Identification of the subtype-selective Sirt5 inhibitor balsalazide through systematic SAR analysis and rationalization via theoretical investigations

Article abstract of DOI:10.1016/j.ejmech.2020.112676

We report here an extensive structure-activity relationship study of balsalazide, which was previously identified in a high-throughput screening as an inhibitor of Sirt5. To get a closer understanding why this compound is able to inhibit Sirt5, we initially performed docking experiments comparing the binding mode of a succinylated peptide as the natural substrate and balsalazide with Sirt5 in the presence of NAD⁺. Based on the evidence gathered here, we designed and synthesized 13 analogues of balsalazide, in which single functional groups were either deleted or slightly altered to investigate which of them are mandatory for high inhibitory activity. Our study confirms that balsalazide with all its given functional groups is an inhibitor of Sirt5 in the low micromolar concentration range and structural modifications presented in this study did not increase potency. While changes on the N-aroyl-β-alanine side chain eliminated potency, the introduction of a truncated salicylic acid part minimally altered potency. Calculations of the associated reaction paths showed that the inhibition potency is very likely dominated by the stability of the inhibitor-enzyme complex and not the type of inhibition (covalent vs. non-covalent). Further in-vitro characterization in a trypsin coupled assay determined that the tested inhibitors showed no competition towards NAD⁺ or the synthetic substrate analogue ZKsA. In addition, investigations for subtype selectivity revealed that balsalazide is a subtype-selective Sirt5 inhibitor, and our initial SAR and docking studies pave the way for further optimization.

Full text of DOI:10.1016/j.ejmech.2020.112676

Journal Pre-proof
Identification of the subtype-selective Sirt5 inhibitor balsalazide through systematic
SAR analysis and rationalization via theoretical investigations
Carina Glas, Johannes C.B. Dietschreit, Nathalie Wössner, Lars Urban, Ehab Ghazy,
Wolfgang Sippl, Manfred Jung, Christian Ochsenfeld, Franz Bracher
PII:
S0223-5234(20)30648-6
DOI:
https://doi.org/10.1016/j.ejmech.2020.112676
EJMECH 112676
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 8 May 2020
Revised Date: 17 July 2020
Accepted Date: 18 July 2020
Please cite this article as: C. Glas, J.C.B. Dietschreit, N. Wössner, L. Urban, E. Ghazy, W. Sippl, M.
Jung, C. Ochsenfeld, F. Bracher, Identification of the subtype-selective Sirt5 inhibitor balsalazide
through systematic SAR analysis and rationalization via theoretical investigations, European Journal of
Medicinal Chemistry, https://doi.org/10.1016/j.ejmech.2020.112676.
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Identification of the subtype-selective Sirt5 inhibitor balsalazide
through systematic SAR analysis and rationalization via theoretical
investigations
Carina Glas a, Johannes C. B. Dietschreit b, Nathalie Wössner c, Lars Urban a, Ehab Ghazy d, Wolfgang
Sippl d, Manfred Jung c, Christian Ochsenfeld b, Franz Bracher a,*
a
Department of Pharmacy – Center for Drug Research, Ludwig-Maximilians University, Butenandtstraße 5-13, 81377
Munich, Germany
b
Chair of Theoretical Chemistry – Department of Chemistry, Ludwig-Maximilians University, Butenandtstraße 7, 81377
Munich, Germany
c
Institute of Pharmaceutical Sciences, Albert-Ludwigs-University Freiburg, Albertstraße 25, 79104 Freiburg im Breisgau,
Germany
d
Institute for Pharmacy, Martin-Luther-University Halle-Wittenberg, Wolfgang-Langenbeck-Straße 4, 06120 Halle (Saale),
Germany
Keywords: balsalazide, sirt5, sirtuins, structure-activity relationships, inhibitor, histone deacylases.
ABSTRACT: We report here an extensive structure-activity relationship study of balsalazide, which was previously identified in a
high-throughput screening as an inhibitor of Sirt5. To get a closer understanding why this compound is able to inhibit Sirt5, we
initially performed docking experiments comparing the binding mode of a succinylated peptide as the natural substrate and
balsalazide with Sirt5 in the presence of NAD+. Based on the evidence gathered here, we designed and synthesized 13 analogues of
balsalazide, in which single functional groups were either deleted or slightly altered to investigate which of them are mandatory for
high inhibitory activity. Our study confirms that balsalazide with all its given functional groups is an inhibitor of Sirt5 in the low
micromolar concentration range and structural modifications presented in this study did not increase potency. While changes on the
N-aroyl-β-alanine side chain eliminated potency, the introduction of a truncated salicylic acid part led to similar potency.
