9,10 By controlling their acetylation status,
SirT1 acts as a metabolic stress sensor that integrates
fluctuations in intracellular NAD+ concentrations11−13 and
signaling from upstream kinases, for example, protein kinase
A,14 into an adequate response.
Deacetylation of lysine residues by sirtuins involves the
stoichiometric cleavage of NAD+ into nicotinamide (NAM)
and O-acetyl-ADP-ribose (OAADPR).15 NAD+ is bound to a
canonical Rossman fold domain, and the NAM moiety is
accommodated in the C-pocket upon binding of the peptide
substrate.16 Cleavage of NAM leads to the formation of a
carbenium ion, which subsequently attacks the carbonyl-
oxygen of the acetyl group. The intermediate undergoes an
Received: January 6, 2021
Published: April 20, 2021
© 2021 The Authors. Published by
American Chemical Society
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Figure 1. Structures of compounds.
Figure 2. Inhibition of SirT1 by initial hits. (A) Inhibition of SirT1 by nine initial hit compounds (1 μM) and selisistat (1 μM) measured with
FLucK529ac and Fluor-de-Lys (B) SirT-Glo assays with 1 and 10 μM compounds. (C) In vitro deacetylation assay of acetylated histone proteins
(0.25 mg/mL) by purified SirT1 (100 nM) in the presence of indicated compounds (10 μM) or DMSO. Compound I was tested in a separate
experiment. (D) Dose-dependent inhibition of histone deacetylation by 4.3 (E) and selisistat. Uncropped images of the blots in Figure S1. (E)
Thermal denaturation of SirT1 (SYPRO orange, Tm: 41.5 °C, and 1 mg/mL) bound to different combinations of NAD+ (1 mM), H3K18ac histone
peptide (1 mM), and selisistat or 4.3 (0.1 mM each). Error bars represent the standard deviation of triplicate measurements.
more specific SirT1 inhibitors may therefore become valuable
as tools in basic research and for the development of improved
drugs.27 Here, we performed a high-throughput screening
(HTS) for inhibitors of recombinant human SirT1 using firefly
luciferase acetylated at active site lysine K529 (FLucK529ac)
as the substrate.28 Thereby, we identified and optimized a
promising new class of highly potent and specific SirT1
inhibitors with a benzoxazine scaffold. Because of their
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a
Scheme 1
a
(a) K2CO3, acetone, 75 °C, 22 h, and 67%; (b) LiOH, H2O/tetrahydrofuran (THF), RT, 1 h, and 93%; (c) NH4OH, EtOH, 80 °C, 20 h, and
75%; (d) RX, K2CO3, NaI, acetone, 65 °C or RCHO, NaBH(OAc)3, DCM, and RT; (e) RCOCl, NEt3, DCM, and 0 °C to RT; (f) RSO2Cl, NEt3,
dioxane, and 100 °C; (g) RNCO, NEt3, DCM, and 0 °C to RT.
pronounced selectivity for SirT1 over other sirtuins, we
decided to name this new inhibitor class Sosbo (for “sirtuin
one selective benzoxazine”).
Structure−Activity Relationship (SAR) Analysis of
Benzoxazine Compounds. We decided to focus our further
investigation and hit optimization on the benzoxazine scaffold
of 4.3. The general synthesis of the dihydro-1,4-benzoxazine
scaffold is outlined in Scheme 1.31,32 2-Aminophenol is
alkylated with ethyl 2,3-dibromopropanoate in the presence
of K2CO3 as a base in 67% yield. The ester 1 is either
hydrolyzed to the carboxylic acid 2 using LiOH or directly
transformed to the amide 3 by treatment with NH4OH in
ethanol in 75% yield.
Amide 3 serves as a platform for the introduction of various
substituents at the 4 position. Reaction with alkyl halides or
reductive amination with aldehydes led to amines 4.1−4.40,
reaction with acid chlorides led to amides 5.1−5.6, reaction
with sulfonyl chlorides led to sulfonamides 6.1−6.3, and
reaction with isocyanates led to ureas 7.1−7.3 (Table 1). First
of all, we resynthesized the hit compound 4.3 by alkylation of 3
with tert-butyl (4-(chloromethyl)thiazol-2-yl)carbamate, Boc-
deprotection, and acetylation. The activity of compound 4.3
could be confirmed, whereas the aminothiazole 4.2 and the N-
Boc aminothiazole 4.1 showed lower activities. Subsequently,
we replaced the thiazole moiety by various substituted
aromatic, as well as heteroaromatic rings. The presence of an
aromatic ring proved to be essential for activity, as hydrogen
(3), methyl (4.4), propargyl (4.17), or tert-butyl acetate
(4.20) in the 4 position showed no activity.
Electron-withdrawing functional groups, for example, nitro
(4.21 and 4.22) or cyano (4.27 and 4.28) in meta- and ortho-
positions, decreased the IC50 to 150−430 nM, whereas
increased bulk (e.g., 4.14, 4.18, 4.19, and 4.38) and
electron-donating groups (e.g., 4.23 and 4.29) reduced the
potency of the compounds. N-acetyl substitution in ortho-,
meta-, or para-positions (4.24, 4.25, and 4.26) did not reach
the activity of N-acetyl thiazole 4.3. Halogen substituents such
as chloride, bromide, and iodide or a methyl ester in the ortho-
position (4.32, 4.33, 4.34, and 4.39) showed all activity in the
submicromolar region. Interestingly, the introduction of two
trifluoromethyl groups turned 4.9 into a potent and quite
specific inhibitor of SirT2. The linkage between the
benzoxazine core and the aromatic substituent was also very
important. Changing the functional group from an amine to an
amide (5.1−5.6), a sulfonamide (6.1−6.3), or a urea (7.1−
RESULTS AND DISCUSSION
■
Identification of a New Class of SirT1 Inhibitors. Using
a recently developed sirtuin activity assay based on
FLucK529ac,28 we screened a library of approximately
170,000 compounds for inhibitors of SirT1. From the primary
screen, 2800 compounds were retested for SirT1 inhibition
with FLucK529ac and for direct inhibition of luciferase, which
narrowed down the list of candidate compounds to 135. Pan-
assay interference compound (PAINS) filtering29 and testing
against redox-cycling behavior30 further reduced the number to
115 initial hits. These were subsequently tested for their
selectivity toward SirT1 compared to SirT2 and SirT3 (Table
S1).
Nine initial hits were selected for further analysis based on
their potency and selectivity (Figure 1). With an IC50 value of
620 nM, dihydro-1,4-benzoxazine 4.3 (E) showed remarkable
potency to inhibit SirT1, while SirT2 and SirT3 were not
affected up to 60 μM. This inhibitory activity is only 13 times
lower and more selective than the gold standard of SirT1
inhibitors, selisistat,22 which we used as a reference compound.
