Structure-based discovery of new selective small-molecule sirtuin 5 inhibitors

Article abstract of DOI:10.1111/cbdd.13077

Human sirtuin 5 (SIRT5) is a protein deacylase regulating metabolic pathways and stress responses and is implicated in metabolism-related diseases. Small-molecule inhibitors for SIRT5 are sought as chemical tools and potential therapeutics. Herein, we proposed a customized virtual screening approach targeting catalytically important and unique residues Tyr102 and Arg105 of SIRT5. Of the 20 tested virtual screening hits, six compounds displayed marked inhibitory activities against SIRT5. For the hit compound 19, a series of newly synthesized (E)-2-cyano-N-phenyl-3-(5-phenylfuran-2-yl)acrylamide derivatives/analogues were carried out structure–activity relationship analyses, resulting in new more potent inhibitors, among which 37 displayed the most potent inhibition to SIRT5 with an IC₅₀ value of 5.59?±?0.75?μM. The biochemical studies revealed that 37 likely acts via competitive inhibition with the succinyl-lysine substrate, rather than the NAD⁺ cofactor, and it manifested substantial selectivity for SIRT5 over SIRT2 and SIRT6. This study will aid further efforts to develop new selective SIRT5 inhibitors as tools and therapeutics.

Full text of DOI:10.1111/cbdd.13077

DR QIANG CHEN (Orcid ID : 0000-0002-2398-4482)
DR GUO-BO LI (Orcid ID : 0000-0002-4915-6677)
PROFESSOR YONG WU (Orcid ID : 0000-0003-0719-4963)
Article type
: Research Article
Structure-based discovery of new selective small molecule sirtuin 5 inhibitors
Sha Liua,†, Sen Jia,†, Zhu-Jun Yua, Hua-Li Wanga, Xu Chenga, Wei-Jian Lia, Li Jinga, Yamei
Yua, Qiang Chena, Ling-Ling Yangb, Guo-Bo Lia,*, Yong Wua,*
a Key Laboratory of Drug Targeting and Drug Delivery System of Ministry of Education,
West China School of Pharmacy, and Laboratory of Biotherapy and Cancer Center, West
China Hospital, West China Medical School, Sichuan University, Chengdu 610041, China.
b College of Food and Bioengineering, Xihua University, Sichuan 610039, China
†S. Liu and S. Ji contributed to equally this work.
*Corresponding author: liguobo@scu.edu.cn (G.-B. Li); wyong@scu.edu.cn (Y. Wu).
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/cbdd.13077
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Abstract
Human sirtuin 5 (SIRT5) is a protein deacylase regulating metabolic pathways and stress
responses, and is implicated in metabolism-related diseases. Small molecule inhibitors for
SIRT5 are sought as chemical tools and potential therapeutics. Herein we proposed a
customized virtual screening approach targeting catalytically important and unique residues
Tyr102 and Arg105 of SIRT5. Of the 20 tested virtual screening hits, 6 compounds displayed
marked inhibitory activities against SIRT5. For the hit compound 19, a series of new
synthesized (E)-2-cyano-N-phenyl-3-(5-phenylfuran-2-yl)acrylamide derivatives/analogues
were carried out structure-activity relationship analyses, resulting in new more potent
inhibitors, among which 37 displayed the most potent inhibition to SIRT5 with an IC50 value
of 5.59 0.75 μM. The biochemical studies revealed that 37 likely acts via competitive
inhibition with the succinyl-lysine substrate, rather than the NAD+ cofactor, and it manifested
substantial selectivity for SIRT5 over SIRT2 and SIRT6. This study will aid further efforts to
develop new selective SIRT5 inhibitors as tools and therapeutics.
Keywords: Sirtuins; Deacylase; SIRT5; Virtual screening; Structure-activity relationship
(SAR)
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Introduction
Sirtuins are nicotinamide adenine dinucleotide (NAD+)-dependent lysine deacylases, which
are highly conserved from bacteria to human.[1,2] There are seven human sirtuin isoforms,
SIRT1-7, which are divided into four classes: SIRT1-3 belong to class I, SIRT4 to class II,
SIRT5 to class III, and SIRT6-7 to class IV.[2] Originally, sirtuins were known as
deacetylases acting on acetyl-lysine substrates from histones and non-histone proteins such as
p53 and β-tubulin.[1,2] Recent studies revealed that sirtuins could remove other acyl groups,
including propionyl,[3] butyryl,[3] malonyl,[4] glutaryl,[5] succinyl,[4] crotonyl,[6] and myristoyl
[7] moieties. Although all sirtuins have a conserved catalytic core domain, they possess
different deacylation activities other than deacetylation activity. For example, SIRT5 can
catalyze desuccinylation, demalonylation and deglutarylation more efficiently than
deacetylation,[4,5] whereas SIRT6 preferentially hydrolyzes long-chain fatty acyl groups, such
as myristoyl-lysine residues.[7-9] SIRT4 can remove lipoic acid and biotin residues from
active site lysines both in vitro and in vivo,[10] while SIRT1-3 seems to be decrotonylases.[6,8]
Owing to the multifaceted activity on various substrate proteins, sirtuins are implicated in
many biological processes such as metabolic regulation, transcriptional regulation, genome
stability, and cell survival.[1,2,11-13]
SIRT5, which mainly localizes in the mitochondrial matrix and preferentially hydrolyzes
acidic acyl modifications, was found to play a pivotal role in mitochondrial metabolism, e.g.,
amino acid degradation, the tricarboxylic acid cycle, and fatty acid metabolism.[14,15] Park et
al recently revealed that SIRT5 represses biochemical activity of, and cellular respiration
through pyruvate dehydrogenase complex (PDC) and succinate dehydrogenase (SDH)[14]
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;