Calculations of the associated reaction paths showed that the inhibition potency is very likely dominated by the stability of the
inhibitor-enzyme complex and not the type of inhibition (covalent vs. non-covalent). Further in-vitro characterization in a trypsin
coupled assay determined that the tested inhibitors showed no competition towards NAD+ or the synthetic substrate analogue
ZKsA. In addition, investigations for subtype selectivity revealed that balsalazide is a subtype-selective Sirt5 inhibitor, and our
initial SAR and docking studies pave the way for further optimization.
succinyl groups from lysine residues, as well as ADP-
ribosylation.[7] Their wide-ranging activity results in an
involvement in many biological processes such as metabolic
or transcriptional regulation, DNA repair, genome stability,
cell survival, aging processes, and stress response, which is
summarized by recent reviews.[7-10] Dysregulation of sirtuins
has been associated with metabolic diseases,[11-12]
cancer,[13-16] and neurodegeneration[17-20], and also life
span extension.[21-22]
In general, targeted modulation of KDACs by small
molecule inhibitors offers an opportunity for both studying the
affected cellular processes (inhibitors as „chemical tools“) and
developing innovative drugs (inhibitors as potential
therapeutics).[5]
In continuation of our previous work on the
development[23-24] and biological characterization[25-28] of
sirtuin inhibitors, we now focused on the isoform Sirt5 which
is, like Sirt3 and Sirt4, mainly located in the mitochondria.[29-
30] To date, the physiological role of Sirt5 is still not fully
understood, but recent studies identified its involvement in
1. Introduction
Posttranslational modifications (PTMs) of proteins have a
strong influence on the regulation of protein function, and
therefore cellular processes. One of the most abundant PTMs
is the bioreversible ε-N-acylation of lysine side chain amino
groups of histones and other cellular proteins.[1] There are 18
human lysine deacylases (KDACs), which are divided into
Zn2+-dependent KDACs consisting of classes I, II, and IV, as
well as NAD+-dependent deacylases constituted by class III
KDACs. The latter are also known as sirtuins due to their
homology to the yeast silent information regulator
(SIR2).[1-3]
2
Sirtuins comprise seven isoforms in mammals (Sirt1 to
Sirt7) with different cellular localization.[4] They are highly
conserved from bacteria to humans.[5] Sirtuins have a
catalytic core that consists of an NAD+-binding Rossmann-
fold subdomain and a small Zn2+-binding module; the active
site is located between those two subdomains.[6] Sirtuins not
only catalyze lysine deacetylation reactions but also the
removal of several other PTMs including myristoyl and

various biochemical pathways.[7] Sirt5 is known to regulate
several metabolic enzymes such as the carbamoylphosphate
synthetase I (CPSI), the rate limiting enzyme of the urea cycle,
which is important in ammonia detoxification.[31-32]
Figure 1: Reported IC50 values of small molecule Sirt5 inhibitors identified by Guetschow et al. in an HTS using MCE.[33]
Furthermore, 3-hydroxy-3-methylglutaryl-CoA synthetase 2
(HMGCS2), involved in ketone body formation, is
Furthermore, selectivity has not been determined sufficiently
for all of them. Relevant published small-molecule Sirt5
inhibitors, including the physiological pan-sirtuin inhibitor
nicotinamide, are shown in Figure S1. There are also several
reported peptidic/peptidomimetic inhibitors.[1, 49, 52-53]
For our research towards novel inhibitors of Sirt5, we
selected a compound which was identified by Guetschow et
al. in 2016[33] via high-throughput screening using microchip
electrophoresis (MCE). The authors screened a library of
1,280 compounds and found the eight promising Sirt5
inhibitors (at least 50 % inhibition of Sirt5 activity at 10 µM,
except antimycin), shown in Figure 1.[33] From those
compounds we selected balsalazide with its published IC50 of
3.9 µM[33] as a lead structure. Other screening hits were not
considered due to their undesired (anti)hormonal (fulvestrant,
a
substrate.[32, 34] Also, Sirt5 plays a role in reducing reactive
oxygen species (ROS) by deacylating proteins such as
SOD1,[35] IDH2, and G6PD[36-37]. Through additional
substrates, Sirt5 is involved in glycolysis[38], tricarboxylic
acid cycle,[38-39] fatty acid metabolism,[38-40] and cellular
respiration[39]. Dysregulation of Sirt5 can cause metabolic
disorders,[41-42] cancer,[43-44] and Alzheimer’s disease or
Parkinson’s disease.[45-46] Consequently, highly selective
and potent low-molecular inhibitors of Sirt5 activity are
urgently needed as chemical tools for in-depth investigation of
the physiological role of Sirt5, as well as exploring this
enzyme as a target for drug therapy.
Previous studies revealed that Sirt5 has only weak
deacetylase activity, but effectively removes acyl residues
derived from dicarboxylic acids (like succinyl or malonyl)
from ε-N-acyl-lysine residues in vitro[39, 47] and in vivo[29,
39]. In contrast to deacetylating KDACs, the main feature in
the active pocket of Sirt5 is an additional binding site for a
negatively charged carboxylate group of the substrate through
an interaction with the supposedly catalytically important and
unique side chains of Arg105 and Tyr102.[5, 47-49]
Therefore, the rational design of selective inhibitors of Sirt5 is
mainly driven by the introduction of an additional, suitably
located carboxylate group for utilization of this crucial
interaction via electrostatic interactions and hydrogen
bonds.[50] Until now, only a few small-molecule Sirt5
inhibitors are known, many of which contain reactive
functional groups (like enones, arylidene(thio)barbiturates)
typical for Pan-assay interference compounds (PAINS).[51]
thyroxine)
or
antimicrobial
activities
(closantel,
methacycline), toxicity (antimycin, anthralin) or stability
issues (probucol, anthralin). Balsalazide (erroneously named
basalazide by Guetschow et al.) is an approved drug which is
actually used for treatment of inflammatory bowel disease.
