Mechanism-Based Inhibitors of the Human Sirtuin 5 Deacylase: Structure–Activity Relationship, Biostructural, and Kinetic Insight

Article abstract of DOI:10.1002/anie.201709050

The sirtuin enzymes are important regulatory deacylases in a variety of biochemical contexts and may therefore be potential therapeutic targets through either activation or inhibition by small molecules. Here, we describe the discovery of the most potent inhibitor of sirtuin 5 (SIRT5) reported to date. We provide rationalization of the mode of binding by solving co-crystal structures of selected inhibitors in complex with both human and zebrafish SIRT5, which provide insight for future optimization of inhibitors with more “drug-like” properties. Importantly, enzyme kinetic evaluation revealed a slow, tight-binding mechanism of inhibition, which is unprecedented for SIRT5. This is important information when applying inhibitors to probe mechanisms in biology.

Full text of DOI:10.1002/anie.201709050

A Journal of the Gesellschaft Deutscher Chemiker
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Accepted Article
Title: Mechanism-based Inhibitors of the Human Sirtuin 5 Deacylase:
Structure-Activity Relationship, Biostructural, and Kinetic Insight
Authors: Nima Rajabi, Marina Auth, Kathrin R. Troelsen, Martin
Pannek, Dhaval Bhatt, Martin Fontenas, Matthew D Hirshey,
Clemens Steegborn, Andreas S. Madsen, and Christian Adam
Olsen
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201709050
Angew. Chem. 10.1002/ange.201709050
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10.1002/anie.201709050
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Mechanism-based Inhibitors of the Human Sirtuin 5 Deacylase:
Structure–Activity Relationship, Biostructural, and Kinetic Insight
Nima Rajabi,[a] Marina Auth,[a] Kathrin R. Troelsen,[a] Martin Pannek,[b] Dhaval P. Bhatt,[c] Martin
Fontenas,[a] Matthew D. Hirschey,[c] Clemens Steegborn,[b] Andreas S. Madsen,[a] and Christian A.
Olsen*[a]
Abstract: The sirtuin enzymes are important regulatory deacylases
in a variety of biochemical contexts and may therefore be potential
therapeutic targets through either activation or inhibition by small
molecules. Here, we describe the discovery of the most potent
dehydrogenase (SDH), and 3-hydroxy-3-methylglutaryl-CoA
synthase 2 (HMGCS2).[8] Additionally, SIRT5 is involved in
detoxification of reactive oxygen species, by deacylating
proteins such as SOD1[9], IDH2, and G6PD.[10] Furthermore,
inhibitor of sirtuin
5
(SIRT5) reported to date. We provide
SIRT5 has been implicated in tumor growth in non-small cell
11]
rationalization of the mode of binding by solving co-crystal structures
of selected inhibitors in complex with both human and zebrafish
SIRT5, which provide insight for future optimization of inhibitors with
more “drug-like” properties. Importantly, enzyme kinetic evaluation
revealed a slow, tight-binding mechanism of inhibition, which is
unprecedented for SIRT5. This is important information when
applying inhibitors to probe mechanisms in biology.
lung cancer[10b,
and has an anti-apoptotic effect in
neuroblastoma cells,[12] highlighting the potential of SIRT5 as a
therapeutic target.
Insight into the NAD+-dependent hydrolytic mechanism of the
sirtuins has been exploited for design of highly potent substrate-
mimicking inhibitors that contain thioamide or thiourea
functionalities, forming stalled intermediates with ADPR in the
sirtuin active sites (Scheme 1A).[13]
Sirtuins are a family of NAD+-dependent silent information
regulator 2 (Sir2) enzymes that catalyze the removal of acyl
groups from e-N-amino groups of lysine residues in the
proteome.[1] The human genome codes for seven different sirtuin
isoforms (SIRT1–7), which are classified according to sequence
similarity and localize to different cellular compartments.[2]
Recently, it has become evident that different enzyme isoforms
exhibit preference for different e-N-acyllysine posttranslational
3]
modifications (PTMs).[1c,
Thus, e-N-acetyllysine (Kac)
functionalities are targeted primarily by SIRT1 and 6 in the
nucleus, SIRT2 in the cytoplasm, and SIRT3 in the
mitochondria.[3a] In addition, long chain acyl groups, such as e-N-
myristoyllysine (Kmyr), are also cleaved by SIRT1–3 and 6.[4]
SIRT5 has been shown to selectively cleave e-N-
carboxyacyllysine derivatives based on malonate (Kmal),[5]
succinate (Ksuc),[5b] and glutarate (Kglu).[6] Recently, the ability
of SIRT4 to cleave the negatively charged e-N-(3-
methylglutaryl)lysine (Kmg) and e-N-(3-methylglutaconyl)lysine
(Kmgc) has also been demonstrated.[7]
Although the role of SIRT5 is not fully understood, it has
been shown to regulate several metabolic enzymes, e.g.,
carbamoyl phosphate synthetase
1
(CPS1), succinate
[a]