dysregulated PDC or SDH activity is linked to type 2 diabetes and cancer.[13,16-18] Another
study showed that SIRT5 could positively regulate glycolysis via demalonylation of
glycolytic enzymes, providing a possible link to cancer metabolism.[15,19] Lu et al found that
SIRT5 is overexpressed in human non-small-cell lung cancer and associated with poor
outcomes.[20] All of these studies indicate that SIRT5 is likely a potential molecular target for
the treatment of metabolism-related disorders through interference with enzymes involved in
metabolism pathways. Small molecule inhibitors for SIRT5 are thus sought as valuable
chemical tools and potential therapeutics. However, currently almost all the reported SIRT5
inhibitors[21,22], such as nicotinamide, suramin, GW5074, sirtinol, cambinol, and
thiobarbiturates (Supporting Information Figure S1), suffer from poor selectivity, particularly
displaying inhibition against other sirtuin isoforms. Therefore, it is presently desirable to
develop new selective SIRT5 inhibitors. In recent years, considerable efforts have been made
to develop new virtual screening approaches,[23-26] which could substantially improve the
efficiency of identifying new hit/lead compounds for SIRT5.
Superimposition of SIRT5 structure with other sirtuin isoforms reveals that SIRT5 has a
similar overall domain organization and fold to SIRT1-3, but different with SIRT6 and
SIRT7 (Supporting Information Figure S2); Tyr102 and Arg105 located in the catalytic core
domain are the unique residues of SIRT5 (Supporting Information Figure S2a), which are
important for specifically recognizing acidic acyl-lysine substrates.[4,27,28] We thus proposed a
customized virtual screening method to screen the compounds that are likely to interact with
Tyr102 and Arg105. We then used a fluorogenic small-molecule substrate to test the
inhibitory activities against SIRT5 for the hit compounds from virtual screening.
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Subsequently, we carried out structure-activity relationship studies for new synthesized
(E)-2-cyano-N-phenyl-3-(5-phenylfuran-2-yl)acrylamide derivatives/analogues with aim of
identifying new potent SIRT5 inhibitors. For the most potent inhibitor, we further examined
the effects of NAD+/substrate concentrations to the inhibitory potency and tested the
selectivity to other sirtuin isoforms.
1. Results
1.1. Customized virtual screening.
Since Tyr102 and Arg105 are catalytically important and unique residues of SIRT5
(Supporting Information Figure S2), we developed a customized virtual screening method to
search for compounds that are likely to specifically interact with Tyr102 and Arg105. In the
virtual screening protocol, Autodock vina docking program[29] was first used to generate
possible binding poses for the enquired compounds, followed by the use of protein-ligand
interaction fingerprint (IFP)-based method[25,26] to filter the compounds potentially having
hydrogen-bonding/electrostatic interactions with Tyr102 and Arg105 (Details see
Experimental section). We employed this virtual screening protocol to screen against our
in-house compound database that contains more than 15,000 small-molecule synthesized
compounds. A total of 635 compounds were predicted with Vinascore lower than -6.5
kcal/mol and to have hydrogen-bonding/electrostatic interactions with Tyr102 and Arg105.
These compounds were further inspected visually to check whether the predicted binding
poses are reasonable and to select diverse compounds as potential SIRT5 inhibitors. Finally,
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20 hit compounds (Figure 1) were selected for subsequent biochemical test; to the best of our
knowledge, all of these compounds have not been reported as sirtuin inhibitors so far. The
predicted binding modes of the selected compounds (1-20) are shown in Figure 1. We
observed that although these compounds may have different binding modes with SIRT5, all
of them are likely to form hydrogen-bonding/electrostatic interactions with Tyr102 and
Arg105 (Figure 1).
1.2.In vitro inhibitory activities of virtual screening hits against SIRT5.
We then tested the inhibitory activities of the 20 selected hits from virtual screening
against recombinant human SIRT5 proteins (Details regarding protein expression and
purification see Experimental section) at 100 μM using a fluorogenic small-molecule
substrate (SuBKA)-coupled trypsin assay[28] (Supporting Information Figure S3a) similar as
that established by Jung et al[22]. The concentrations of NAD+ and SuBKA used for the
activity test are 200 μM and 10 μM, respectively. The results revealed that most of the 20
tested compounds displayed inhibition against SIRT5, and half of them had >30% inhibition
(Table 1). Compounds 2, 6, 9, 11, 18, and 19 manifested inhibition rates with 51.3% 5.5%,
61.3% 5.3%, 70.03% 0.56%, 77.26% 1.38%, 94.3% 0.53%, and 97.14% 1.03%,
respectively (Table 1). We observed that the most potent compounds 18 and 19 displayed
dose-dependent inhibitory activities to SIRT5 (Figure 2a-b), with IC50 values of 18.30 0.88
μM and 9.26 2.09 μM, respectively; both are more potent than the known SIRT5 inhibitor
Nicotinamide (IC50 = 45.7 7.42 μM, Figure 2c). Notably, 18 and 19 are predicted to have
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similar binding modes with SIRT5 (Figure 1r and 1s); in particular, their carboxylate group is
likely positioned to make hydrogen-bonding/electrostatic interactions with Tyr102 and
Arg105 (Figure 1r and 1s). These results indicated the customized virtual screening method,
which searched for compounds likely to interact with Tyr102 and Arg105, is a useful strategy
for the identification of new SIRT5 inhibitors.
1.3.Structure-activity relationship studies.
With aim of identifying more potent SIRT5 inhibitor, we next synthesized new
(E)-2-cyano-N-phenyl-3-(5-phenylfuran-2-yl)acrylamide (the core scaffold of compound 19)
derivatives/analogues with different amide-N-substituents (moiety A) and
furan-5-substituents (moiety B) (compounds 21-39 in Table 2; 1H/13C NMR spectra of these
compounds shown in Supporting Information Figure S6-S24) and carried out
structure-activity relationship (SAR) analyses for these new synthesized compounds.
The route used for the synthesis of 21-39 are summarized in Scheme 1. The commercially
available mono-substituted or poly-substituted aniline (40) was reacted with 2-cyanoacetic
acid (41) in the presence of PCl5 to give the substituted 2-cyano-N-phenylacetamide (42).
Meanwhile, suzuki cross-coupling reactions of (5-formylfuran-2-yl)boronic acid (43) with
substituted iodobenzene (44) led to the production of substituted
5-phenylfuran-2-carbaldehydes (45). Then, target compounds (21-23) and ester-containing
intermediates were obtained by the condensation reactions between compounds 42 and 45 at
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80
were directly converted to carboxyl-containing target compounds 24-39 via hydrolysis in the
presence of NaOH at 60 for 0.5 h.
in the presence of piperidine as a catalyst. Finally, the ester-containing intermediates
The inhibitory activities (IC50) of 21-39 against SIRT5 are in Table 2. We observed that
compounds 21-23, with a moiety A of 3,4-dimethylbenzene and a moiety B of
p-methylbenzoate (21), p-benzonitrile (22) or p-nitrobenzene (23), displayed lower inhibitory
activities against SIRT5 than 25 (IC50 = 22.7 2.61 μM, Table 2), which has a moiety A of
3,4-dimethylbenzene and a moiety B of p-benzoic acid. Compared with 25, 24 (IC50 = 100.6
8.98 μM), containing a moiety B of m-benzoic acid, also showed a weaker inhibitory
potency to SIRT5. These results indicated that the p-benzoic acid as moiety B may provide
important pharmacophore features for binding with SIRT5; as observed by molecular
docking simulations, the carboxylate group of the p-benzoic acid moiety is likely to be
positioned to interact with residues Tyr102 and Arg105. Compounds 26 and 27, bearing
substituted p-benzoic acid as moiety B, showed comparable or slight weaker inhibitory
potency to SIRT5 (with IC50 of 23.6 2.70 μM and 57.4 19.21 μM, respectively, Table 2),
indicating that substituents on the phenyl of the p-benzoic acid (moiety B) do not seem to be
able to markedly improve the inhibitory activity. We thus reserved the p-benzoic acid as
moiety B, and synthesized compounds 28-39 with various substituents as moiety A. We
observed that compounds 28-34, with C/N/O-containing substituents, showed varied
inhibitory activities (IC50) from 8.03 μM to 40.9 μM (Table 2), which are better than
Nicotinamide (IC50 = 45.7 7.42 μM, Figure 2c); 34 has relatively good inhibitory activity
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with an IC50 value of 8.03 0.70 μM, slightly better than the virtual screening hit compound
19 (IC50 = 9.26 0.88 μM, Figure 2b). Interestingly, 35 (IC50 = 62.2 7.44 μM) with an
m-benzoic acid as moiety A displayed an obviously less activity compared with 34 (IC50 =
8.03 0.70 μM). Compounds 36-39, containing halo-substituted moiety A, showed a slight
better inhibitory activity than compound 25 (IC50 = 22.7 2.61 μM, Table 2), excepting
compound 39 (IC50 = 37.9 6.13 μM, Table 2). Notably, compound 37 bearing
2-fluorobenzonitrile as moiety A manifested an IC50 value of 5.59 0.75 μM (Figure 3a),
which is better than the hit compound 19 (Table 2). The molecular docking studies indicated
that, similar to 19 (Figure 1s), the p-benzoic acid moiety of 37 likely forms
hydrogen-bonding and electrostatic interactions with Tyr102 and Arg105, and the amide
linkage makes hydrogen bonds with Leu227 and Tyr255 (Figure 3b); additionally, the
fluorine of 37 seems have halogen-bonding interactions with Asn226 (Figure 3b). We further
used the LEADOPT program[30] to predict the ADMET properties (a total of 12 kinds of
ADMET properties involved in LEADOPT) for all of the synthesized compounds. The
results showed that all of these compounds have ADMET score ≥ 9. Taken together, the SAR
studies revealed that the p-benzoic acid as moiety B is likely to provide essential
pharmacophore features to bind with SIRT5, and it is possible to improve the inhibitory
potency via modifications of moiety A.
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1.4.Compound 37 likely acts as a succinyl-lysine substrate competitive inhibitor.
For compound 37 (IC50 = 5.59 0.75 μM, Figure 3a), we tested the inhibitory potency in
the presence of 0.1% Triton X-100 to check whether it inhibits SIRT5 enzymes through
colloid-like aggregations[31]. We observed that 37 has similar SIRT5 inhibitory activity in the
presence and absence of 0.1% Triton X-100, probably excluding the possibility that 37 is a
promiscuous aggregate. We next examined the effects of the concentrations of NAD+
cofactor and SuBKA substrate (Supporting Information Figure S3a) on the inhibition potency
of 37 against SIRT5. The results are shown in Figure 4a-b and Table 3. No obvious
difference between inhibitory activities of 37 against SIRT5 for different concentrations of
NAD+ was observed (Figure 4a and Table 3), suggesting that 37 probably does not bind to the
NAD+ pocket, thereby having no competitive relationship with the NAD+ cofactor. In
contrast, we observed that the inhibition potency of 37 to SIRT5 is apparently affected by
different concentrations of the SuBKA substrate (Figure 4b). When treated with 300 μM, 100
μM, 33 μM, and 11 μM of SuBKA, the inhibitory activities (IC50) of 37 to SIRT5 are 28.35
2.47 μM, 28.02 2.02 μM, 14.93 1.90 μM, and 6.64 0.82 μM, respectively (Table 3),
implying that 37 might be a SuBKA-competitive inhibitor of SIRT5. Comparison of the
predicted binding mode of 37 with the crystal structure of SIRT5:sucH3K9 (PDB ID:
4F4U)[27] revealed that 37 likely has a similar binding mode with the peptide substrate
sucH3K9 (Figure 4c); both have hydrogen-bonding interactions with Tyr102, Arg105,
Leu227, and Tyr255, electrostatic interactions with Arg105, and hydrophobic interactions
with Phe223 (Figure 4c). These results indicated that 37 likely mimics the sucH3K9 substrate
and acts via competitive inhibition with the succinyl-lysine substrate of SIRT5.
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1.5. Compound 37 displayed no or very weak inhibitory activity against SIRT2 and SIRT6.
With aim of examining the selectivity of compound 37 to other sirtuin isoforms, we next
tested the inhibitory activity against class I enzyme SIRT2 and class IV enzyme SIRT6. The
Benzyl-Lys(Acetyl)-AMC (AcBKA, Supporting Information Figure S3b) and
Ac-TARKmy-AMC (Supporting Information Figure S3c) were used as substrates for SIRT2
and SIRT6, respectively (Details see Experimental section).[28] We observed that 37
displayed no inhibition against SIRT2 even at the highest concentration of 600 μM
(Supporting Information Figure S4a). Similarly, no obvious inhibitory activity to SIRT6 was
observed for 37 (Supporting Information Figure S4b). We performed molecular docking
studies for 37 with SIRT2 and SIRT6. No key interactions between 37 and SIRT2 and SIRT6
were observed for top-ranking docking poses (Figure S5). The results indicated that 37 has a
good selectivity for SIRT5 over SIRT2 and SIRT6.
2. Discussion
This work clearly revealed the potential of a customized virtual screening method
targeting specific active site features for the identification of hit compounds for SIRT5.
Coupled with the fluorogenic substrate-based assays, several new hit compounds were
identified, among which the (E)-2-cyano-N-phenyl-3-(5-phenylfuran-2-yl)acrylamide
compound 19 displayed most potent inhibition to SIRT5. The limited SAR studies revealed
that it is possible to improve the potency of
(E)-2-cyano-N-phenyl-3-(5-phenylfuran-2-yl)acrylamide inhibitors towards SIRT5. The SAR
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studies also resulted in a potent SIRT5 inhibitor, compound 37, with an IC50 value of 5.59
0.75 μM. Further biochemical studies found that 37 likely acts as a succinyl-lysine substrate,
rather than NAD+, competitive inhibitor, and manifests substantial selectivity for SIRT5 over
SIRT2 and SIRT6. Overall, this study provides an effective in silico approach for rapid
identification of hit compounds for SIRT5, and will aid further efforts to develop new
selective SIRT5 inhibitors as tools and therapeutics.
3. Experimental section
4.1. Virtual screening details. The customized virtual screening protocol employed
molecular docking simulations and docking pose analyses, which are sequentially described
below.
4.1.1 Docking simulations using AutoDock Vina. The in-house chemical database, containing
more than 15,000 small-molecule compounds, was used as the screening database. The
chemical structures (pdf format) were converted to mol2 format using OpenBabel [32], and
then further converted to pdbqt format for AutoDock Vina [29] using the Raccoon script
(http://autodock.scripps.edu/resources/raccoon).
An X-ray crystal structure of human SIRT5 in complex with a bicyclic intermediate
(PDB ID: 4F56)[27] was used as the docking template. The bicyclic intermediate, water
molecules, and other solvent molecules were removed. The protein model was then assigned
with Gasteiger-Marsili charges and added non-polar hydrogens using AutoDockTools
(http://autodock.scripps.edu/resources/adt). The binding site was set as a square grid. The
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grid center was set to coordinates of [x, y, z = -7.85, -3.03, 14.21] and the grid size was set to
23Å × 23Å × 23Å encompassing the entire SIRT5 catalytic core domain. The number of
docking poses was set as 10, and the other parameters for Vina were set as default.
4.1.2 Docking pose analyses. With the aim of searching for compounds likely to interact
with catalytic important residues Tyr102 and Arg105, we carried out docking poses
analyses using an interaction fingerprint (IFP)-based method. The IFP method involves eight
types of protein-ligand interactions, including hydrogen-bond donor, hydrogen-bond
acceptor, positively charge, negatively charge, face-to-face π-π stacking interactions,
edge-to-face π-π stacking interactions, and hydrophobic interactions as that in our previously
reported methods[26,30,33,34]. In this study, we filtered the compounds that potentially form
hydrogen-bonding and electrostatic interactions (negatively charges) with Tyr102 and
Arg105. The filtered compounds were further inspected visually to check whether the
predicted docking poses are reasonable and to select structurally diverse compounds. To the
end, a total of 20 compounds were selected and tested against SIRT5 activity in vitro (see
Table 1); all these compounds (purity: >98%) were purchased from Enamine Ltd and used
without further purification.
4.2. Protein cloning, expression, and purification. The human SIRT5 (residues 34-269)
were PCR-amplified and cloned into the PET28 vector resulting in a construct with
N-terminal His-tag and a TEV protease cleavage site. Proteins were expressed in E. coli
Transetta(DE3) cells overnight at 16 , and induced for overexpression using isopropyl
β-D-1-thiogalactopyranoside (IPTG, 0.3 mM final concentration) at an OD600 of 0.6~0.8. The
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cells were harvested and resuspended in lysis buffer(20 mM Tris–HCl, 250 mM NaCl, pH
8.0), and then lysed by a ultrahigh-pressure homogenizer. The cellular debris was removed by
centrifugation at 15,000 r/min for 30 min. The supernatant was applied onto an Ni-NTA
column (Roche), followed by extensive washing with 20 volumes of 20 mM Tris-HCl pH
8.0, 250 mM NaCl with 10 mM imidazole to remove nonspecifically binding proteins.
Recombinant proteins were eluted with 20 mM Tris-HCl pH 8.0, 250 mM NaCl with 250
mM imidazole, and then further exchanged to the buffer of 50 mM Tris-HCl pH 8.0, 150 mM
NaCl. The resulted SIRT5 proteins were pooled and concentrated to ~10 mg/ml and stored at
-80 °C. All purification steps were monitored by SDS-PAGE and the concentration was
determined by NannoDrop 2000 spectro photometer (Thermo Scientific). Human SIRT225-389
and SIRT61-355 proteins were purified similarly as described above.
4.3. Fluorescence-based inhibition assays. The inhibition activities of compounds against
recombinant human SIRT5 were performed with a fluorescence-based assay similar as that
established by Jung et al[22,28]. The fluorogenic substrate Benzyl-Lys(Succinyl)-AMC
(SuBKA, Supporting Information Figure S3a) was used. All the enzymes and substrates were
dissolved in the assay buffer: 50 mM Tris, 137 mM NaCl, 2.7 mM KCl, pH 8.0. The assay
was performed in 96-well black microplate (Corning No. 3603) with a reaction volume of 60
μL per well. Reaction wells contained SIRT5 proteins (0.2 μM), SuBKA (10 μM), NAD+
(200 μM), and/or the compounds at different concentrations. Control wells containing
SuBKA, NAD+, and/or compounds (with the same concentrations as that in reaction wells) in
assay buffer were included in each plate with aim of excluding the interference of fluorescent
compounds. The reactions were incubated for 2 h at 37
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and 140 rpm, and then stopped by