This compound has very poor bioavailability from the gut,
which is desirable for treatment of inflammatory bowel
disease[54], but is a major handicap for intended systemic
therapy of other diseases. Consequently, balsalazide is not a
suitable compound for simple “drug repurposing”[55-56], but
needs significant optimization in the course of attempted
development as a drug for systemic use.
2. Results and discussion
Chemistry
2

In a first step, we intended to verify the inhibitory activity
of balsalazide on Sirt5. Therefore, we synthesized this
compound (Scheme 1) and determined its IC50 in a Sirt5
fluorometric enzyme assay (Figure 2).
Figure 2: Inhibition of Sirt5 desuccinylase activity by balsalazide
determined using the FLUOR DE LYS® SIRT5 fluorometric drug
discovery assay kit; AFU = absolute fluorescent units.
To get a closer understanding why this compound is able to
inhibit Sirt5, we analyzed the interaction of a co-crystallized
succinyl-lysine based peptide as a reported Sirt5 substrate
(PDB 3RIY) (Figure 3a) and carried out docking calculations
for the inhibitor balsalazide (Figure 3b) in the presence of
NAD+.
As mentioned above, the main binding feature of substrate
proteins to Sirt5 is the interaction between a carboxylate group
with the side chains of Arg105 and Tyr102. This is also
clearly reproduced by our docking calculations (docking
scores and interaction energies of all docked inhibitors are
listed in Table S2 in the Supporting Information). According
to these, not only the succinylated substrate peptide, but also
balsalazide is able to interact with the protonated basic Arg105
of Sirt5 via its carboxylate group. In addition, balsalazide
shows three hydrogen bond interactions with a hydroxy group
in the ribose subunit of NAD+ as well as with the backbone
carbonyl groups of Val221 and Glu225 (Figure 3b). No
significant interactions with the azo linker were detected by
the docking calculations. Hence, we concluded that the side
chain of balsalazide, derived from β-alanine with its
carboxylate group, significantly contributes to the affinity, and
in consequence its inhibitory activity, whereas, the role of the
salicylic acid part of balsalazide (which also bears a
carboxylate group) remained to be elucidated.
Scheme 1: Synthesis of balsalazide as representative for the azo
coupling procedure employed for analogues carrying a free phe-
nolic hydroxy group in p-position (1 and 5 - 13). For the prepara-
tion of 1, phenol was used as the coupling partner in the azo
coupling instead of salicylic acid (SA). For 8, 9, and 10 an addi-
tional final deprotection step was required. For thiourea 12 the
side chain was constructed after azo coupling. Detailed proce-
dures and yields are given in the Supporting Information.
For the synthesis of balsalazide we followed published
procedures [57-58] starting from 4-nitrobenzoyl chloride. The
β-alanine derived side chain was constructed with 56 % yield.
Subsequent hydrogenation gave primary amine S2 in 75 %
yield. The final azo coupling could be performed under
standard azo coupling conditions without the need of further
purification. Balsalazide was obtained with 85 % yield and
was then tested for its inhibitory activity against Sirt5.
We found that balsalazide is indeed an inhibitor of Sirt5
with an IC50 of 5.3 µM in our assay system (Figure 2).
Figure 3: Interaction of the co-crystallized succinyl-lysine based substrate peptide with Sirt5 (a) and docking results obtained for the small
molecule inhibitor balsalazide (b) in the presence of NAD+. In both, the terminal carboxylate group interacts with side chain groups of
3

Arg105 (R105; guanidinium) and Tyr102 (Y102; phenol). The benzamide group of the side chain in balsalazide as well as the aliphatic
amide in the succinylated peptide further interact with a hydroxy group in the ribose subunit of NAD+ (colored salmon) as well as with the
backbone carbonyl group of Val221 (V221). Hydrogen bonds are shown as black lines and distances are given in Å.
To further investigate which functional groups of
balsalazide are crucial for high inhibitory activity, we
systematically designed a series of analogues, in which single
functional groups were either deleted or minimally altered. As
shown in Figure 4, we first removed functional groups from
the salicylic acid part of the molecule, resulting in phenol 1,
benzoic acid 2, and phenyl analogue 3. Further variations
affected the N-aroyl-β-alanine side chain: truncated
derivatives, the carboxylic acid 4, and primary amide 5 are
missing the β-alanine unit (with the putatively crucial distal
carboxylate group), whereas in compound 6 only the distal
carboxylic acid is deleted. The role of the benzamide moiety
was investigated by replacing the carboxamide with an
ethylene unit (7), by creating a secondary benzylamine (8),
and a benzyl ether (9). Deletion of the amide nitrogen led to
the aromatic ketone 10 and the secondary alcohol 11. Another
analogue (12) was designed inspired by a publication of Zang
et al. from 2015 [59], who postulated thiourea containing
inhibitors to be highly effective “warheads” for Sirt5
inhibition (in this context see also a subsequent study by
Rajabi et al. [60]). In comparison to natural N-succinylated
substrates of Sirt5, the amide in balsalazide is inverted. Thus,
we further synthesized an analogue of balsalazide (13)
carrying an inverted amide in the form of an N-succinylated
aromatic amine.