Surprisingly, we did not detect any inhibitory activity of 4.3
using the Fluor-de-Lys assay (Figure 2A). We therefore used
an orthogonal SirT-Glo assay (Promega) to test the selected
set of compounds (Figure 2B and Table S2). This assay
depends on the release of luciferin from the C-terminus of a
peptide upon deacetylation. Again we observed SirT1
inhibition only by a subset of the compounds, suggesting
that inhibition by most of these compounds is substrate-
dependent.
To test the SirT1 inhibition on a physiological substrate, we
performed deacetylation reactions with isolated human
histones (Figure 2C). Only selisistat and 4.3 were able to
prevent deacetylation in a dose-dependent manner (Figure
2D). Thermal shift assays showed that the interaction of 4.3
with SirT1 depends on the presence of a substrate peptide and
is enhanced by NAD+ (Figure 2E). Selisistat stabilized SirT1
against heat denaturation similar to 4.3, indicating that they
might act by the same mechanism.
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Table 1. SAR Investigation of the Substituents R1 and R2 with IC50 Values Given as Arithmetic Means of Four Technical
Replicates
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Table 1. continued
a
Measured separately using an inhibitor concentration range of 0.003−60 μM.
a
Scheme 2
a
(a) BnBr, K2CO3, NaI, acetone, 65 °C, 18 h, and 76%; (b) LiOH, H2O/THF, RT, 18 h, and 94%; and (c) NR1R2, HATU, DIPEA, DMF, and RT.
7.3) completely abolished SirT1 inhibition. Eventually, we
probed the SAR of the carboxamide group in the 2-position of
the benzoxazine (Scheme 2).
To this end, ester 1 was N-alkylated with benzyl bromide
and K2CO3 as a base in 76% yield. The ester was hydrolyzed
using LiOH, and the resulting carboxylic acid was coupled with
methylamine, dimethylamine, and piperidine to afford the
amides 10.1, 10.2, and 10.3. Neither ester 8 or carboxylic acid
9 nor any of the secondary or tertiary amides showed any
activity, highlighting the importance of a primary amide group
(Table 1).
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Finally, we were interested in the different activities of the
enantiomers of the dihydro-1,4-benzoxazines. Therefore, we
chose compound 4.27 as the most active and selective one.
The enantiomers of 4.27 were separated on a chiral stationary
phase, and their activity against SirT1, SirT2, and SirT3 was
tested. Enantiomer-2 of 4.27 inhibited SirT1 with an IC50
value of 110 nM, about half the activity of the racemate,
whereas the IC50 of enantiomer-1 of 4.27 was approximately
400-fold higher. Neither of the enantiomers showed significant
activity against the other sirtuins up to 60 μM.
Benzoxazines Inhibit SirT1 in Cultured Human Cells.
SirT1 counteracts activation of p53 by reverting acetylation of
Lys-382.9,10 The acetylation stabilizes p53 against ubiquitin-
dependent proteasomal degradation in vivo. Inhibition of
SirT1 by selisistat blocks deacetylation of this residue and leads
to increased p53 levels in the presence of genotoxic
compounds such as etoposide.33 In contrast, the pan-specific
sirtuin inhibitor NAM does not enhance p53 acetylation in
cultured mammalian cells.33 We treated MDA-MB-231 cells, a
human breast adenocarcinoma cell line with intact p53, with
etoposide and increasing concentrations of selisistat, 4.22, or
4.27 (Figure 3). All three compounds induced a strong
increase in the level of p53 K382ac, indicating that the
benzoxazines are cell permeable and active in vivo.
indicating that they bind similar to selisistat to the extended C-
pocket.34
Molecular Docking of 3,4-Dihydro-2H-1,4-Benzoxa-
zines into SirT1. At first, the docking routine was validated by
redocking of the selisistat analogue into the crystal structure of
its complex with SirT1 (PDB-ID 4I5I).35 The docked and
crystallized poses of the ligand within the binding pocket of
SirT1 show excellent overlap with a root mean square
deviation (RMSD) of 0.408 Å (Figure S3). Thus, the applied
docking procedure with binding site definition including
structural water molecules and cofactor NAD+ is suitable for
providing reliable docking poses for SirT1-inhibitors. To
rationalize the inhibitory activity of 3,4-dihydro-2H-1,4-
benzoxazines, the structures were docked into the crystal
structure of SirT1.
When looking at the binding mode of the selisistat analogue
and the cofactor NAD+ within the active site of SirT1, it
becomes obvious that several structural water molecules play
important roles for binding of the selisistat analogue to amino
acids of the binding pocket and cofactor NAD+ (Figure 5).
Therefore, the usage of NAD+ and structural water molecules
as the integral part of the binding pocket appears perfectly
justified. Docking of 21 benzoxazine analogues shows similar
trends for the GBVI/WSA dG docking score of MOE and
pIC50-values of the compounds with a Pearson correlation
coefficient of r = −0.57 (Table S3 and Figure S4). In addition,
the docking poses of the benzoxazine core of most potent
compounds (IC50-values <5 μM) show excellent overlap, while
larger variations are observed for the substituents in the 4-
position (Figure 5B and Figure S5).
The benzoxazine core snuggles perfectly into the binding
pocket of SirT1 showing considerable overlap with the
selisistat analogue in the crystal structure of SirT1 (Figure
5A). Moreover, the selisistat analogue, as well as the 3,4-
dihydro-2H-1,4-benzoxazines, binds very similarly to SirT1 by
direct hydrogen bonds between an amide nitrogen and the side
chain carboxyl group of D348, as well as between I347 and
different ligand atoms and indirectly by hydrogen bonds
through structural water molecule 702 to the backbone
carbonyl oxygens of A262 and P271 (Figure 5A and Figure
S6).
Figure 3. Benzoxazines inhibit SirT1 in vivo. Etoposide (50 μM for
20 h) preincubated MDA-MB-231 cells were treated for 3 h with
DMSO, NAM (2 mM), selisistat, 4.22, or 4.27 (0.5, 1.25, or 5 μM).
Acetylation of p53 K382 was subsequently measured by western
blotting using modification-specific antibodies (biological replicates of
the experiment are shown in Figure S2).
In addition, selisistat, as well as the benzoxazine core, forms
hydrogen bonds to another structural water 717, which is
connected with two different oxygen atoms of cofactor NAD+
by further hydrogen bonds (Figure 5A). Because of the perfect
and reproducible steric fit of the benzoxazine core into the
binding pocket, the positions of both bridging water molecules,
702 and 717, are also highly conserved. In addition and very
similar to the selisistat analogue, the benzoxazine core shows
Pi-Pi interactions with F273 and F297 (Figure S6).