the addition of 60 μL of a solution containing 3~4 U·μL-1 trypsin (Sigma-Aldrich No. T8003)
and 8 mM nicotinamide, followed by further incubation for 20 min at 37 and 140 rpm.
Fluorescence intensity was measured using a microplate reader (BioTek Cytation 3, λex = 390
nm, λem = 460 nm). The IC50 determination was performed with compound concentrations
varying from 600 μM to 0.03 μM in 3-fold dilution. All determinations were performed in
ꢅꢂ
triplicates. The inhibition rate was calculated using the formula: ꢀꢁℎ% = 100 − ꢂ
×
ꢃꢄ
ꢄ
ꢂ ꢅꢂ
ꢃ
ꢆ
100, where ꢇ denotes fluorescence in a reaction well containing compounds, ꢇ denotes
ꢈꢉ
ꢈ
fluorescence in a reaction well without compounds, ꢇ denotes fluorescence in a control well
ꢉ
containing compounds, and ꢇꢊ denotes fluorescence in a control well without compounds.
The IC50 values were obtained using Graphpad Prism software (La Jolla, CA).
4.3.1 NAD+/substrate competitive assay. To investigate the inhibition mechanism of 37
against SIRT5, we tested the effects of varied concentrations of SuBKA or NAD+ to the
inhibition potency. With different concentrations of NAD+ (800 μM, 400 μM, 200 μM, 100
μM, and 50 μM), the IC50 values were tested under the conditions of SIRT5 (0.2 μM),
SuBKA (200 μM) and 37 (600 μM ~ 0.03 μM). When treated with different concentrations of
SuBKA (300 μM, 100 μM, 33 μM, and 11 μM), the IC50 values were obtained with SIRT5
(0.2 μM), NAD+ (200 μM) and 37 (600 μM ~ 0.03 μM).
4.3.2 Selectivity against SIRT2 and SIRT6. With aim of examining the selectivity to other
sirtuin isoforms, 37 was tested against SIRT2 and SIRT6. The substrates
Benzyl-Lys(Acetyl)-AMC (AcBKA, Supporting Information Figure S3b) and
Ac-TARKmy-AMC (Supporting Information Figure S3c) were used for SIRT2 and SIRT6
activity test, respectively. The SIRT2 proteins (0.5 μM) were mixed with AcBKA (20 μM),
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NAD+ (200 μM) or/and different concentrations of 37 (600 μM ~ 0.03 μM). The SIRT6
proteins (0.2 μM) were mixed with Ac-TARKmy-AMC (10 μM), NAD+ (200 μM) or/and
different concentrations of 37 (600 μM ~ 0.03 μM). The activity test methods for SIRT2 and
SIRT6 are similar with that described above for SIRT5.
4.4. Chemical synthesis. All the reagents were purchased from market (e.g., Sigma-Aldrich
agency, Shanghai, China; Juhui Chemical, Chengdu, China) and were used without further
purification. Reactions were monitored by thin layer chromatography (TLC) on Merck silica
gel 60 F-254 thin layer plates. All the target compounds were purified to >95% purity, as
determined by high-performance liquid chromatography (HPLC). HPLC analysis was
performed on a Waters 2695 HPLC system equipped with a Kromasil C18 column (4.6 mm ×
250 mm, 5 μm). 1H NMR and 13C NMR spectra were recorded on a Bruker AV-400
spectrometer at 400 MHz and 100 MHz, respectively. Chemical shifts (δ) are quoted in parts
per million (ppm) relative to tetramethylsilane (TMS) as an internal standard. Low-resolution
and high-resolution mass spectral (MS) data were acquired on an Agilent 1100 series LC-MS
instrument with UV detection at 254 nm in electrospray ionization (ESI) mode.
General procedure 1: PCl5-mediated amide formation. To a cooled solution of
2-cyanoacetic acid (41, 1.0g, 11.7 mmol) in dichloromethane (20 ml), phosphorus
pentachloride (PCl5, 2.5g, 12 mmol) was slowly added. The reaction mixture was stirred at 40
for 0.5 h, and then substituted anilines (11.5 mmol ) were added to the reaction at room
temperature. Subsequently, the mixture was warmed to 40 and stirred for 2h. Upon
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completion of the reaction as determined by TLC, the organic solvent was concentrated to
dryness and the residue was dispersed into 50 ml ice water. At this time, a brown solid was
formed and collected by filtration. The crude product was washed by saturated NaHCO3 (10
ml) and anhydrous ether (20 ml) to give substitued 2-cyano-N-phenylacetamide (42,
80-92%). These products were taken up for the next step without any purification.
General procedure 2: Suzuki cross-coupling reaction. A mixture of
(5-formylfuran-2-yl)boronic acid (43, 1.5 equiv), substituted iodobenzene (44, 1.0 equiv),
Na2CO3 (2.0 equiv) and Pd(PPh3)2Cl2 (0.1 equiv) in MeCN/H2O (5 mL/ 1 mmol) was stirred
at 60 for 1.0 h under a nitrogen atmosphere. Upon completion of the reaction as
determined by TLC, the mixture was filtered and the filtrate was concentrated in vacuo. The
products were purified by column chromatography with appropriate eluents (PE/EA). Yield:
67-88%.
General procedure 3: Piperidine-mediated condensation reaction reaction. To a solution
of substitued 2-cyano-N-phenylacetamides (42, 1.0 equiv) substitued
5-phenylfuran-2-carbaldehydes (45, 1.0 equiv) in EtOH (5ml/1.0mmol), catalytic amount of
piperidine was added. Then the reaction was allowed to warm to 80
and stirred for 2 h.
After completion (monitored by TLC), the mixture was concentrated under reduced pressure.
The residues were purified by column chromatography with appropriate eluents (PE/EA) to
give target compound 21-23 and ester-containing intermediates. Yield: 75-92%.
General procedure 4: Hydrolytic reaction of ester. The ester-containing intermediates
(1.0 equiv) were taken in THF/MeOH (6 mL/mmol), and NaOH (1.0 equiv) was added. The
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reaction mixture was refluxed for ~0.5 h at 60 . After completion (monitored by TLC), the
mixture was concentrated and acidified with 1 M HCl (pH 4-5). During the acidification, a
buff solid was formed and collected by filtration. The filter cake was washed with a small
amount of water and dried in a vacuum oven to give the product 24-39. Yield: 65-78%.
ACKNOWLEDGEMENTS
This work was supported by the National Natural Science Foundation of China (Grant No.
81502989 and 81373259), the Scientific Research Foundation of Sichuan University (Grant
No. 20822041A4193), and the China Postdoctoral Science Foundation Funded Project (Grant
No. 2015M570789).
CONFLICT OF INTEREST
The authors declare no conflict of interest.
SUPPORTING INFORMATION
Additional Supporting Information may be found online in the supporting information tab for
this article.
This article is protected by copyright. All rights reserved.