The syntheses of these analogues, all of which still contain
the core azo group, were performed in the broadest sense
following the synthetic procedure of balsalazide depicted in
Scheme 1 with an azo coupling of an in-situ prepared
arenediazonium salt and an electron-rich arene as the central
step. For the preparation of the aniline precursors needed for
the formation of the arenediazonium salts, synthetic protocols
derived from literature were used (for details see Supporting
Information). For analogues 8, 9, and 10 simple additional
deprotection steps (ester hydrolysis for 8 and 10, Boc
deprotection for 9) were required after the azo coupling. In
case of 12, the thiourea containing side chain had to be
introduced after azo coupling due to instability under azo
coupling conditions. Only in 2 and 3 the azo unit had to be
constructed via a different route as shown in Scheme 2.
In contrast to analogues bearing a phenolic OH group,
compounds 2 and 3 had to be synthesized using a Mills
reaction that comprises the oxidation of an aniline to an
aromatic
nitroso
derivative
using
oxone®
(i.e.,
2KHSO5•KHSO4•K2SO4), followed by condensation with
building block S2 (see Scheme 1) to give the azobenzenes.
All synthetic procedures are described in detail in the
Supporting Information (Part A).
Inhibitory activity
To compare the inhibitory activity of all synthesized
derivatives with balsalazide, we used an enzyme-based
fluorometric assay system according to manufacturer’s
instructions. The assay procedure has two steps: In a first step
the FLUOR DE LYS®-Succinyl substrate is incubated with
human recombinant Sirt5, co-substrate NAD+, and the
potential inhibitor. In a second step the desuccinylated
FLUOR DE LYS®-Succinyl is treated with the FLUOR DE
LYS® developer to produce a fluorophore (λex = 390 nm, λem
=
460 nm; see Supporting Information).
All compounds, including balsalazide and the known Sirt5
inhibitors nicotinamide and suramin, were tested at a final
concentration of 50 µM. The results shown in Figure 5 (and
Figure
4 for easy structural comparison) reveal that
balsalazide is in fact a potent inhibitor of Sirt5, and some of
the synthesized analogues (1, 2, 3) show comparable
inhibitory activity. The known Sirt5 inhibitors nicotinamide
and suramin are less active than balsalazide. On a second look,
one can clearly identify that the carboxylic acid in the β-
alanine-derived side chain has to be present and in an
appropriate distance from the aromatic ring (see poorly active
analogues 4, 5, and 6 missing this carboxylate), which
strongly supports the postulated binding mode via interaction
of a carboxylate with Arg105 and Tyr102 (Error! Reference
source not found.).
Variations of the benzamide unit in the side chain also lead
to reduced inhibitory activity (7, 8, 9, 11) with no major
differences between variants with no hetero atoms (7), an
ether as a hydrogen bond acceptor (9), a secondary amine as a
hydrogen bond donor and acceptor (8), or a secondary alcohol
(11). With keto analogue 10, we observed the weakest drop in
activity in this subgroup, which is in accordance with the
postulated hydrogen bond of the corresponding amide
carbonyl group of balsalazide to NAD+ (Error! Reference
source not found.). The thiourea-based side chain in 12 did in
our study, in contrast to previous claims[59], not lead to
higher affinity. The inverted amide in 13 did also lead to
reduced inhibitory activity, once again supporting the
postulated relevance of the hydrogen bond to NAD+.
Interesting results were further obtained from the
compounds containing changes on the salicylic acid part of the
lead structure. The inhibitory activity of benzoic acid 2 (E/Z
ratio at the azobenzene unit of 90:10) and phenyl analogue 3
(E/Z ratio of 89:11) dropped considerably, but not to the extent
observed before for the modifications of the β-alanine-derived
side chain. This leads to the conclusion that changes on the
salicylic acid part have a weaker impact on Sirt5 inhibition.
Surprisingly, phenol 1 showed less significant loss of activity,
Scheme 2: Mills coupling reactions employed for analogues 2
and 3.
4

further demonstrating that changes on the salicylic acid part
are tolerated to some extent.
Figure 4: Balsalazide and its synthesized analogues to investigate structure-activity relationships with Sirt5 inhibition in % at 50 µM/IC50
values provided below each structure.
cence units; λex = 390 nm, λem = 460 nm; number of biological
⃝
replicates stated with circles ( ) for each bar.
The highly conserved activity of phenol 1 compared to 2
and 3 can, at least in part, be attributed to the fact that para-
hydroxy azobenzenes show fast thermal cis-to-trans
isomerisation in protic polar solvents[61] and therefore appear
configurationally stable in the E-configuration.
This also confirms the binding mode seen in the docking
calculations for E-configured balsalazide (Figure 3b). With 1
we could clearly verify that the carboxylic acid relevant for
high affinity binding is indeed the one in the β-alanine side
chain, not the one in the salicylic acid subunit.
To summarize, the compounds that showed no major drop
in inhibitory activity compared to the screening hit balsalazide
were 1, 2, and 3. Those compounds were, along with one
compound (negative control) that showed very poor inhibition
(8), further tested for inhibition of Sirt1, 2, and 3 at 10, 50, and
100 µM using an acetylated synthetic substrate[62] to identify
their subtype selectivity over class I sirtuins. The results
Figure 5: Residual Sirt5 enzyme activity after 1 h incubation with
the inhibitors in % (Finh/Fctrl). The known non-selective inhibitors
nicotinamide and suramin were included as positive controls.