In contrast to the benzoxazine core, the substituents in the
4-position show a considerable shift. However, it is noteworthy
that many analogues are still able to form hydrogen bonds to
structural water molecule 732, although with larger positional
variations (Figure 5B and Figure S5). This water molecule is
also fixed to SirT1 by hydrogen bonds to Y317 and Q320. The
substituents of the benzoxazine analogues are accommodated
in the upper region of the binding pocket of SirT1 close to the
entrance (Figure S7).
Benzoxazines Are Uncompetitive Inhibitors of SirT1.
Selisistat is an uncompetitive inhibitor with respect to
NAD+.22,34 The partial structural similarity of the new SirT1
inhibitors to selisistat prompted us to analyze their mode of
inhibition. Therefore, we measured apparent Vmax and KM
values for selisistat, 4.22, and 4.27 in dependence of the NAD+
concentration (Figure 4A). In all three cases, we observed a
reduction of Vmax and KM characteristic of uncompetitive
inhibition. Hence, selisistat and benzoxazines may share a
common mechanism of inhibition. Next, we tested binding of
the inhibitors to SirT1−3 in dependence of substrates by
thermal shift assays (Figure 4B). None of the inhibitors
showed any stabilization of SirT2 or SirT3 at a concentration
of 90 μM, demonstrating their pronounced specificity.
Selisistat stabilized SirT1 only in the presence of NAD+, in
line with it being part of the binding pocket of the inhibitor.35
The additional presence of substrate peptide (H3K18ac)
further stabilized the complex, suggesting that SirT1 is able to
simultaneously bind selisistat and both substrates. Compounds
4.3 and 4.27 showed the same pattern of SirT1 stabilization,
Interestingly, the access to the active site is rather restricted
because of a very narrow entrance area according to the crystal
structure of SirT135 (PDB-ID 4I5I, Figure S7). Obviously, the
adjacent region of the upper part of the binding pocket has to
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Figure 4. Kinetic characterization of SirT1 inhibition by 4.22 and 4.27. (A) Michaelis−Menten kinetics of selisistat, 4.22, and 4.27 measured using
the continuous FLucK529ac assay. KM, Ki, Vmax, and error range were determined for different inhibitor concentrations by fitting the data using the
Michaelis−Menten model of GraphPad Prism 8. (B) Thermal denaturation of SirT1−3 bound to different combinations of NAD+, acetylated
histone peptide (H3K18ac for SirT1/3 and H4K16ac for SirT2) and selisistat, 4.27, or 4.3.
be rather flexible to enable the access of compounds such as
selisistat or 4.27. This expected behavior is consistent with
larger B-factors particularly in the loop between I316 and S324
and the docking poses of 3,4-dihydro-2H-1,4-benzoxazine
analogues with a broader range of topologies for the
substituents in the 4-position, as well as the pronounced
adaptability of structural water 732, which is attached to Y317
and Q320 in the putative flexible loop.
Altogether, the 3,4-dihydro-2H-1,4-benzoxazines and selisi-
stat show large structural overlap and very similar molecular
interactions. The cofactor NAD+ and three structural water
molecules (702, 717, and 732), as well as Pi-Pi interactions
with F273 and F297, are essential for the molecular
recognition of 3,4-dihydro-2H-1,4-benzoxazine analogues.
Finally, we compared the binding properties of the two
enantiomers of 4.27 (Figure S8). The docking score of the R-
enantiomer (−6.7) is significantly poorer than that of the S-
enantiomer (−7.8). Although the benzoxazine core of both
enantiomers shows significant overlap, the constraints of the
binding pocket and the different configurations at the chiral C-
atom enforce a considerable distortion of the oxazine ring in
the R-enantiomer of 4.27. This results in the formation of only
one hydrogen bond to D348 for the R-enantiomer of 4.27,
while the S-enantiomer shows three hydrogen bonds to D348
and structural water molecules 717 and 732. Therefore, we
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Figure 5. Molecular docking of 3,4-dihydro-2H-1,4-benzoxazines into SirT1. (A) Overlay of the crystallized selisistat analogue (green ball and
stick) and docked 4.27 (magenta ball and stick) within the binding pocket of SirT1 (PDB-ID 4I5I). Amino acids that are important for the
recognition of the ligand and cofactor are shown and colored (green: crystal structure and magenta: docked and energy minimized complex). The
elongated lower active site pocket is occupied by cofactor NAD+. The two slightly different conformations of NAD+ and adjacent amino acids result
from energy minimization of the docking result. Structural water molecules are displayed as solid beads and annotated according to the
corresponding PDB data set. Hydrogen bonds are shown as dark green dotted lines. Additional hydrophobic interactions are not shown for the sake
of clarity. (B) Overlay of docking poses of most potent SirT1 inhibitors. Best compounds, 4.21, 4.22, and 4.27, and corresponding structural water
molecules are colored in dark brown, magenta, and green, respectively. The positions of structural water molecules in different docking poses are
̈
highlighted by red dotted circles. Water molecules are shown as beads and numbered with a leading W. Distances in Ångstrom between heavy
atoms of hydrogen bonds are indicated as dark green numbers.
suggest that 4.27 ent-2, which is the more potent SirT1
inhibitor, is the S-enantiomer.
Hence, Sosbo inhibitors will be a valuable tool to investigate
the cellular role of SirT1. This is important to identify potential
side effects of selisistat.
CONCLUSIONS
The mode of SirT1 binding by Sosbo inhibitors is likely to
be similar to that of selisistat. Both act as uncompetitive
inhibitors that bind to the enzyme−substrate complex. The
docking studies suggest that Sosbo compounds are extended
C-site inhibitors.34 Structural studies on the SirT1/Sosbo
complex are essential for their further optimization.
■
Using a new lysine deacetylase assay based on FLucK529ac, we
identified a new class of compounds with excellent inhibitory
properties for SirT1. The traditional Fluor-de-Lys assay would
not have identified the initial hit (Figure 2A), emphasizing the
benefit of orthogonal assays in compound screening. The SAR
analysis revealed critical functionalities of the inhibitor and
allowed us to improve their activity.
Our initial studies with cultured human cells proved their
ability to pass the plasma membrane and to act on cellular
targets. At the same time, we observed no apparent toxicity at
concentrations sufficient to inhibit deacetylation of p53.
EXPERIMENTAL SECTION
■
Plasmids and Strains. Strains and plasmids used in this study are
listed in Table S4.