N. Rajabi, M. Auth, K. R. Troelsen, M. Fontenas, Prof. Dr. A. S.
Madsen, Prof. Dr. C. A. Olsen
Center for Biopharmaceuticals & Department of Drug Design and
Pharmacology
University of Copenhagen
Universitetsparken 2, DK-2100, Copenhagen (Denmark)
E-mail: cao@sund.ku.dk
[b]
[c]
M. Pannek, Prof. Dr. C. Steegborn
Universität Bayreuth
Lehrstuhl Biochemie und Forschungszentrum für Biomakromoleküle
Universitätsstraße. 30, 95447 Bayreuth (Germany)
Dhaval P. Bhatt, Prof. Dr. M. D. Hirschey
Duke University Medical Center
Sarah W. Stedman Nutrition and Metabolism Center
4321 Medical Park Drive, Durham, NC 27704 (United States)
Scheme 1. (A) Sirtuin hydrolytic mechanism. (B) Previous mechanism-based
inhibitor. (C) Inhibitor optimization in this study. X = O or S, NAD+ = reduced
nicotinamide adenine dinucleotide, ADP = adenosine diphosphate, Cbz =
carboxybenzyl.
Supporting information for this article is given via a link at the end of
the document. X-ray: diffraction data and coordinates have been
deposited with the wwPDB (www.wwpdb.org) under accession
numbers 6ENX (zSIRT5/10), 6EO0 (zSIRT5/29), 6EQS (hSIRT5/29).
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Scheme 2. Subset of the structure–activity relationship study, measuring compound potency against recombinant SIRT5 as previously described.[6a, 14] Potencies
are given as IC50 values or %-inhibition at the highest tested concentration (see Figure S1 for dose-response curves). Please consult the Supporting Information
for a list of additional compounds and their potencies (Scheme S1) as well as synthetic Schemes S2–S25.
Taking advantage of the acyl-substrate specificity of SIRT5, this
strategy has been successfully adapted to selectively inhibit this
isozyme (Scheme 1B).[6b, 15] Here, we performed an extensive
iterative structure–activity relationship (SAR) study, evaluating
more than 70 compounds, which furnished SIRT5-selective
inhibitors exhibiting nanomolar affinity via a slow, tight-binding
mechanism. These are the most potent SIRT5 inhibitors
reported to date; however, our study also highlights the
necessity for thorough assessment of inhibitor mechanism and
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calls into question the application of IC50 values as the sole
measure of potency for these chemotypes.
Furthermore, alkylation of the C-terminal amide was beneficial
for potency and extending the structure with an i+2 amino acid
resulted in a slight increase in potency in one case (15).
However, to limit the peptidic nature of the ligand, we chose 10
for further SAR. Next, we explored modifications of the PTM and
the e-amide bond (18–29, Scheme 2, yellow area). Inspired by
work on Kac surrogates by Cole and Denu, introducing
hydrazide[16] and urea[17] functionalities, respectively, we
designed compounds 20 and 21, as well as extended the series
with semicarbazide 22 and carbamate 23. Compound 24 was
inspired by work on fluorinated acetamides,[17-18] and inverted
As starting point, we chose e-N-thioglutaryllysine over e-N-
thiosuccinyllysine due to the lower KM value of glutarylated
substrates.[6a] The a-amino group was kept Cbz protected to
address the C-terminal, first by introduction of a series of amines
(1–6 Scheme 2 and Supporting Scheme S1). Inspired by
examination of co-crystal structures of SIRT5 with a peptide
substrate (PDB 3RIY and 4GIC), we then extended the series to
di- and tripeptides (7–17), addressing the importance of side
chain bulkiness, stereochemistry, and presence of backbone
secondary amide (Scheme 2, gray area). The two latter proved
important with a preference for L-configuration at position i+1 (10
vs 11), while the steric bulk of the side chain had minor effect
(12 vs 14).
amide (25) as well as Glu(Cbz) 26 have been introduced as side
15c]
chains previously.[6b,
Finally, 3-methylglutaryl-mimicking[7]
analogues (27 and 28) were prepared along with compound 29,
the thiourea analogue of 10. Collectively, this exercise showed
that
thioamide-
and
thiourea-based
compounds were the most potent and thus,
compound 29 was chosen for individual
optimization of the N-terminal (30–44, Scheme
2, green area) and modifications to the C-
terminal N-alkyl group (45–47, Scheme 2,
cyan area).