Figure Legends
Figure 1. (a-t) The predicted binding modes of the hit compounds 1-20. Compounds are
shown in green sticks and dash lines represent hydrogen-bonding interactions.
Figure 2. (a-b) The inhibitory activity (IC50) curves of the virtual screening hits 18 and 19,
and (c) the known SIRT5 inhibitor Nicotinamide.
Figure 3. (a) The inhibitory activity (IC50) curves of 37 against SIRT5. (b) View of the
predicted binding mode of 37 with SIRT5.
Figure 4. (a-b) The inhibitory activity (IC50) curves of 37 against SIRT5 obtained with
different concentrations of NAD+ cofactor and SuBKA substrate. (c) Superimposition of the
predicted binding mode of 37 with the crystal structure of SIRT5:sucH3K9 (PDB ID:
4F4U)[27] reveals that 37 has a similar binding mode with sucH3K9.
Scheme 1. Reagents and conditions: (a) PCl5, CH2Cl2, 40 , 2h; (b) substituted iodobenzene,
Na2CO3, Pd(PPh3)2Cl2, MeCN:H2O=1:1, 60 , 1h; (c) Piperidine, EtOH, 80 , 2h; (d)
NaOH, THF/MeOH, 60 , 0.5h.
References
[1] B. Chen, W. Zang, J. Wang, Y. Huang, Y. He, L. Yan, J. Liu, W. Zheng, Chem. Soc.
Rev. 2015, 44, 5246.
[2] R. H. Houtkooper, E. Pirinen, J. Auwerx, Nat. Rev. Mol. Cell Biol. 2012, 13, 225.
This article is protected by copyright. All rights reserved.