Expression and Purification of the SirT1, SirT2, and SirT3
Catalytic Domain. BL21(DE3) was transformed with the pBK-His6-
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SirT2/3 (50 μg/mL kanamycin, kan) or pCDF-His6-SirT1 (50 μg/
mL spectinomycin, spec) plasmid. A 10 mL LB (50 μg/mL kan or
spec) overnight culture was incubated at 37 °C with shaking 200 rpm.
One liter of culture (LB, 50 μg/mL kan or spec) was inoculated with
the overnight culture and grown at 37 °C and 200 rpm to an OD600 of
0.8 (SirT1) or 0.3 (SirT2/3). Expression was induced by addition of
0.5 mM IPTG (SirT1) or 0.2% arabinose (SirT2/3) and culture-
shifted to 18 °C (SirT1) or 30 °C (SirT2/3). The cells were
harvested by centrifugation, and the pellet was washed with PBS and
stored at −20 °C. For purification, the cell pellet was thawed on ice
and resuspended in sirtuin wash buffer (20 mM Tris pH 8, 200 mM
NaCl, 20 mM imidazole, and 1 mM DTT) supplemented with 0.2
mM PMSF, lysozyme (0.5 mg/mL), and DNAse (∼1 mg). The
digested cells were lysed by two passages through a pneumatic cell
disintegrator. The lysate was cleared by centrifugation (20 min,
20.000 rpm, and 4 °C), and 500 μL Ni2+-NTA resin was added to the
supernatant. The suspension was agitated for 1 h (SirT1/3) or 24 h
(SirT2) at 4 °C after which the beads were collected by passing the
suspension over a plastic column with a frit. The beads were washed
with 40 mL of sirtuin wash buffer, and the protein was eluted with 4
mL of sirtuin wash buffer supplemented with 200 mM imidazole. The
buffer was exchanged for sirtuin gel filtration buffer (20 mM Tris pH
8, 50 mM NaCl, and 10 mM DTT) before the concentrated protein
was applied to an HILoad 26/60 Superdex 200 size-exclusion
chromatography column (GE healthcare) equilibrated with gel
filtration buffer. By monitoring the UV absorption at 280 nm,
fractions containing protein were identified and analyzed by sodium
dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The
pure fractions were concentrated, aliquoted, and flash-frozen in liquid
nitrogen for storage at −80 °C.
FLucK529ac Lysine Deacetylase Assay. FLucK529ac was
produced by genetic code expansion to install acetyllysine at the
active site residue K529 of Firefly Luciferase as described previously.28
Deacetylase activities were measured in continuous format. Briefly,
the reaction mixture was prepared in triplicate in a 96-well plate each
well containing 100 nM SirT1, diluted FLucK529ac (ca. 270 nM),
and 100−1600 nM sirtuin inhibitor in a final volume of 40 μL sirtuin
buffer 2 (25 mM Tris/HCl pH 8.0, 137 mM NaCl, 2.7 mM KCl, 1
mM MgCl2, and 1 mM GSH). Fifty microliters of 2× luciferase buffer
(40 mM tricine, 200 μM EDTA, 7.4 mM MgSO4, 2 mM NaHCO3, 34
mM DTT, 0.5 mM ATP, and 0.5 mM luciferin and pH 7.8) were
added before the reaction was started by addition 10 μL of 10-fold
concentrated NAD+ (0−5000 μM). The reaction mixture was
preincubated for 2 min at room temperature before the luminescence
of each well was recorded over 30 min at room temperature. The
KDAC activity was calculated from the slope of the linear reaction
phase. The kinetic parameters and error ranges were retrieved by
fitting the data using the GraphPad Prism 8 (v8.4.3) standard
Michaelis−Menten function with the preset parameters.
sirtuin buffer containing 25 mM Tris/HCl pH 8.0, 137 NaCl, 2.7 mM
KCl, 1 mM MgCl2, and 1 mM GSH were added and incubated for 15
min at RT. Four microliters of 2 mM NAD+ in sirtuin buffer were
added to start the reaction. After 30 min at RT, 8 μL of 2x luciferase
buffer were added to detect luciferase activity. After 10 min,
luminescence was detected with a SpectraMax Paradigm plate reader.
Fluor-de-Lys Assay. The reaction composition and conditions
used were identical to conditions used in the FLucK529ac lysine
deacetylase assay with the luciferase replaced by 10 μM Fluor-de-Lys
peptide (Ac-Gly-Gly-Lys(ac)-AMC). All inhibitors were used at a
concentration of 1 μM. The reaction was stopped by addition of an
equal volume of 2× trypsin solution (0.1 mg/mL trypsin in sirtuin
buffer 2). The fluorescence of the liberated coumarin (ex. 355 nm,
em. 460 nm) was quantified after 5 min at RT using a FluoStar
Omega Microplate Reader (BMG Labtech).
SirT-Glo Assay. Compounds were dispensed with the highest
assay concentration of 10 μM in eight dilution steps and a factor of 3
into dry white 384-well plates (Corning 4513). Eight microliters of 15
nM SirT1 in 25 mM Tris/HCl pH 8.0, 137 mM NaCl, 2.7 mM KCl, 1
mM MgCl2, 1 mM GSH, and 1 mM NAD+ were added and incubated
for 45 min at RT. Eight microliters of Glo-buffer with substrate and
developer were added according to the manufacturer’s protocol
(Promega). After 10 min, luminescence was detected with a
SpectraMax Paradigm plate reader.
ADP-Glo Control Assay (for Direct FLuc Inhibition). Compounds
were dispensed as above. Eight microliters of kinase detection buffer
with a substrate was added followed by incubation for 45 min at RT.
Eight microliters of sirtuin buffer with 25 μM ADP and 1 mM NAD+
was added, and after 10 min, luminescence was detected with a
SpectraMax Paradigm plate reader.
Histone Deacetylation Assay. The deacetylation reactions were
set up on ice in a 30 μL volume of sirtuin buffer 2 containing 250 μg/
mL acid extracted histones,36 100 nM SirT1, 1 mM NAD+, and
varying concentration of inhibitors (10 mM stock solutions in
DMSO). The reaction mixture was incubated for 30 min at 30 °C,
and the reaction was stopped by addition of 10 μL 4× SDS-sample
buffer. Ten microliters of the deacylation reactions were separated on
an 18% SDS-PAGE and transferred to a PVDF membrane, and
histone acetylation was detected using pan-specific acetyl-lysine
antibody (Pan-AcK, PTM Biolabs, #105, 5% BSA/TBST 1:2000).