Fluorescence measured upon incubation with the co-solvent
DMSO alone (no inhibitor) was set to 100 % enzyme activity. All
inhibitors were tested at 50 µM final concentration. F = fluores-
5

revealed some subtype selectivity for all tested balsalazide
analogues (Table 1 and Table S1).
antimicrobial effects (tested against diverse bacteria and fungi)
in our routine testing (see Supporting Information).
Table 1: Summarized enzyme inhibition of balsalazide, selected
analogues, nicotinamide, and suramin against Sirt1, 2, 3, and 5 at
*
50 µM. n.i. = less than 10 % inhibition, a = assay system devel-
oped by Heltweg et al.[63], b = FLUOR DE LYS® SIRT5 fluo-
rometric drug discovery assay kit.
Inhibition (at 50 µM compound con-
centration)
Compound
Sirt1a
n.i.*
n.i.
Sirt2a
Sirt3a
Sirt5b
83 %
73 %
63 %
62 %
27 %
balsalazide
11 %
26 %
21 %
16 %
19 %
n.i.
1
16 %
n.i.
2
3
n.i.
n.i.
n.i.
8
n.i.
11 %
nicotina-
mide
suramin
21 %
95 %
65 %
72 %
48 %
10 %
67 %
53 %
In detail, negative control 8, which was in this set the
weakest inhibitor of Sirt5 (27 % at 50 µM) was even less
potent on Sirt1, 2, and 3. For the inhibitors 2 and 3, which lack
the phenolic OH group, we saw 63 % and 62 % Sirt5
inhibition at 50 µM, respectively. Both compounds showed no
inhibition of Sirt1 and Sirt3 even at 100 µM inhibitor
concentration (see Table S1). Furthermore, with 21 % and
16 % inhibition at 50 µM (and less than 25 % inhibition at
100 µM), 2 and 3 had very low inhibitory activity against
Sirt2. Phenol 1 showed relatively strong Sirt5 inhibition (73 %
at 50 µM) but was slightly less subtype selective than the
other tested active analogues. At 50 µM balsalazide exhibited
very good selectivity for Sirt5. No inhibition of Sirt1 and Sirt3
and only marginal (11 %) Sirt2 inhibition was observed at this
concentration. Therefore, balsalazide is still the best inhibitor
of Sirt5 with 83 % at 50 µM and subtype selectivity over
Sirt1, 2, and 3.
To further investigate the inhibition of Sirt5 by balsalazide,
competition towards the cofactor NAD+ and the synthetic
substrate Z-Lys(succinyl)-7-amino-4-methylcoumarin (ZKsA)
were tested in a trypsin-coupled assay (Figure 6). For
comparison, also compound 1 as the most potent analogue of
balsalazide and compound 8 as a negative control were
examined. As expected, since 8 does not exhibit noteworthy
inhibition of Sirt5, substrate conversion remained unchanged
as compared to a positive control containing only DMSO
instead of an inhibitor. For varying NAD+ concentrations as
well as varying ZKsA concentrations, application of
balsalazide and 1 at 50 µM lead to overall decreased ZKsA
conversion. For competitive inhibitors conversion would
converge towards that of the DMSO control with increasing
concentrations of NAD+ and ZKsA, respectively, which was
not observed for neither balsalazide nor 1. Therefore, there is
no competition between these inhibitors and NAD+ or ZKsA.
This finding is in accordance with our docking studies,
especially for NAD+, which was postulated to engage in a
hydrogen bond with balsalazide. In general, it is possible that
balsalazide does compete with larger peptide substrates even
though it could not be displaced by the small molecule
substrate ZKsA.
Figure 6: In-vitro characterization of balsalazide, phenolic ana-
logue 1, and negative control 8 by evaluation of competition with
NAD+ or the synthetic Sirt5 substrate ZKsA; (a) inhibitors (50
µM) and enzyme were incubated with increasing concentrations
of NAD+ , showing that the inhibitors are not competitive towards
NAD+; (b) inhibitors (50 µM) and enzyme were incubated with
increasing concentrations of the synthetic substrate ZKsA, show-
ing that the inhibitors are not competitive towards ZKsA.
Quantum-chemical calculations
As we have investigated the reaction mechanism of Sirt5 in
a previous study[64], we aimed to classify the mechanism of
inhibition by the inhibitors developed in this project. The
desuccinylation reaction proceeds via several steps (see
Scheme 3 and Supporting Information for illustrated reaction
steps with selected inhibitors).
The first step is the cleavage of the glycosidic bond in
NAD+ by a nucleophilic attack of the substrate’s amide
carbonyl oxygen. The second step is a base-mediated
cyclization to a cyclic orthocarboxylic acid derivative. For a
detailed investigation of the reaction mechanism see, e.g.,
Zhou et al.[65] and von der Esch et al.[64], or Wang et al. for
Sirt2[66].
In addition to these results, neither balsalazide nor the
analogues shown in Figure
4 showed cytotoxic or
6

Scheme 3: Exemplary reaction step 1 and step 2 for the reaction
catalyzed by Sirt5 with an N-succinyl lysine model substrate.