[3] Y. Chen, R. Sprung, Y. Tang, H. Ball, B. Sangras, S. C. Kim, J. R. Falck, J. Peng, W.
Gu, Y. Zhao, Mol. Cell. Proteomics 2007, 6, 812.
[4] J. Du, Y. Zhou, X. Su, J. J. Yu, S. Khan, H. Jiang, J. Kim, J. Woo, J. H. Kim, B. H.
Choi, B. He, W. Chen, S. Zhang, R. A. Cerione, J. Auwerx, Q. Hao, H. Lin, Science
2011, 334, 806.
[5] M. Tan, C. Peng, Kristin A. Anderson, P. Chhoy, Z. Xie, L. Dai, J. Park, Y. Chen, H.
Huang, Y. Zhang, J. Ro, Gregory R. Wagner, Michelle F. Green, Andreas S. Madsen, J.
Schmiesing, Brett S. Peterson, G. Xu, Olga R. Ilkayeva, Michael J. Muehlbauer, T.
Braulke, C. Mühlhausen, Donald S. Backos, Christian A. Olsen, Peter J. McGuire,
Scott D. Pletcher, David B. Lombard, Matthew D. Hirschey, Y. Zhao, Cell Metab.
2014, 19, 605.
[6] X. Bao, Y. Wang, X. Li, X.-M. Li, Z. Liu, T. Yang, C. F. Wong, J. Zhang, Q. Hao, X.
D. Li, eLife. 2014, 3, e02999.
[7] X. Zhang, S. Khan, H. Jiang, M. A. Antonyak, X. Chen, N. A. Spiegelman, J. H.
Shrimp, R. A. Cerione, H. Lin, Nat. Chem. Biol. 2016, 12, 614.
[8] J. L. Feldman, J. Baeza, J. M. Denu, J. Biol. Chem. 2013, 288, 31350.
[9] H. Jiang, S. Khan, Y. Wang, G. Charron, B. He, C. Sebastian, J. Du, R. Kim, E. Ge, R.
Mostoslavsky, H. C. Hang, Q. Hao, H. Lin, Nature 2013, 496, 110.
This article is protected by copyright. All rights reserved.