SirT1 Inhibitor Screening. HTS was carried out using the
compound library of the Compound Management and Screening
Center of the Max Planck Society, including ca. 170,000 commercial
and proprietary compounds. Primary screening was carried out in
1536 well format at 12.5 μM yielding 4760 primary hits with residual
SirT1 activity below 50%. After filtering for compound purity, 2800
compounds were retested for SirT1 activity in FLucK529ac lysine
deacetylase and luciferase assays in quadruplicate. Eight hundred
forty-two compounds were confirmed for SirT1 activity (mean
residual activity of <50%) while after filtering for luciferase inhibition
135 hits remained. After additional PAINS filtering and testing against
redox-cycling behavior, 115 hits remained, which were selectively
profiled for SirT1, 2, and 3 in dose-response mode.
Redox-Cycling Compound Method. To exclude compounds
that inactivate SirT1 through redox-cycling behavior, redox-cycling
assays were carried out according to Tarnowski et al. with slight
modifications.30 Compounds were dispensed into dry black 384-well
plates (Corning 4514). For the resazurin assay, 5 μM resazurin
solution in 25 mM Tris/HCl pH 8.0, 137 mM NaCl, 2.7 mM KCl, 1
mM MgCl2, 1 mM GSH, 3 μM DTT, and 1 mM NAD+ was added.
For the Amplex Ultrared assay, 25 μM Amplex Ultrared, 0.001 U/mL
horseradish peroxidase, and 25 U/mL superoxide dismutase solution
in 25 mM Tris/HCl pH 8.0, 137 mM NaCl, 2.7 mM KCl, 1 mM
MgCl2, 1 mM GSH, 3 μM DTT, and 1 mM NAD+ were added. After
30 min, fluorescence was detected with a Paradigm Spectramax reader
(EX535/EM595).
HTS Format. Compounds were dispensed into dry white 1536-well
plates (Corning 3729). Two microliters of a mixture of 30 nM Sirt1
and diluted firefly luciferase (ca. 90 nM; 1:3000) FLucK529ac in 25
mM Tris/HCl pH 8.0, 137 mM NaCl, 2.7 mM KCl, and 1 mM
MgCl2 were added and incubated for 15 min at RT. Two microliters
of 2 mM NAD+ were added to start the reaction. After 30 min, 4 μL
of 2× luciferase buffer were added to detect luciferase activity. After
10 min, luminescence was read with an Envision reader
(PerkinElmer).
Luciferase Counter-Assay. Compounds were dispensed into dry
white 384-well plates (Corning 4513). Eight microliters of 0.5 nM
firefly luciferase (Sigma, #L9506) in 25 mM Tris/HCl pH 8.0, 137
mM NaCl, 2.7 mM KCl, 1 mM GSH, 3 μM DTT, and 1 mM NAD+
were added and incubated for 30 min at RT. Eight microliters of
luciferin solution were added to start the reaction. After 30 min,
luminescence was read with a Paradigm Spectramax reader.
IC50 Measurements. Compounds were dispensed with the highest
assay concentration of 60 μM in 8 or 12 dilution steps as indicated
and a factor of 3 into dry white 384-well plates (Corning 4513). Four
microliters of either 30 nM SirT1 or 47 nM SirT2 or 103 nM SirT3
and diluted firefly luciferase FLucK529ac (ca. 90 nM; 1:3000) in
PAINS Filtering. The PAINS filtering was performed computa-
tionally according to Baell et al.,29 and the structural features were
taken in SMARTS encoding from Saubern et al.37 In addition,
structural features described by Dahlin et al.38 were manually
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transformed into SMARTS and also removed. The PAINS filter was
implemented as a Pipeline Pilot protocol that takes a list of
compounds as input, filters out PAINS-containing structures by
SMARTS-based substructure matching, and outputs only the PAINS-
free compounds.
Chemical Synthesis and Characterization. An overview of the
synthetic strategy of Sosbo compounds is shown in Schemes 1 and 2.
Details on synthetic procedures and chemical characterization can be
found in the Supporting Information. All test compounds were greater
than 95% pure as determined using an Agilent 1100 Series HPLC
equipped with a EC50/3 Nucleodur C18 Gravity column 1.8 μm
from Macherey Nagel.
GloMelt Thermal Shift Assay. SirT1 or SirT3 (8 μM) was added
into 384-well plates (LightCycler 480 Multiwell Plate 384, white) in
buffer (25 mM Tris/HCl pH 8.0, 137 mM NaCl, 2.7 mM KCl, and 1
mM GSH) with 1 mM NAD+ and 0.25 mM histone peptide H3K18ac
as indicated. SirT2 (16 μM) was added into 384-well plates
(Lightcycler 480,384-well plate, white) in buffer (25 mM Tris/HCl
pH 8.0, 137 mM NaCl, 2.7 mM KCl, and 1 mM GSH) with 1 mM
NAD+ and 0.5 mM histone peptide H4K16ac as indicated. Selisistat
(30 μM) or 4.27/4.3 (90 μM) was added followed by incubation for
20 min at RT. Upon addition of GloMelt dye (Biotium) into buffer
(25 mM Tris/HCl pH 8.0, 137 mM NaCl, 2.7 mM KCl, and 1 mM
GSH), thermal shift assays were run with a LightCycler 480 II
(ROCHE) according to the manufacturer’s protocol. Protein melting
curves were analyzed, and ΔTm was calculated with LightCycler
Thermal Shift Analysis software (ROCHE).
(PDB-ID 4I5I) was obtained from the RCSB Protein Data Bank and
subjected to the Quickprep procedure of MOE 2019 including 3D-
protonation for subsequent docking. The partial charges of all protein
and ligand atoms were calculated using the implemented Amber14
force field. The docking site was defined by the surrounding amino
acids and structural water molecules within a radius of 5 Å. Structural
water molecules are characterized by limited mobility and therefore
resolvable by X-ray crystallography, which is caused by at least two or
three hydrogen bonds to adjacent amino acids and bound ligands.
Cofactor NAD+ is also included in the definition of the binding
pocket because it is connected to the selisistat analogue inhibitor by
conventional hydrogen bonds via structural water molecule 717 in the
crystal structure of the SirT1 (PDB-ID 4I5I). Molecular docking was
performed choosing the triangle matcher for placement of the ligand
in the binding site and ranked with the London dG scoring function.
The best 30 poses were passed to refinement and energy minimization
in the pocket using the induced fit method, and the 10 best poses
were rescored using the GBVI/WSA dG scoring function. Chimera
from the University of California UCSF was used to calculate the
RMSD value between crystallized and redocked SirT1 ligand (PDB-
ID 4I5I).
ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00017.