Shown are only the reactants and His158 (the only protein residue
that actively takes part in the reaction). The used reaction coordi-
nate is highlighted in red. Other reaction arrows are not explicitly
included in the reaction coordinate.
Figure 7: Reaction curves of the first two reaction steps (reaction coordinate I and reaction coordinate II correspond to the red arrows in
Scheme 3) of the deacylation reaction catalyzed by Sirt5. The paths were using adiabatic mapping in a QM/MM setup using B3LYP-
D3/def2-svp. As substrates we considered a) the model substrate, b) the lead structure balsalazide, c) balsalazide with an inverted peptide
bond (13), and d) the thiourea derivative 12 that acts likely as covalent inhibitor.
To make our approach comparable within this study, we
computed the energy of the reaction for an analogue of the
natural substrate (an ε-N-succinylated peptide), balsalazide,
the balsalazide analogue with an inverted side chain amide
group (13) (which in its structure is closer related to
succinylated protein substrates), and the thiourea analogue
(12) in the same manner.
We employed the docking result of each compound to the
crystal structure of Sirt5 (PDB: 3RIY) as starting point. For all
four substrates, we calculated the reaction curve for the first
two reaction steps via adiabatic mapping in a QM/MM setting
using B3LYP-D3/def2-svp (for computation details see
Supporting Information). The results are shown in Figure 7.
The computed activation barrier of the first step is relatively
high for all four compounds, but due to the identical
computational protocol among the four investigated
compounds they can easily be compared.
The peptide substrate clearly shows a stable intermediate
after both reaction steps (Figure 7a), whereas balsalazide does
not seem to form a stable intermediate, as expected for a non-
covalent inhibitor (Figure 7b). The simulations of the
virtually inactive compound 13 suggest that it is also an
inhibitor due to its extremely high first barrier (Figure 7c),
whereas we expected it to have a lower barrier and be
deacylated like the natural substrate (due to its N-succinyl
motif closely related to natural substrates of Sirt5). The
thiourea analogue (12) is apparently an irreversible inhibitor,
as it forms an extremely stable product after the first reaction
step. This is in complete accordance with the results of
He[52], Zhou[65], and Zang[59] who suggest thio-derivatives
as reactive warheads for the active site of sirtuins.
The higher strain due to the bulkiness of the benzene ring in
13 as compared to the small lysine side chain of the
succinylated peptide is likely the reason for the higher reaction
barrier of 13. In the case of balsalazide, the increased distance
between the side chain amide carbonyl group and NAD+, due
to the inversion of the peptide bond (compared to the natural
N-succinyl substrate), adds to the instability of the first
intermediate, rendering it a non-covalent inhibitor.
enzyme complex and not the aspect whether the inhibitor
binds covalently or not. However, those interactions cannot be
analyzed by the present computations.
3. Conclusion
In conclusion, we selected balsalazide from a high-
throughput screening of potential Sirt5 inhibitors based on its
drug-like structure and first inhibitory activity results. After
having identified a potential binding mode via docking
calculations, we performed extensive structure-activity
relationship studies. By designing and synthesizing 13
analogues along with balsalazide we investigated which
functional groups are crucial for high inhibitory activity. In
each analogue, single functional groups were either deleted or
minimally altered. With these analogues we performed
fluorescence based enzyme assays for determination of their
inhibitory activity against Sirt5 and also to identify their
subtype selectivity over sirtuins 1, 2, and 3. We could reveal
that no structural feature of balsalazide should be
lightheadedly changed in order to retain Sirt5 inhibitory
activity. Changes on the salicylic acid part are tolerated to
some extent, whereas even minor modifications on the β-
alanine-derived side chain lead to complete loss of potency.
Further in-vitro characterization was conducted in a trypsin
coupled assay to determine the inhibitors’ (balsalazide, phenol
1 and negative control 8) competition towards the cofactor
NAD+ and the synthetic substrate ZKsA. The inhibitors
showed no competition towards NAD+ or ZKsA.
Through calculations of the reaction paths of selected
compounds, we concluded that the inhibition strength is very
likely dominated by the interaction of functional groups
increasing the stability of the inhibitor-enzyme complex and
not the aspect what kind of inhibitor (covalent/non-covalent) it
is.
Our study now reveals that balsalazide with all its given
functional groups is a potent inhibitor of Sirt5 with an IC50 of
5.3 µM with proven subtype selectivity over Sirt1, 2, and 3.
This means balsalazide has a higher potency than most
published inhibitors which do not exceed IC50 values of 45
µM and lies within the range of Sirt5 inhibitors by Liu et al.
and Maurer et al. (IC50 values < 10 µM). However, these latter
potent Sirt5 inhibitors either lack subtype selectivity or have
not been investigated thoroughly in this aspect. Balsalazide
does, in contrast to these inhibitors, not contain reactive
functional groups typical for Pan-assay interference
The comparison to the experimental results presented in the
previous section suggests that the mode of inhibition or the
amount by which the activation barrier is raised is not decisive
for the compound’s property as inhibitor. The inhibition
strength is very likely dominated by the interaction of
functional groups increasing the stability of the inhibitor-
7

compounds (PAINS) and therefore represents a useful
chemical tool for investigation of the physiological roles of
Sirt5.