[10] Rommel A. Mathias, Todd M. Greco, A. Oberstein, Hanna G. Budayeva, R.
Chakrabarti, Elizabeth A. Rowland, Y. Kang, T. Shenk, Ileana M. Cristea, Cell 2014,
159, 1615.
[11] M. S. Bonkowski, D. A. Sinclair, Nat. Rev. Mol. Cell Biol. 2016, 17, 679.
[12] L. Yang, X. Ma, C. Yuan, Y. He, L. Li, S. Fang, W. Xia, T. He, S. Qian, Z. Xu, G. Li,
Z. Wang, Eur. J. Med. Chem. 2017, 134, 230.
[13] S. Xu, C.-X. Liu, W. Xu, L. Huang, J.-Y. Zhao, S.-M. Zhao, Signal Transduct. Target.
Ther. 2017, 2, 16035.
[14] J. Park, Y. Chen, D. X. Tishkoff, C. Peng, M. Tan, L. Dai, Z. Xie, Y. Zhang, B. M.
Zwaans, M. E. Skinner, D. B. Lombard, Y. Zhao, Mol. Cell 2013, 50, 919.
[15] Y. Nishida, Matthew J. Rardin, C. Carrico, W. He, Alexandria K. Sahu, P. Gut, R.
Najjar, M. Fitch, M. Hellerstein, Bradford W. Gibson, E. Verdin, Mol. Cell 2015, 59,
321.
[16] M. A. Selak, S. M. Armour, E. D. MacKenzie, H. Boulahbel, D. G. Watson, K. D.
Mansfield, Y. Pan, M. C. Simon, C. B. Thompson, E. Gottlieb, Cancer Cell 2005, 7,
77.
[17] A. M. Cervera, J. P. Bayley, P. Devilee, K. J. McCreath, Mol. Cancer 2009, 8, 89.
[18] E. H. Smith, R. Janknecht, L. J. Maher, 3rd, Hum. Mol. Genet. 2007, 16, 3136.
[19] A. Chalkiadaki, L. Guarente, Nat. Rev. Cancer 2015, 15, 608.
This article is protected by copyright. All rights reserved.