Full blots, replicate experiments, redocking of the
selisistat analogue ligand, correlation of docking scores,
overlay of docking poses, 2D interactions, docking poses
of benzoxazine analogues, overlay of enantiomers,
GBVI/WSA dG docking score, strains and plasmids,
and detailed chemical procedures and characterization of
compounds (PDF)
Table S1: Selectivity of initial hits toward SirT1
compared to SirT2 and SirT3 (XLSX)
Table S2: Orthogonal SirT-Glo assay results (XLSX)
Molecular formula strings (CSV)
SYPRO Orange Thermal Shift Assay. All tested conditions were
prepared in triplicate with each replicate containing 1 mg/mL purified
SirT1,
GKAPRK[Ac]QLATK-COOH),
1 mM NAD+,
1 mM H3K18ac peptide (NH2-
0.1 mM selisistat or 4.3, and
10× SYPRO Orange in 20 μL sirtuin buffer 2. The change in
fluorescence was measured in a rtPCR cycler using the following
program: 20 °C 10 min, 360× [20 °C, 10 s, FRET Mode, +0.2 °C/
Cycle], hold 8 °C. The experimental data were fitted with SciDavis
(version 1.22) using a Boltzmann equation, determining the Tm of
each replicate.
Treatment of MDA-MB-231 Cells with SirT1 Inhibitors and
Western Blotting Analysis. MDA-MB-231 cells were grown in
RPMI 1640 medium supplemented with GlutaMAX (Gibco), 10%
FBS (Sigma-Aldrich), and 100 U/mL streptomycin−penicillin
(Gibco). For treatment, 2 × 105 cells were seeded per well in a six-
well plate containing 2 mL growth medium. The next day, 50 μM
etoposide was added to induce p53 acetylation, and after 20 h, the
medium was exchanged against 2 mL growth medium containing
DMSO (2 μL), NAM (2 mM, in water), or inhibitor (1000× stock
solution in DMSO). The medium was removed after 3 h, and the cells
were washed with 1× PBS. The cells were lysed inside the wells by
addition of 200 μL 231 lysis buffer (10 mM Tris−HCl pH 7.4, 150
mM NaCl, 15% glycerol, 1% Triton-X-100, 1× Pierce Protease
Inhibitor Tablets (Thermo Scientific), and 0.05 mg/mL DNase I).
The lysis was incubated for 20 min on ice. SDS was added to a
concentration of 1% and incubated for another 10 min at room
temperature while being agitated to solubilize all proteins. The lysate
was collected and placed in a sonication bath for 10 min at RT to
reduce its viscosity and then centrifuged for 10 min at 18,000 rpm.
The supernatant (160 μL) was mixed with 40 μL 5× SDS-sample
buffer, and 45 μL of each sample were separated on a 4−12% Bis-Tris
NuPAGE Bolt gel (Novex). The immunoblot analysis was carried out
using specific antibodies for p53 (AP6266b, abcepta, and 1:1000 in
3% BSA/PBS 0.1% Tween20), p53 K382Ac (#2525, Cell Signaling,
1:1000 in 5% BSA/TBS 0.1% Tween20), and H3 (ab1791, Abcam,
1:3000 in 3% BSA/TBS). The signal was developed using the
antirabbit-HRP secondary antibody (A6154, Sigma-Aldrich, 1:10000
in 5% Milk/TBS) and ECL Select (GE Healthcare) western blotting
detection agent.
An overview of Z′-factors for the high-throughput screen
(XLSX)
Docking pose (PDB-Sosbo_4.2) (PDB)
Docking pose (PDB-Sosbo_4.3) (PDB)
Docking pose (PDB-Sosbo_4.6) (PDB)
Docking pose (PDB-Sosbo_4.7) (PDB)
Docking pose (PDB-Sosbo_4.8) (PDB)
Docking pose (PDB-Sosbo_4.10) (PDB)
Docking pose (PDB-Sosbo_4.16) (PDB)
Docking pose (PDB-Sosbo_4.21) (PDB)
Docking pose (PDB-Sosbo_4.22) (PDB)
Docking pose (PDB-Sosbo_4.23) (PDB)
Docking pose (PDB-Sosbo_4.27) (PDB)
AUTHOR INFORMATION
Corresponding Author
■
Heinz Neumann − Department of Structural Biochemistry,
Max Planck Institute of Molecular Physiology, Dortmund
44227, Germany; Department of Chemical Engineering and
Biotechnology, University of Applied Sciences Darmstadt,
Darmstadt 64295, Germany; orcid.org/0000-0001-
9657-1913; Phone: +496151.16-38203;
Email: heinz.neumann@h-da.de
Authors
Molecular Docking. Modeling, preparation, and visualization of
structural data as well as molecular docking were performed using
MOE 2019 software (Chemical Computing Group ULC, Canada).
The crystal structure of SirT1 in complex with a selisistat analogue
Martin Spinck − Department of Structural Biochemistry, Max
Planck Institute of Molecular Physiology, Dortmund 44227,
Germany
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Article
Matthias Bischoff − Compound Management and Screening
Center, Dortmund, Dortmund 44227, Germany
Philipp Lampe − Compound Management and Screening
Center, Dortmund, Dortmund 44227, Germany
Franz-Josef Meyer-Almes − Department of Chemical
Engineering and Biotechnology, University of Applied Sciences
Darmstadt, Darmstadt 64295, Germany; orcid.org/
0000-0002-1001-3249
REFERENCES
■
(1) Imai, S. I.; Guarente, L. NAD+ and sirtuins in aging and disease.
Trends Cell Biol. 2014, 24, 464−471.
(2) Blum, C. A.; Ellis, J. L.; Loh, C.; Ng, P. Y.; Perni, R. B.; Stein, R.
L. Sirt1 modulation as a novel approach to the treatment of diseases
of aging. J. Med. Chem. 2011, 54, 417−432.
(3) Cantó, C.; Auwerx, J. Caloric restriction, SIRT1 and longevity.
Trends Endocrinol. Metab. 2009, 20, 325−331.
(4) Vaquero, A.; Scher, M.; Lee, D.; Erdjument-Bromage, H.;
Tempst, P.; Reinberg, D. Human SirT1 interacts with histone H1 and
promotes formation of facultative heterochromatin. Mol. Cell 2004,
16, 93−105.
Sonja Sievers − Compound Management and Screening
Center, Dortmund, Dortmund 44227, Germany
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.1c00017
(5) Nemoto, S.; Fergusson, M. M.; Finkel, T. SIRT1 functionally
interacts with the metabolic regulator and transcriptional coactivator
PGC-1α. J. Biol. Chem. 2005, 280, 16456−16460.
Author Contributions
∥M.S. and M.B. contributed equally.