(E)-5-((4-((2-Carboxyethyl)carbamoyl)phenyl)diazenyl)-2-
hydroxybenzoic acid (balsalazide)[57]
S2 (100 mg, 0.480 mmol) was suspended in H2O (1.1 mL),
before conc. HCl (0.14 mL) was added. The mixture was
cooled to 0 °C and a cold solution of NaNO2 (34.1 mg, 0.495
mmol) in water (0.25 mL) was added dropwise. The mixture
was stirred for 1 h and then added dropwise to a solution of
salicylic acid (68.3 mg, 0.495 mmol) dissolved in water (0.83
mL) containing sodium hydroxide (40.3 mg, 1.01 mmol) and
sodium carbonate (81.4 mg, 0.768 mmol) at 0 °C. The final
However, balsalazide is not a promising candidate for
application as a drug targeting Sirt5, due to its poor water
solubility, minimal absorption from the gut, and liability for
enzymatic degradation by gut bacteria under reductive
cleavage of the azo moiety[54]. Therefore, we intend to
further vary functional groups on our lead structure and
evaluate these inhibitors to not only increase the inhibitory
activity while maintaining subtype selectivity but also to
change the structure into a drug candidate with appropriate
physicochemical properties and high physiological stability
for systemic use.
reaction mixture was adjusted to pH 8 with 2
N aq. NaOH and
stirred for 3 h, then poured into 0.5 aq. HCl at 0 °C. The
N
precipitate obtained was collected by filtration, washed with
water and suction dried to give balsalazide (146 mg, 0.409
mmol, 85 %) as
a
brown solid. Rf: 0.53
(EtOAc/hexanes+AcOH 30:70+1). m.p.: 239 °C (literature
254 – 255 °C[57]). 1H-NMR (500 MHz, (CD3)2SO): δ (ppm) =
12.29 (s, 2H, 1-COOH and 3’’-COOH), 8.70 (t, J = 5.4 Hz,
1H, CONH), 8.36 (d, J = 2.6 Hz, 1H, 6-H), 8.09 (dd, J = 8.9,
2.6 Hz, 1H, 4-H), 8.02 (d, J = 8.5 Hz, 2H, 3’-H and 5’-H),
7.92 (d, J = 8.5 Hz, 2H, 2’-H and 6’-H), 7.15 (d, J = 8.9 Hz,
1H, 3-H), 3.49 (td, J = 7.1, 5.4 Hz, 2H, 1’’-H), 2.54 (t, J = 7.1
Hz, 2H, 2’’-H). 13C-NMR (126 MHz, (CD3)2SO): δ (ppm) =
172.9 (C-3’’), 171.2 (1-COOH), 165.5 (CONH), 164.3 (C-2),
153.3 (C-1’), 144.4 (C-5), 136.1 (C-4’), 128.9 (C-4), 128.5 (C-
3’ and C-5’), 126.1 (C-6), 122.1 (C-2’ and C-6’), 118.5 C-3),
114.2 (C-1), 35.7 (C-1’’), 33.7 (C-2’’). IR: ṽ (cm-1) = 3067,
1702, 1658, 1634, 1539, 1450, 1302, 1198, 856, 752. HR-MS
4. Experimental
Full and detailed experimental synthetic and biological
protocols as well as description of computational methods and
docking results can be found in the Supporting Information.
Representative compound synthesis, balsalazide[57-58].
3-(4-Nitrobenzamido)propanoic acid (S1)[57-58]
β-Alanine (2.00 g, 22.4 mmol) was added to a solution of
1
N NaOH (50 mL) at 0 °C. After the addition of finely
powdered 4-nitrobenzoyl chloride (4.17 g, 22.4 mmol) the
solution was stirred for 30 min at 0 °C and was then allowed
to warm up to rt over a period of 2 h. Subsequently, excessive
starting material was filtered off and the filtrate was adjusted
-
(ESI): m/z = [M-H]- calcd for C17H14N3O6 : 356.0888; found:
to pH 2-3 through the addition of 1
N aq. HCl at 0 °C. The
356.0889. Purity (HPLC): 210 nm: >95 %; 254 nm: >95 %.
resulting precipitate was collected, washed with water and
dried under reduced pressure. The crude product was
recrystallized from hot acetone to give S1 (3.20 g, 13.4 mmol,
60 %) as a white solid. Rf: 0.53 (EtOAc/hexanes+AcOH
FLUOR DE LYS® SIRT5 fluorometric drug discovery
assay kit. For screening the inhibitory activity of synthesized
inhibitors against recombinant human Sirt5 a commercially
available Sirt5 fluorometric drug discovery kit (BML-AK513)
from ENZO LIFE SCIENCES (ELS) AG (Lausen,
Switzerland) was used. Fluorescence was measured on a
FLUOstar Omega microplate reader (BMG Labtech,
Ortenberg, Germany) (excitation wavelength: 355 nm,
emission wavelength: 460 nm). Graphs were generated using
GraphPad Prism 8.0 software (La Jolla, CA).