Co-crystal structures of NAD+-derived
intermediates of both lead compounds 10 and
29 in complex with SIRT5 from either zebrafish
(zSIRT5) or man (hSIRT5) were solved
(Supporting Table S1). These structures
revealed detailed insight into the binding
modes of the two compounds (Figure 1A–D).
Both structures of zSIRT5 contained the
compound (10 or 29) bound as bicyclic
intermediate with ADP-ribose, similar to
structure III in Scheme 1A (mixed with a
fraction bound as intermediate II in Scheme
1A for compound 29), and with expected
interactions of the carboxylate with Y98 and
R101 (Figure 1A,B and Supporting Figure S2).
Slight structural deviations between the
complexes were observed in a Zn-domain loop,
the co-factor binding loop, and helix a3,
presumably due to the subtle differences in the
ligand acyl groups. Interestingly, the structure
of 29 in complex with hSIRT5 revealed only
the ADP-ribose-1′-thioimidate intermediate II of
Scheme 1 and no bicyclic intermediate (Figure
1C). However, the protein chains of the
hSIRT5 and zSIRT5 complexes with 29 were
Figure 1. Co-crystal structures resulting from incubation of zSIRT5 with NAD+
and inhibitors 10 or 29 as well as hSIRT5 with NAD+ and inhibitor 29. (A)
Superposition of co-crystal structures of zSIRT5 with either bicyclic
intermediate III (compound 10 shown in pale blue) or an indistinguishable
mixture of bicyclic intermediate III and ADP-ribose-1′-thioimidate intermediate
II (compound 29 shown in pale green). (B) Active site zoom of the zSirt5
almost identical (rmsd 0.31 Å for 225 Ca atoms), and the reason
for partially stalling at different intermediate states remains to be
elucidated. Whereas thioamides have been co-crystallized with
sirtuins previously,[19] these are the first structures with thiourea-
based inhibitors. It is reassuring to observe examples for both
intermediates II and III, confirming that this functionality behaves
similarly to thioamides. Important interactions of the compound
with hSIRT5 were again the glutaryl carboxylate with Y102 and
R105 as well as the e-NH to backbone carbonyl of V221 and
additional backbone–backbone amide interactions (Figure 1C).
Four SIRT5 chains with variations in the rotation of the Cbz and
complexes in panel
A with interactions between protein and ligand
represented as dashed lines. (C) The active site of co-crystal structure of
hSIRT5 and ADP-ribose-1′-thioimidate intermediate II with compound 29.
Hydrogen bonding interactions between protein and ligand are shown as
dashed lines. (D) Surface view of the hSIRT5 complex containing the ADPR–
29 intermediate, showing the different positions of the Cbz and indole moieties,
while the rest of the ligand is tightly bound.
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indole moieties of the ligand were resolved (Figure 1D). These
varying end group conformations are influenced by crystal
packing, and the indole positions indicate a flexibility in the
SIRT5 complex that is in agreement with the minor effect of this
group observed in the SAR.
Furthermore, the observed flexibility of the Cbz group as well
as its lack of specific interactions with the protein surface (Figure
1D) indicated that a variety of functionalities could be tested in
the continued SAR. Thus, for the ease of synthesis, we decided
to investigate the N-terminal relative to compound 30, which is
devoid of the C-terminal i-propyl group, allowing for ready
preparation of the series 30–44 by solid-phase synthesis. We
included amide, urea, and sulphonamide analogues of the Cbz
group, including various lengths (30–37). Due to the potency of
In parallel, we briefly re-investigated the importance of steric
bulk at the C-terminus, now in the context of compound 29 (45–
47, Scheme 2, cyan area). Combining the results of these two
series, we prepared compounds 48–50 in a final iteration,
providing compound 49 as the most potent inhibitor with an
improvement in IC50 value of >100-fold from compound 1. Not
surprisingly based on the well-documented substrate specificity
of SIRT5, selected compounds (29 and 48–50) exhibited
excellent selectivity for SIRT5 over SIRT1–3 and 6 (Figure 2A).