(6) Rodgers, J. T.; Lerin, C.; Haas, W.; Gygi, S. P.; Spiegelman, B.
M.; Puigserver, P. Nutrient control of glucose homeostasis through a
complex of PGC-1α and SIRT1. Nature 2005, 434, 113−118.
(7) Brunet, A.; Sweeney, L. B.; Sturgill, J. F.; Chua, K. F.; Greer, P.
L.; Lin, Y.; Tran, H.; Ross, S. E.; Mostoslavsy, R.; Cohen, H. Y.; Hu,
L. S.; Cheng, H. L.; Jedrychowski, M. P.; Gygi, S. P.; Sinclair, D. A.;
Alt, F. W.; Greenberg, M. E. Stress-Dependent Regulation of FOXO
Transcription Factors by the SIRT1 Deacetylase. Science 2004, 303,
2011−2015.
Author Contributions
Conceptualization, H.N. and S.S.; methodology, M.S., M.B.,
and P.L., investigation, M.S., M.B., P.L., F.-J.M.-A., and H.N.;
data curation, S.S.; writing − original draft, F.-J.M.-A. and
H.N.; writing − review and editing, all authors; supervision,
H.N. and S.S.; project administration, H.N. and S.S.; and
funding acquisition, H.N.
(8) Motta, M. C.; Divecha, N.; Lemieux, M.; Kamel, C.; Chen, D.;
Gu, W.; Bultsma, Y.; McBurney, M.; Guarente, L. Mammalian SIRT1
represses Forkhead transcription factors. Cell 2004, 116, 551−563.
(9) Luo, J.; Nikolaev, A. Y.; Imai, S. I.; Chen, D.; Su, F.; Shiloh, A.;
Guarente, L.; Gu, W. Negative control of p53 by Sir2α promotes cell
survival under stress. Cell 2001, 107, 137−148.
(10) Vaziri, H.; Dessain, S. K.; Eaton, E. N.; Imai, S.-I.; Frye, R. A.;
Pandita, T. K.; Guarente, L.; Weinberg, R. A. HSIR2 SIRT1 functions
as an NAD-dependent p53 deacetylase. Cell 2001, 107, 149−159.
(11) Cantó, C.; Gerhart-Hines, Z.; Feige, J. N.; Lagouge, M.;
Noriega, L.; Milne, J. C.; Elliott, P. J.; Puigserver, P.; Auwerx, J.
AMPK regulates energy expenditure by modulating NAD+ metabo-
lism and SIRT1 activity. Nature 2009, 458, 1056−1060.
(12) Revollo, J. R.; Grimm, A. A.; Imai, S. I. The NAD biosynthesis
pathway mediated by nicotinamide phosphoribosyltransferase regu-
lates Sir2 activity in mammalian cells. J. Biol. Chem. 2004, 279,
50754−50763.
(13) Fulco, M.; Cen, Y.; Zhao, P.; Hoffman, E. P.; McBurney, M.
W.; Sauve, A. A.; Sartorelli, V. Glucose restriction inhibits skeletal
myoblast differentiation by activating SIRT1 through AMPK-
mediated regulation of Nampt. Dev. Cell 2008, 14, 661−673.
(14) Gerhart-Hines, Z.; Dominy, J. E.; Blättler, S. M.; Jedrychowski,
M. P.; Banks, A. S.; Lim, J. H.; Chim, H.; Gygi, S. P.; Puigserver, P.
The cAMP/PKA pathway rapidly activates SIRT1 to promote fatty
acid oxidation independently of changes in NAD+. Mol. Cell 2011, 44,
851−863.
(15) Borra, M. T.; O’Neill, F. J.; Jackson, M. D.; Marshall, B.;
Verdin, E.; Foltz, K. R.; Denu, J. M. Conserved enzymatic production
and biological effect of O-acetyl-ADP-ribose by silent information
regulator 2-like NAD+-dependent deacetylases. J. Biol. Chem. 2002,
277, 12632−12641.
(16) Zhao, K.; Harshaw, R.; Chai, X.; Marmorstein, R. Structural
basis for nicotinamide cleavage and ADP-ribose transfer by NAD
+-dependent Sir2 histone/protein deacetylases. Proc. Natl. Acad. Sci.
U. S. A. 2004, 101, 8563−8568.
(17) Tanner, K. G.; Landry, J.; Sternglanz, R.; Denu, J. M. Silent
information regulator 2 family of NAD-dependent histone/protein
deacetylases generates a unique product, 1-O-acetyl-ADP-ribose. Proc.
Natl. Acad. Sci. U. S. A. 2000, 97, 14178−14182.
(18) Sauve, A. A.; Schramm, V. L. Sir2 regulation by nicotinamide
results from switching between base exchange and deacetylation
chemistry. Biochemistry 2003, 42, 9249−9256.
(19) Tissenbaum, H. A.; Guarente, L. Increased dosage of a sir-2
gene extends lifespan in Caenorhabditis elegans. Nature 2001, 410,
227−230.
Notes
The authors declare the following competing financial
interest(s): Heinz Neumann and Martin Spinck hold a patent
on the lysine deacetylase assay used in this manuscript.
ACKNOWLEDGMENTS
■
We thank Petra Geue for technical assistance and Sascha Gentz
for peptide synthesis. This project was financially supported by
the Behrens-Weise Foundation and the Max-Planck-Institute
of Molecular Physiology.
ABBREVIATIONS
■
AMC, 7-amino-4-methylcoumarin; ATP, adenosine triphos-
phate; Boc, benzyloxycarbonyl; DCM, dichloromethane;
DIPEA, N,N-Diisopropylethylamine; DMF, dimethylforma-
mide; DMSO, dimethyl sulfoxide; DNAse, desoxyribonuclease;
DTT, dithiothreitol; DTT, dithiothreitol; EDTA, ethyl-
enediaminetetraacetic acid; EtOH, ethanol; FBS, fetal bovine
serum; FLuc, Firefly Luciferase; FOXO, Forkhead box protein
O; FRET, fluorescence resonance energy transfer; GSH,
glutathione; H3K18ac, histone H3 K18 acetylation;
H4K16ac, histone H4 K16 acetylation; HATU, 1-[Bis-
(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]-
pyridinium 3-oxide hexafluorophosphate; HTS, high-through-
put screening; i.a., inter alia; IC50, half-maximal inhibitory
concentration; KDAC, lysine deacetylase; KM, Michaelis−
Menten constant; NAD+, nicotinamide adenine dinucleotide;
NAM, nicotinamide; NTA, nitrilotriacetic acid; OAADPR, O-
acetyl-ADP-ribose; PAINS, pan-assay interference compounds;
PBS, phosphate buffered saline; PGC1-α, eroxisome prolifer-
ator-activated receptor gamma coactivator 1-alpha; PKA,
protein kinase A; PMSF, phenylmethylsulfonyl fluoride;
PVDF, polyvinylidene difluoride; RMSD, root mean square
deviation; RT, room temperature; rtPCR, real-time polymerase
chain reaction; SAR, structure−activity relationship; SDS-
PAGE, sodium dodecyl sulfate polyacrylamide gel electro-
phoresisv; Sosbo, sirtuin one selective benzoxazine; STAC,
sirtuin-activating compound; THF, tetrahydrofuran; Tris,
tris(hydroxymethyl)aminomethane; Vmax, maximum enzy-
matic rate
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(20) Kaeberlein, M.; McVey, M.; Guarente, L. The SIR2/3/4
complex and SIR2 alone promote longevity in Saccharomyces
cerevisiae by two different mechanisms. Genes Dev. 1999, 13,
2570−2580.
(36) Spinck, M.; Neumann-Staubitz, P.; Ecke, M.; Gasper, R.;
Neumann, H. Evolved, selective erasers of distinct lysine acylations.
Angew. Chem., Int. Ed. 2020, 59, 11142−11149.
(37) Saubern, S.; Guha, R.; Baell, J. B. KNIME workflow to assess
PAINS filters in SMARTS format. Comparison of RDKit and indigo
cheminformatics libraries. Mol Inf. 2011, 30, 847−850.
(21) Rogina, B.; Helfand, S. L. Sir2 mediates longevity in the fly
through a pathway related to calorie restriction. Proc. Natl. Acad. Sci.
U. S. A. 2004, 101, 15998−16003.
(38) Dahlin, J. L.; Nissink, J. W. M.; Strasser, J. M.; Francis, S.;
Higgins, L.; Zhou, H.; Zhang, Z.; Walters, M. A. PAINS in the assay:
Chemical mechanisms of assay interference and promiscuous
enzymatic inhibition observed during a sulfhydryl-scavenging HTS.
J. Med. Chem. 2015, 58, 2091−2113.
(22) Napper, A. D.; Hixon, J.; McDonagh, T.; Keavey, K.; Pons, J.
F.; Barker, J.; Yau, W. T.; Amouzegh, P.; Flegg, A.; Hamelin, E.;
Thomas, R. J.; Kates, M.; Jones, S.; Navia, M. A.; Saunders, J. O.;
DiStefano, P. S.; Curtis, R. Discovery of indoles as potent and
selective inhibitors of the deacetylase SIRT1. J. Med. Chem. 2005, 48,
8045−8054.
(23) Robinette, T. M.; Nicholatos, J. W.; Francisco, A. B.; Brooks, K.
E.; Diao, R. Y.; Sorbi, S.; Ricca, V.; Nacmias, B.; Brieno-Enríquez, M.
̃
A.; Libert, S. SIRT1 accelerates the progression of activity-based
anorexia. Nat. Commun. 2020, 11, 1−10.
(24) Broussy, S.; Laaroussi, H.; Vidal, M. Biochemical mechanism
and biological effects of the inhibition of silent information regulator 1
(SIRT1) by EX-527 (SEN0014196 or selisistat). J. Enzyme Inhib. Med.
Chem. 2020, 35, 1124−1136.
(25) Ekblad, T.; Schuler, H. Sirtuins are unaffected by PARP
̈
inhibitors containing planar nicotinamide bioisosteres. Chem. Biol.
Drug Des. 2016, 87, 478−482.
(26) Peck, B.; Chen, C. Y.; Ho, K. K.; Di Fruscia, P.; Myatt, S. S.;
Charles Coombes, R.; Fuchter, M. J.; Hsiao, C. D.; Lam, E. W. F.
SIRT inhibitors induce cell death and p53 acetylation through
targeting both SIRT1 and SIRT2. Mol. Cancer Ther. 2010, 9, 844−
855.
(27) Wössner, N.; Alhalabi, Z.; González, J.; Swyter, S.; Gan, J.;
Schmidtkunz, K.; Zhang, L.; Vaquero, A.; Ovaa, H.; Einsle, O.; Sippl,
W.; Jung, M. Sirtuin 1 inhibiting thiocyanates (S1th)a new class of
isotype selective inhibitors of NAD+ dependent lysine deacetylases.
Front. Oncol. 2020, 10, 1−15.
(28) Spinck, M.; Ecke, M.; Sievers, S.; Neumann, H. Highly sensitive
lysine deacetylase assay based on acetylated firefly luciferase.
Biochemistry 2018, 57, 3552−3555.
(29) Baell, J. B.; Holloway, G. A. New substructure filters for
removal of pan assay interference compounds (PAINS) from
screening libraries and for their exclusion in bioassays. J. Med.
Chem. 2010, 53, 2719−2740.
(30) Tarnowski, M.; Barozet, A.; Johansson, C.; Eriksson, P. O.;
Engkvist, O.; Walsh, J.; Nissink, J. W. M. Utility of resazurin,
horseradish peroxidase, and NMR assays to identify redox-related
false-positive behavior in high-throughput screens. Assay Drug Dev.
Technol. 2018, 16, 171−191.
(31) Bourlot, A. S.; Sánchez, I.; Dureng, G.; Guillaumet, G.;
Massingham, R.; Monteil, A.; Winslow, E.; Pujol, M. D.; Mérour, J. Y.
New substituted 1,4-benzoxazine derivatives with potential intra-
cellular calcium activity. J. Med. Chem. 1998, 41, 3142−3158.
(32) Touzeau, F.; Arrault, A.; Guillaumet, G.; Scalbert, E.; Pfeiffer,
B.; Rettori, M. C.; Renard, P.; Mérour, J. Y. Synthesis and biological
evaluation of new 2-(4,5-dihydro-1H-imidazol-2-yl)-3,4-dihydro-2H-
1,4-benzoxazine derivatives. J. Med. Chem. 2003, 46, 1962−1979.
(33) 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.
(34) 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, E2772−E2781.
(35) Zhao, X.; Allison, D.; Condon, B.; Zhang, F.; Gheyi, T.; Zhang,
A.; Ashok, S.; Russell, M.; MacEwan, I.; Qian, Y.; Jamison, J. A.; Luz,
J. G. The 2.5 Å crystal structure of the SIRT1 catalytic domain bound
to nicotinamide adenine dinucleotide (NAD+) and an indole (EX527
analogue) reveals a novel mechanism of histone deacetylase
inhibition. J. Med. Chem. 2013, 56, 963−969.
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