[20] W. Lu, Y. Zuo, Y. Feng, M. Zhang, Tumour Biol. 2014, 35, 10699.
[21] L. Yang, X. Ma, Y. He, C. Yuan, Q. Chen, G. Li, X. Chen, Sci. China Life Sci. 2017,
60, 249.
[22] B. Maurer, T. Rumpf, M. Scharfe, D. A. Stolfa, M. L. Schmitt, W. He, E. Verdin, W.
Sippl, M. Jung, ACS Med. Chem. Lett. 2012, 3, 1050.
[23] E. Cichero, P. D'Ursi, M. Moscatelli, O. Bruno, A. Orro, C. Rotolo, L. Milanesi, P.
Fossa, Chem. Biol. Drug Des. 2013, 82, 718.
[24] E. Cichero, S. Espinoza, S. Franchini, S. Guariento, L. Brasili, R. R. Gainetdinov, P.
Fossa, Chem. Biol. Drug Des. 2014, 84, 712.
[25] G.-B. Li, L.-L. Yang, Y. Yuan, J. Zou, Y. Cao, S.-Y. Yang, R. Xiang, M. Xiang,
Methods 2015, 71, 158.
[26] G.-B. Li, M. I. Abboud, J. Brem, H. Someya, C. T. Lohans, S.-Y. Yang, J. Spencer, D.
W. Wareham, M. A. McDonough, C. J. Schofield, Chem. Sci. 2017, 8, 928.
[27] Y. Zhou, H. Zhang, B. He, J. Du, H. Lin, R. A. Cerione, Q. Hao, J. Biol. Chem. 2012,
287, 28307.
[28] H.-L. Wang, S. Liu, Z.-J. Yu, C. Wu, L. Cheng, Y. Wang, K. Chen, S. Zhou, Q. Chen,
Y. Yu, G.-B. Li, RSC Adv. 2017, Accepted
[29] O. Trott, A. J. Olson, J. Comput. Chem. 2010, 31, 455.
This article is protected by copyright. All rights reserved.

[30] G.-B. Li, S. Ji, L.-L. Yang, R.-J. Zhang, K. Chen, L. Zhong, S. Ma, S.-Y. Yang, Eur. J.
Med. Chem. 2015, 93, 523.
[31] B. Y. Feng, A. Shelat, T. N. Doman, R. K. Guy, B. K. Shoichet, Nat. Chem. Biol. 2005,
1, 146.
[32] N. M. O'Boyle, M. Banck, C. A. James, C. Morley, T. Vandermeersch, G. R.
Hutchison, J. Cheminformatics 2011, 3, 33.
[33] G.-B. Li, L.-L. Yang, W.-J. Wang, L.-L. Li, S.-Y. Yang, J. Chem. Inf. Model. 2013, 53,
592.
[34] G.-B. Li, L.-Y. Huang, H. Li, S. Ji, L.-L. Li, S.-Y. Yang, RSC Adv. 2016, 6, 61137.
This article is protected by copyright. All rights reserved.

Figure 1. (a-t) The predicted binding modes of the hit compounds 1-20. Compounds ar
e
shown in green sticks and dash lines represent hydrogen-bonding interactions.

Figure 2. (a-b) The inhibitory activity (IC50) curves of the virtual screening hits 18 and 19,
and (c) the known SIRT5 inhibitor Nicotinamide.

Figure 3. (a) The inhibitory activity (IC50) curves of 37 against SIRT5. (b) View of the
predicted binding mode of 37 with SIRT5.
Figure 4. (a-b) The inhibitory activity (IC50) curves of 37 against SIRT5 obtained with
different concentrations of NAD+ cofactor and SuBKA substrate. (c) Superimposition of the
predicted binding mode of 37 with the crystal structure of SIRT5:sucH3K9 (PDB ID:
4F4U)[27] reveals that 37 has a similar binding mode with sucH3K9.

NH2
O
O
a
R
CN
R
CN
HO
N
H
B
CN
A
H
41
40
42
N
c or c,d
O
R'
R
O
21-39
b
R'
OHC
O
OHC
B(OH)2
O
I
R'
43
44
45
O
O
O
O
O
21: A=
22: A=
B=
B=
B=
B=
B=
28: A=
B=
N
H
34: A=
B=
B=
OH
OH
O
O
N
O
O
O
35: A=
HO
B=
B=
29: A=
30: A=
OH
OH
NO2
23: A=
24: A=
O
O
O
F3C
Cl
36: A=
37: A=
B=
B=
OH
O
OH
COOH
O
O
F
O
O
31: A=
32: A=
B=
B=
OH
OH
25: A=
26: A=
O
N
OH
O
O
O
O
F3C
O
38: A=
B=
B=
OH
OH
OH
N
O
F
Cl
H
O
O
O
N
27: A=
B=
B=
B=
33: A=
39: A=
O
OH
OH
OH
Cl
Scheme 1. Reagents and conditions: (a) PCl5, CH2Cl2, 40 , 2h; (b) substituted iodobenzene,
Na2CO3, Pd(PPh3)2Cl2, MeCN:H2O=1:1, 60 , 1h; (c) Piperidine, EtOH, 80 , 2h; (d)
NaOH, THF/MeOH, 60 , 0.5h.
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Table 1. Chemical structures and inhibitory activities of the 20 hit compounds identified by
customized virtual screening.
Cpd. ID
M. W.
241.24
Chemical Structure
Inh%@100μM
17.41 13.83
1
242.23
261.28
51.3 5.5
2
3
28.91 24.12
282.30
284.27
293.28
307.32
311.38
313.28
321.26
21.54 10.87
18.70 12.73
61.3 5.3
4
5
6
7
17.43 1.82
24.54 1.93
70.03 0.56
11.08 11.2
8
9
10
332.36
77.26 1.38
11
337.35
349.41
9.2 3.80
12
13
15.43 2.23
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351.33
354.58
387.19
401.73
-5.54 1.37
33.66 8.07
34.24 14.39
30.85 2.33
14
15
16
17
420.85
437.79
94.3 0.53
18
N
O
N+
OH
H
N
97.14 1.03
19
20
-O
O
O
O
Cl
488.42
122.13
73.75 8.91
70.57 5.68
Nicotinamid
e
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Table 2. The inhibitory activities (IC50) of new
(E)-2-cyano-N-phenyl-3-(5-phenylfuran-2-yl)acrylamide derivatives/analogues against
SIRT5. s
ADMET scorea
9
Cpd. ID
Moiety A
Moiety B
IC50 (μM)
9.26 0.83
19
133.4 34.32
82.7 32.23
72.1 7.83
9
9
9
21
22
23
100.6 8.98
22.7 2.61
23.6 2.70
10
9
24
25
26
10
57.4 19.21
40.9 5.91
36.9 6.98
9
9
27
28
29
10
30
31
10.8 1.62
32.3 3.33
10
9
24.3 5.93
10
32
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