We were then interested in gaining insight into the kinetic
behaviour of our most potent compound (49) along with
intermediate lead compounds 10 and 29 as well as compound 1
and patented compound V (Scheme 1) as a control. To achieve
this, we first performed a continuous assay[14] to establish
37 and the high abundance of sulphonamides in approved drugs, whether the inhibition occurred at steady-state kinetics. Not
we prepared analogues 38–44 as well.
surprisingly, since this has been reported previously for
mechanism-based inhibitors of
SIRT1,[20] compounds 10, 29 and 49
exhibited slow, tight-binding kinetics
(Figure 2B and Supporting Figure S3).
Interestingly, the less potent compounds
1 and V behaved like standard fast-on–
fast-off inhibitors (Figure 2B and
Supporting Figure S3), indicating that
the change in mechanism is not
associated with the thioamide or
thiourea amide bond surrogate, but
rather developing as the backbone-
interacting part of the molecule gains
affinity.
Nevertheless, slow binding could be
associated with interaction with NAD+
and enzyme or enzyme alone, so we
performed pre-incubation experiments
of selected inhibitors and SIRT5 with or
without NAD+ to address these
scenarios and to evaluate whether
compounds 19–25 exhibited slow-
binding as well (Figure 2C). Not
surprisingly, the slow-binding behaviour
of compounds 10, 19, 29 and 49 was
depending on the presence of NAD+,
indicating that it involves formation of
the stalled intermediate. However, it is
intriguing that optimization of the
scaffold’s contribution to affinity imposes
a change in mechanism of inhibition.
Interestingly,
the
urea-containing
compound 21 also exhibited NAD+-
dependent slow-binding, which may
revive the use of this functionality for
future inhibitor design. The remaining
compounds did not exhibit slow-binding
within the time-frame of the pre-
incubation experiments (Figure 2C).
Figure 2. Biochemical evaluation in vitro. (A) Selectivity of compounds 29 and
48–50, measured at an inhibitor concentration of 10 µM. Acyllysines of
substrates used are indicated for individual sirtuins. (B) Progression curves
and data fitting for inhibition of recombinant SIRT5 by compounds 1, 29, and
49. (C) Preincubation experiments of compounds 10, 19–25, 29, and 49.
Finally, we tested an ethylester prodrug version of
compound 29 (Et-29) for its ability to affect the degree of lysine
glutarylation in cells. However, it is unfortunately not trivial to
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Park, J. M. Burneskis, J. C. Barrett, I. Horikawa, Mol. Biol. Cell 2005, 16,
4623-4635.
detect changes in either lysine malonylation, succinylation, or
glutarylation even compared to the control CRISPR-Cas9 SIRT5
knockout cell line (Supporting Figures S4 and S5). Further
optimization of the experimental design and subsequent
evaluation of these compounds in a cellular context will be of
immediate future interest.
In summary, we describe mechanism-based inhibitors of
sirtuin 5 that exhibit up to a 100-fold increase in IC50 values
compared to a patented reference compound included in our
assays. Importantly, we show that kinetic analyses of inhibitors
of these enzymes is important for appropriate comparison of
potencies as we disclose the first examples of slow, tight-binding
behaviour for SIRT5 inhibitors. This calls for more thorough
investigation mechanism-based inhibitors of all sirtuins. We also
describe structural information for the binding mode of thiourea-
based sirtuin inhibitors for the first time, which provides
important insight for future inhibitor design.
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(04115; C.S.), the Lundbeck Foundation (Group Leader
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(2011-01-0169, 2013-01-0333, and CF15-011; C.A.O.), the
Novo Nordisk Foundation (NNF15OC0017334; C.A.O.), and the
European Research Council (ERC-CoG-725172 – SIRFUNCT;
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Conflict of interest
The authors declare no conflict of interest.
Keywords: enzyme inhibitors • sirtuins • posttranslational
modifications • deacylases • drug discovery
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Entry for the Table of Contents
Medicinal Chemistry
COMMUNICATION
SAR study of mechanism-based
inhibitors, combined with structural
insight from X-ray co-crystal
structures provide potent inhibitors
of the sirtuin 5 hydrolase. Kinetic
investigations furthermore reveal
unprecedented slow, tight-binding
behaviour of several compounds.
Nima Rajabi,[a] Marina Auth,[a] Kathrin
R. Troelsen,[a] Martin Pannek,[b]
Dhaval P. Bhatt,[c] Martin Fontenas,[a]
Matthew D. Hirschey,[c] Clemens
Steegborn,[b] Andreas S. Madsen,[a]
and Christian A. Olsen*[a]
Page No. – Page No.
Mechanism-based Inhibitors of
the Human Sirtuin 5 Deacylase:
Structure–Activity Relationship,
Biostructural, and Kinetic Insight
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