1
30:70+1). m.p.: 163 °C. H-NMR (500 MHz, (CD3)2SO): δ
(ppm) = 12.26 (s, 1H, COOH), 8.87 (t, J = 5.4 Hz, 1H,
CONH), 8.34 – 8.29 (m, 2H, 3’-H and 5’-H), 8.08 – 8.03 (m,
2H, 2’-H and 6’-H), 3.48 (td, J = 7.1, 5.4 Hz, 2H, 3-H), 2.54
(t, J = 7.0 Hz, 2H, 2-H). 13C-NMR (126 MHz, (CD3)2SO): δ
(ppm) = 172.8 (C-1), 164.6 (CONH), 149.0 (C-4’), 140.0 (C-
1’), 128.7 (C-2’ and C-6’), 123.5 (C-3’ and C-5’), 35.8 (C-3),
33.5 (C-2). IR: ṽ (cm-1) = 3374, 2526, 1714, 1625, 1590, 1545,
1519, 1408, 1348, 1304, 1277, 1207, 878, 851, 727, 608. HR-
Associated Content
Supporting Information accompanies this paper: (A)
chemical synthesis; (B) biological data; (C) docking; (D)
QM/MM simulations; (E) NMR spectra.
MS (ESI): m/z = [M-H]- calcd for C10H9N2O5 : 237.0517;
found: 237.0519. Purity (HPLC): 210 nm: >95 %; 254 nm:
>95 %.
-
3-(4-Aminobenzamido)propanoic acid (S2)[58]
S1 (2.69 g, 11.3 mmol) was dissolved in dry MeOH (38
mL) and Pd/C (10 wt% on activated carbon, 269 mg) was
added under N2 atmosphere. Hydrogenation was performed
under 1 bar H2 pressure at rt for 3 h. Subsequently, the
reaction mixture was filtered through a pad of celite and the
solvent was removed in vacuo to give S2 (1.76 g, 8.44 mmol,
75 %) as a white solid. Rf: 0.17 (MeOH/DCM 5:95). m.p.: 153
Author information
Corresponding Author
* F.B.: franz.bracher@cup.uni-muenchen.de
ORCIDs
Carina Glas: 0000-0002-5014-4921
1
°C. H-NMR (400 MHz, (CD3)2SO): δ (ppm) = 12.19 (s, 1H,
Johannes C.B. Dietschreit: 0000-0002-5840-0002
Nathalie Wössner: 0000-0002-4402-2442
Wolfgang Sippl: 0000-0002-5985-9261
Manfred Jung: 0000-0002-6361-7716
Christian Ochsenfeld: 0000-0002-4189-6558
Franz Bracher: 0000-0003-0009-8629.
COOH), 8.02 (t, J = 5.5 Hz, 1H, CONH), 7.57 – 7.52 (m, 2H,
2’-H and 6’-H), 6.54 – 6.49 (m, 2H, 3’-H and 5’-H), 5.70 –
5.42 (m, 2H, NH2), 3.39 (td, J = 7.2, 5.5 Hz, 2H, 3-H), 2.46 (t,
J = 7.2 Hz, 2H, 2-H). 13C-NMR (101 MHz, (CD3)2SO): δ
(ppm) = 173.0 (C-1), 166.2 (CONH), 151.6 (C-4’), 128.6 (C-
2’ and C-6’), 121.1 (C-1’), 112.5 (C-3’ and C-5’), 35.4 (C-3),
34.1 (C-2). IR: ṽ (cm-1) = 1714, 1618, 1595, 1557, 1514, 1340,
1319, 1213, 1197, 1183, 840. HR-MS (ESI): m/z = [M-H]-
Author Contributions
C.G. performed synthesis, enzyme assays, coordinated data
assembly and wrote the manuscript. J.D. performed quantum-
chemical calculations. N.W. performed enzyme assays. L.U.
-
calcd for C10H11N2O3 : 207.0775; found: 207.0775. Purity
(HPLC): 210 nm: >95 %; 254 nm: >95 %.
8

that is overexpressed in thyroid carcinoma cell lines and tissues. Br. J.
Cancer 2002, 86 (6), 917-923, doi: 10.1038/sj.bjc.6600156.
performed synthesis. E.G. performed docking calculations. W.S.
supervised modelling and docking calculations. M.J. supervised
enzyme assays. C.O. supervised quantum-chemical calculations.
F.B. designed the project, supervised synthesis and wrote the
manuscript.
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C.; McLean, P. J.; Young, A. B.; Abagyan, R.; Feany, M. B.; Hyman, B.
T.; Kazantsev, A. G., Sirtuin 2 Inhibitors Rescue α-Synuclein-Mediated
Toxicity in Models of Parkinson's Disease. Science 2007, 317 (5837), 516,
doi: 10.1126/science.1143780.
Notes
The authors declare no competing interests.
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Acknowledgements
This
research
was
funded
by
the
Deutsche
Forschungsgemeinschaft
(DFG, German Research Foundation)
- SFB 1309 - 325871075 (Chemical Biology of Epigenetic
Modifications). MJ, WS and NW thank the DFG for funding
(Ju295/14-1, Si868/15-1; MJ and NW additionally thank
235777276/GRK1976).
We thank Anna Niedrig for determination of HPLC purities
and Martina Stadler for MTT and agar diffusion tests.
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Highlights:
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Docking identifies binding mode of balsalazide with Sirt5 in the presence of NAD+
Balsalazide is a potent subtype-selective Sirt5 inhibitor
Extensive structure-activity relationship study with 13 synthesized azobenzenes
Calculation of associated reactions paths
Sirt5 inhibition is very likely dominated by stability of inhibitor-enzyme complex

Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests: