A Set of Highly Sensitive Sirtuin Fluorescence Probes for Screening Small-Molecular Sirtuin Defatty-Acylase Inhibitors

Small-Molecular Sirtuin Defatty-Acylase Inhibitors

Letter

A Set of Highly Sensitive Sirtuin Fluorescence Probes for Screening

Yuya Nakajima, Mitsuyasu Kawaguchi,* Naoya Ieda, and Hidehiko Nakagawa*

Cite This: ACS Med. Chem. Lett. 2021, 12, 617 −624

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sı Supporting Information

ABSTRACT: Human sirtuins (SIRT1−7) regulate not only

deacetylation but also deacylation of fatty acid-derived acyl

moieties (defatty-acylation) at the ε-amino group of lysine

residues. SIRT-subtype-speciﬁc defatty-acylase activity modulators

are needed for detailed investigation of the biological roles of these

enzymes, and to ﬁnd suitable small molecules, we require

appropriate screening systems. Here, we designed and synthesized

a set of SIRT defatty-acylase activity probes with various quencher

moieties and peptide sequences based on our previously developed

one-step FRET-based SIRT probe SFP3, using improved method-

ology. Scanning of this set of probes with SIRT isozymes revealed

that certain probe/isozyme combinations showed especially high

responses. To illustrate the utility of the combinations thus

identiﬁed, we applied compound 18/SIRT2 for inhibitor screening of a large chemical library. This enabled us to discover a new

small molecule SIRT2-speciﬁc defatty-acylase inhibitor.

KEYWORDS: sirtuin, ﬂuorescence probes, defatty-acylase activity, screening, inhibitors

uman sirtuins (SIRT1−7) are a family of NAD -

acylase activity plays an important role in cancer proliferation.

However, so far, there has been no report on the physiological

roles of the defatty-acylase activities of SIRT1 and SIRT3.

Therefore, SIRT-subtype-speciﬁc modulators are required both

as tools for basic research to understand the physiological

functions of SIRTs and as candidate therapeutic agents.

So far, SIRT ﬂuorescent probes employing various detection

methods have been developed. Generally, they involve trypsin

digestion of a C-terminal lysine residue after the SIRT

dependent histone deacetylases. SIRTs are involved in

metabolic regulation, stabilization of genomic DNA, stress

−6

responses, and even aging,

and SIRT modulators are

−10

considered to have potential therapeutic value.

For a long

time, it was thought that SIRTs only catalyze deacetylation

reactions of histones and the adenine diphosphate (ADP)-

ribosylation reaction. However, more recently, it was found

that SIRTs can remove various acyl groups, including

propionyl, butyryl, crotonyl, succinyl, hexanoyl, octanoyl,

decanoyl, dodecanoyl, myristoyl, palmitoyl, lipoyl, and benzoyl

from histones and many other protein sub-

SIRT1−3, which have relatively potent deacetylase

activity, can also remove long-chain fatty acyl groups at the ε-

22,23

enzymatic reaction.

However, this requirement is problem-

atic for screening purposes because trypsin inhibitors generate

false-positive signals. Therefore, a one-step procedure for

detection of SIRT activity would be preferable. Some one-step

probes employing an intramolecular reaction mechanism,

1,12

groups,

strates.

3,14

11,12

amino group of lysine residues in vitro.

which aﬀords a ﬂuorescence increase after deacylation of a

Protein fatty-acylation is crucial for anchoring proteins to

the cell membrane and plays important roles in cell signaling

and protein−protein interactions. Early studies of protein fatty

acylation were focused on N-terminal glycine myristoylation

Among human SIRTs, SIRT6

is a prominent defatty-acylase, which regulates secretion of

24−26

lysine residue, have been reported,

but unfortunately, the

background ﬂuorescence signal gradually increases even in the

absence of enzymes. Dai and coworkers developed a Forster

resonance energy transfer (FRET)-based ﬂuorescence probe

15,16

and cysteine palmitoylation.

containing a 2-aminobenzoylamide group at the acyl side chain

17−19

tumor necrosis factor α (TNF-α) and exosomes.

Recent

Received: January 8, 2021

Accepted: March 9, 2021

Published: March 11, 2021

work suggests that the defatty-acylase activity of SIRT2 toward

the ε-amino group of lysine residues regulates the localization

of K-Ras4a oncoprotein and promotes cellular trans-

formation via interaction with A-Raf. In addition to K-

Ras4a, the defatty-acylase activity of SIRT2 regulates the

activity of RalB and cell migration. Therefore, SIRT2 defatty-

2021 American Chemical Society

ACS Medicinal Chemistry Letters

ACS Med. Chem. Lett. 2021, 12, 617−624

Letter

Figure 1. Molecular design strategy and function of newly designed one-step SIRT activity probes employing the FRET mechanism.

as a ﬂuorophore. Its ﬂuorescence is activated by SIRT-

mediated hydrolytic release of the ﬂuorophore moiety, but the

absorption and ﬂuorescence wavelengths of the ﬂuorophore, 2-

aminobenzoylamide, are quite short and may overlap with

those of chemical compounds to be screened. Therefore, there

is still a need for a stable, one-step ﬂuorescence probe with a

longer ﬂuorescence wavelength for chemical screening

purposes.

RESULTS AND DISCUSSION

■

Molecular Design Strategy for New SIRT Activity

Probes. For the development of the new ﬂuorescence probes,

we modiﬁed the original design and synthetic scheme used for

8,29

our previously reported probe, SFP3,

as shown in Figure 1.

In SFP3, the linker and ﬂuorophore were conjugated at the C-

terminus of the H3K9 peptide, which was obtained by solid-

phase synthesis (SPS). However, the linker and ﬂuorophore

moieties were subsequently conjugated in the liquid phase,

which requires an increased number of synthetic steps,

resulting in a low yield and many byproducts. In our new

design, the linker and ﬂuorophore are conjugated at the N-

terminus of the peptide chain, and the scheme was modiﬁed to

allow all the components to be incorporated by means of SPS.

In addition, SFP3 showed unwanted interactions (e.g.,

intramolecular stacking interactions) between the ﬂuorophore

and the quencher group due to its highly ﬂexible propyl linker.

In our new design, the linker structure was changed to a more

rigid cyclohexane ring with the aim of preventing such stacking

interactions and increasing the reactivity with SIRTs.

We have already reported a one-step chemical probe for

SIRT activity, SFP3, which can evaluate the defatty-acylase

activity of SIRT1−3 and SIRT6 (Figure S1). SFP3 is a

FRET-based ﬂuorescence probe containing a 4-(4-

dimethylaminophenylazo)benzoyl (Dabcyl) group as a

quencher, and its ﬂuorescence is activated by SIRT-mediated

hydrolytic release of the Dabcyl moiety, as shown in Figure 1.

The long structure of the Dabcyl group appears to mimic a

long-chain fatty acyl group because the K values of the Dabcyl

28,29

quencher and the myristoyl group are similar.

However,

for purposes such as high-throughput chemical screening and

subtype-speciﬁc imaging of cellular SIRT, it would be useful to

have a set of probes with diﬀerent characteristics. Therefore, in

this work, we set out to build a set of probes incorporating

various peptide sequences and quenchers using improved

synthetic methodology (Figure 1). We evaluated the kinetic

parameters of the obtained probes toward SIRT1−7 and

compared them with the corresponding parameters of SFP3.

We found that certain combinations of probes and isozymes

showed especially high responses, supporting the value of our

approach for developing SIRT-subtype-speciﬁc probes. We

conﬁrmed its practical utility by applying one of the new

probes, 18, which showed especially high sensitivity to SIRT2,

for SIRT2 inhibitor screening. This enabled us to discover a

new small molecule SIRT2 defatty-acylase inhibitor.

According to the above strategy, we designed 13 with a 9-

residue H4K16 peptide as a substrate peptide and Dabcyl-PH

as a quencher, followed by 14 and 15, which have a 7- and an

1-residue H4K16 peptide, respectively. We next designed 16

and 17, which contain the “RIKRY” sequence of peptide-type

SIRT2-selective inhibitors (S2iL8, S2iD7), anticipating that

these sequences would increase the binding aﬃnity to SIRT2.

Our previous ﬁndings indicated that a longer linker structure of

the Dabcyl group increases the reactivity with SIRT. Thus,

we next designed 18 with Dabcyl-BH as a quencher, which is

longer by one carbon atom than Dabcyl-PH. We then

considered the inﬂuence of the electron density of the

quencher group. All the above probes have Dabcyl-type

quenchers with an electron-donating dimethylamino group at

the end of the structure. We thus designed 19, which contains

an H4K16 peptide and a Disperse Red quencher moiety

ACS Med. Chem. Lett. 2021, 12, 617−624

ACS Medicinal Chemistry Letters

Letter

bearing an electron-withdrawing nitro group at the end of the

structure. We also designed 20−23 containing peptide

sequences RIKRY, RalB, H2BK12, and H3K9 with a Disperse

Red quencher. The characteristics of these probes are

summarized in Table 1.

Table 1. Structures of Quenchers and Peptides Used in the

New Probes

quencher

peptide sequence

length

SFP3

Dabcyl-PH

Dabcyl-BH

Disperse Red

H3K9 (QTARKSTGG)

H4K16 (KGGAKRHRK)

H4K16 (GGAKRHR)

H4K16 (GKGGAKRHRKV)

S2iL8 (NFRIKRYSN)

S2iD7 (DYRIKRYHT)

H4K16 (KGGAKRHRK)

S2iD7 (DYRIKRYHT)

RalB (KKSFKERSS)

9 residues

7 residues

11 residues

9 residues

H2BK12 (APAPKKGSK)

H3K9 (QTARKSTGG)

Synthesis of FRET-Based SIRT Activity Probes 13−23.

3−23 were synthesized as shown in Scheme S1. Dabcyl-PH

3), Dabcyl-BH (6), and Disperse Red (10) were synthesized

and conjugated with Fmoc-Lys-OH in 4, 3, and 4 steps,

respectively. Finally, each probe was obtained by Fmoc solid-

phase synthesis on Sieber amide resin, cleaved, and puriﬁed by

preparative reversed-phase HPLC (purity: >95%). The

structures were conﬁrmed by HRMS.

(

Figure 2. (A) Absorption and (B) ﬂuorescence spectra of 1 μM 5-

FITC and 13−23. Data were measured in Tris-buﬀer (50 mM Tris-

HCl (pH 8.0), 150 mM NaCl), containing 0.01% DMSO as a

cosolvent.

Table 2. Photophysical Properties of SFP3, 13−23, and 5-

Photochemical Properties of 13−23. We measured the

absorption and ﬂuorescence spectra and calculated the

ﬂuorescence quantum yields of all the synthesized probes

FITC

−1

^λmax (nm)

ϵ (10 M cm⁻¹

)

λ_e_m(nm)

Φ_F_L

SFP3

496

494

499

496

497

493

494

8.91

4.75

4.26

5.52

3.96

4.69

4.58

4.59

4.34

5.82

5.59

6.31

7.04

517

516

517

516

517

513

515

516

518

0.008

0.053

0.014

0.007

0.021

0.018

0.012

0.015

0.005

0.008

0.010

0.006

0.696

(

Figure 2, Table 2). The absorption spectrum of each probe

showed overlapping absorbance of FITC and the quencher

dye, and the ﬂuorescence spectra indicated that the

ﬂuorescence of each probe was suﬃciently quenched by the

FRET mechanism compared to the reference, 5-FITC (Figure

B).

Reactivity of 13−23 with SIRT1−7. Next, we examined

the reactivity of each probe with SIRT1−7 by means of

enzymatic assay in 96-well half area plates at 37 °C for 1 h. The

values of ﬂuorescence enhancement (fold change) after 1 h are

shown in Table 3 and Figure S2.

Most of the probes reacted with SIRT1, SIRT2, and SIRT3,

and some showed a large ﬂuorescence enhancement (>10-

fold) after 1 h enzymatic reaction. In contrast, none of the

probes reacted signiﬁcantly with SIRT4, 5, or 7, suggesting that

our quencher-hydrolyzing strategy might not be suitable for

detection of their activities. In the case of SIRT6, several

probes, including 22 and 23, showed moderate to high

reactivity compared to SFP3. This result suggests that the

peptide sequence, especially H3K9, may be critical for

improving the binding aﬃnity with SIRT6, and the Disperse

Red quencher may be more suitable than the Dabcyl quencher

in these cases, although the overall reactivity with SIRT6 was

lower than those with SIRT1−3.

-FITC

Data were measured in Tris-buﬀer (50 mM Tris-HCl (pH 8.0), 150

mM NaCl). For determination of Φ , ﬂuorescein in 0.1 N NaOH

(

= 0.85) was used as a ﬂuorescence standard.

with 13 appears to be due to the diﬀerence of linker length in

the quencher moieties because all three probes have the same

peptide chain.

In the case of SIRT1, SFP3 and 13−18 showed large

ﬂuorescence enhancements regardless of the peptide sequence,

suggesting that SIRT1 favors the Dabcyl structure having a

dimethylamino group over the Disperse Red structure having a

nitro group, except for 16 and 17, which have peptide

sequences optimized for SIRT2. In the case of SIRT2, 18 and

19 showed the greatest ﬂuorescence enhancement, suggesting

that SIRT2 precisely recognizes the peptide sequence H4K16.

The linker length of the quencher moiety appears to be a key

18 and 19, whose quenchers are Dabcyl-BH and Disperse

Red, respectively, showed large ﬂuorescence enhancements

with SIRT1−3. Because the linker structures of Dabcyl-BH

and Disperse Red are longer by one carbon atom than that of

Dabcyl-PH, the diﬀerence of reactivity of 18 and 19 compared

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ACS Medicinal Chemistry Letters

selectively reacted with SIRT3 over SIRT1 and SIRT2.

Letter

Table 3. Fluorescence Enhancement (SIRT(+)/SIRT(−))

of 13−23 and SFP3 with SIRT1−7 after Enzymatic Reaction

for 1 h

Overall, these results indicate that our FRET-based

ﬂuorescence probes having a quencher dye as a SIRT-

hydrolyzable motif are suitable for detecting SIRT1−3 and 6.

Moreover, the factors inﬂuencing recognition of the

ﬂuorescence probes as a substrate by each SIRT subtype are

slightly diﬀerent, suggesting that it is possible to design SIRT-

subtype-speciﬁc ﬂuorescence probes by precisely optimizing

their peptide sequences, quencher structures, and linker

lengths.

SIRT1 SIRT2 SIRT3 SIRT4 SIRT5 SIRT6 SIRT7

SFP3

13.84

11.99

10.90

19.74

4.79

5.89

5.45

4.72

6.68

1.85

1.92

12.04

10.34

6.97

5.64

3.83

4.06

2.20

4.00

5.45

6.55

1.92

1.03

0.93

0.92

1.12

1.14

1.11

1.04

1.25

1.20

1.18

1.20

1.08

1.07

1.03

1.02

1.11

1.09

1.06

1.07

1.15

1.05

0.99

1.07

3.60

1.33

1.05

1.04

1.07

1.05

2.21

2.55

1.19

3.36

3.51

9.38

1.04

1.03

1.01

1.07

1.04

1.01

1.00

1.09

1.05

0.99

1.01

1.04

4.76

1.96

Determination of the Kinetic Constants of 13−23

14.23

14.74

5.85

8.86

5.00

12.48

27.23

26.71

26.18

12.31

14.07

with SIRT1−3. To quantitatively evaluate the reactivity of

each probe with SIRT1−3, the Michaelis constant (K ), the

catalytic rate constant (k ), and the speciﬁcity constant (k /

cat

K ) were determined from Michaelis−Menten plots (Figure 3,

Figure S3A −C , Table S1A −C).

6.06

For SIRT1, the k and K values of Dabcyl-type probes

cat

(

SFP3 and 13−18) are larger than those of Disperse Red-type

probes (19−23), suggesting that Dabcyl-type probes are more

eﬃciently hydrolyzed by SIRT1 than Disperse Red-type

probes, though SIRT1 has a higher aﬃnity for the Disperse

Red quencher than for the Dabcyl quencher. As a result, the

diﬀerence of k /K values for the two quenchers is small.

factor for high reactivity. As for SIRT3, interestingly, the

probes with the Disperse Red quencher (19−23) showed

signiﬁcantly higher ﬂuorescence enhancement than those with

the Dabcyl quencher. Notably, among these probes, 20 and 21

cat

Figure 3. Kinetic parameters of the enzymatic reactions of SIRT1−3 with SFP3 and 13−23. (A) k_c_a_tvalues of each probe. (B) K values of each

probe. (C) k /K values of each probe. Asterisks indicate the kinetic parameters of 22 for SIRT1 were not determined. Data for SFP3 were taken

cat

from ref 28.

ACS Medicinal Chemistry Letters

ACS Med. Chem. Lett. 2021, 12, 617−624

Letter

Figure 4. (A) Dependency of the enzymatic reaction rate of 18 on SIRT2 concentration. Results are mean ± SD (n = 3). (B) Time dependence of

the enzymatic reaction of 18 with various concentrations of SIRT2 in the range of 2.92−8.75 nM. Results are mean ± SD (n = 3). (C) Reliability of

the optimized screening conditions, evaluated in 48 wells of a 96-well half area microplate. CV (coeﬃcient of variation) = SD/Av ≤ 10%; S/B =

signal/background ≥3.0; S/N = signal/noise; Z′ factor = 1 − (3 × SD

+ 3 × SD )/(Av −Av ) ≥ 0.5 for reliable screening. SD = standard

00%

100%

deviation, Av = average. (D) Inhibition curve of S2DMi-6 toward SIRT2 obtained with 18. Results are mean ± SD (n = 3).

Indeed, all the newly developed probes have k /K values of

the order of 10 .

^F^o^r^S^I^R^T²^,^t^h^e^Km values of 16 and 17, which contain

peptide sequences derived from SIRT2-selective inhibitors, are

smaller than those of the other probes with H4K16 sequences.

consistent with the results shown in Table 3, and the higher

cat

reactivity of the Disperse Red-type probes with SIRT3 might

be attributed to their higher aﬃnity for SIRT3 compared with

Dabcyl-type probes. It is noteworthy that the k /K value of

cat

2 was smaller than those of other Disperse Red-type probes,

At the same time, the K values of 18 and 19 are smaller than

suggesting that the H2BK12 sequence may not be favorable

and that the peptide sequence can inﬂuence SIRT3 substrate

recognition.

Overall, the eﬀects of quencher structure and peptide

sequence on the reaction rate appear to be relatively large,

whereas the eﬀect of the peptide length is smaller. Although

the newly developed probes have lower reactivity toward

SIRT1 than SFP3, they have similar or higher reactivity toward

SIRT2 and SIRT3. In particular, 20 and 21 exhibited

that of 13, suggesting that the longer linker length on the

quencher is crucial for higher aﬃnity with SIRT2. 20 showed

the highest aﬃnity with SIRT2 However, the k values tend

cat

to be larger for probes with larger K values, and as a result,

there is no signiﬁcant diﬀerence in k /K values among all the

cat

probes.

For SIRT3, the k /K values are of the order of 10 for

cat

most of the Disperse Red-type probes, i.e., about 10 times

larger values than those of the Dabcyl-type probes. This is

ACS Medicinal Chemistry Letters

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Letter

Figure 5. (A) Three-stage screening. (B) Chemical structure of C. (C) Inhibition curves of A, B, and C toward SIRT2 obtained with 18. The

results are mean ± SD (n = 3). (D) Inhibition curve of A toward SIRT2 obtained with p53(Myr)-AMC. The results are mean ± SD (n = 3).

signiﬁcantly higher reactivity than SFP3 with SIRT2 (>24-

fold) and SIRT3 (>154-fold).

Next, we conducted molecular docking simulations of

Dabcyl-PH, Dabcyl-BH, and Disperse Red with the SIRT2

catalytic site (PDB ID: 4X3P) using the CDOCKER algorithm

in Discovery Studio Client v17.2.0.16349 (BIOVIA Inc.)

conﬁrming the reliability of our screening system even with as

low a concentration of SIRT2 as 2.92 nM.

We used the established conditions to examine whether 18

can be applied to determine the half-inhibitory activity (IC50

)

of the reported SIRT defatty-acylase inhibitor S2DMi-6

(

Figure 4D, Figure S6A ).^T^h^e^m^e^a^s^u^r^e^d^I^C50 value was

.007 μM, which is close to the value given in our previous

(Figure S4 ). A comparison of the CDOCKER interaction

report (IC = 0.019 μM, determined by SFP3). In addition,

energies of the three quenchers suggests that the binding

aﬃnities of Dabcyl-BH and Disperse Red are larger than that

^w^e^d ^e ^t ^e ^r ^m ⁱ ⁿ ^e ^d ^t ^h ^e ^I ^C50 value of S2DMi-9 toward SIRT6 by

^u^sⁱⁿ^g²³⁽^F ⁱ ^g ^u ^r ^e ^S ⁶ ⁾^.^T^h^e^m^e^a^s^u^r^e^d^I^C50 value was 0.10 μM,

of Dabcyl-PH, which is consistent with the K values of 13, 18,

which is close to the value given in our previous report (IC =

and 19. Interestingly, all the quenchers share some interactions

.25 μM, determined with SFP3).

Chemical Screening of SIRT2 Defatty-Acylase Inhib-

(

e.g., hydrogen bonding with Val233, and π−π stacking

interaction with Phe190), but only Disperse Red can also form

itors Using 18. Following the HTS validation of 18, we

applied it to screen 9600 compounds from a large chemical

library for nonpeptide novel SIRT2 defatty-acylase inhibitors

a π -cation interaction between its nitro group and Tyr139

Figure S5A-C ).

Validation of an HTS System for SIRT2 Inhibitors

(

Figure 5A).

Using 18. To illustrate the utility of our newly synthesized

probes, we conducted a chemical screening assay to ﬁnd

SIRT2 inhibitors because these would be candidate anticancer

Primary screening was performed in 384-well plates, and all

compounds were tested at 10 μM in the presence of 0.5%

DMSO. This screen yielded three candidate compounds, A, B,

and C, which showed more than 35% inhibition at 10 μM

agents. Based on the results shown in Table 3, we chose 18

as the most suitable probe for SIRT2 defatty-acylase modulator

screening. Initially, we conﬁrmed that the reaction rate of 18

increased linearly with increasing concentration of SIRT

(

Figure 5B, Figure S7A ). We next conducted a second

screening with 18 to conﬁrm dose-dependency and to

eliminate false-positive compounds (Figure 5C) and found

that compound C reproducibly inhibited SIRT2 with an IC₅₀

value of 47.9 μM. We then conducted a third screening to

conﬁrm the SIRT2 defatty-acylase inhibitory activity of the

three hit compounds by using our previously developed two-

(

Figure 4A). We next evaluated the time dependence of the

enzymatic reaction of 18 with various concentrations of SIRT2

Figure 4B) and found that the ﬂuorescence increase was

(

linear for at least 4 h. A low concentration of SIRT2 (2.92 nM)

was suﬃcient for SIRT2 activity detection, reﬂecting the high

reactivity of 18. We further validated the reliability of our

screening system with 18 (Figure 4C) by using 48 wells of a

step defatty-acylase probe, p53(Myr)-AMC (Figure S8).

Among the three candidates, compound C dose-dependently

inhibited SIRT2 demyristoylase activity determined with

p53(Myr)-AMC, and its IC₅₀value was calculated to be 44.4

μM, in good accordance with the value obtained using 18

(Figure 5D, Figure S7B).

6-well half area plate (SIRT2 (+) in 40 wells and SIRT2 (−)

in 8 wells). The CV₁value was less than 10%, the S/B ratio

00%

was more than 3, and the Z′ factor was more than 0.5,

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ACS Medicinal Chemistry Letters

Complete contact information is available at:

Letter

Further evaluations of compound C were conducted to

conﬁrm its selectivity for SIRT2 defatty-acylase activity. We

found that compound C selectively inhibits defatty-acylase

activity of SIRT2 over SIRT1, 3, and 6 (Figure S9A). However,

compound C also inhibited SIRT2 deacetylase activity with an

^I^C⁵0 value of 5.09 μM, indicating that it is a dual inhibitor of

SIRT2 deacetylase and defatty-acylase activities (Figure S9B).

In addition, we determined the IC values of known SIRT2

Structure of SFP3, reactivity of SFP3 and 13−23 with

SIRT1−7, Michaelis−Menten plots for SFP3 and 13−

3 with SIRT1−3, structure of hit compounds A and B

and their SIRT2 defatty-acylase inhibitory activity

determined by p53(Myr)-AMC, docking simulation of

quenchers with SIRT2, selectivity of compound C over

SIRT1, 3, and 6 and deacetylase activity of SIRT2,

inhibitory activities of known SIRT2 inhibitors evaluated

with 18, synthesis of 13−23 and in vitro experimental

procedures (PDF)

inhibitors, nicotinamide, AGK2, SirReal2, and TM, by

using 18 and compared them with those of compound C

(Figure S10). SirReal2 and TM are much more SIRT2

deacetylase-selective than the classical inhibitors nicotinamide

and AGK2. It was found that compound C inhibits both

SIRT2 deacetylase and defatty-acylase activities, indicating that

its defatty-acylase selectivity is similar to those of nicotinamide

and AGK2.

Thus, the screening assay with our newly developed, highly

sensitive ﬂuorescence probe, 18, enabled us to identify

compound C as a nonpeptide, small-molecular SIRT2

defatty-acylase inhibitor.

AUTHOR INFORMATION

■

Corresponding Authors

Mitsuyasu Kawaguchi − Graduate School of Pharmaceutical

Sciences, Nagoya City University, Nagoya, Aichi 467-8603,

Japan; orcid.org/0000-0002-1919-1391 ; Phone: +81-

52-836-3408; Email: mkawaguchi@phar.nagoya-cu.ac.jp;

Fax: +81-52-836-3408

Hidehiko Nakagawa − Graduate School of Pharmaceutical

Sciences, Nagoya City University, Nagoya, Aichi 467-8603,

Japan; orcid.org/0000-0001-8435-4401 ; Phone: +81-

52-836-3407; Email: deco@phar.nagoya-cu.ac.jp;

Fax: +81-52-836-3407

CONCLUSION

■

In this research, we designed and synthesized a set of 11 new

one-step ﬂuorescence probes with various peptide sequences

and quenchers using an improved synthetic scheme that does

not involve liquid-phase steps. Evaluation of these probes with

SIRT isozymes indicated ﬁrst that the length of the quencher

structure and the peptide sequence are more important factors

than peptide sequence length for determining the reaction rate

with SIRT1−3, and second, that the Disperse Red quencher is

selectively recognized by SIRT3.

Authors

Yuya Nakajima − Graduate School of Pharmaceutical Sciences,

Nagoya City University, Nagoya, Aichi 467-8603, Japan

Naoya Ieda − Graduate School of Pharmaceutical Sciences,

Nagoya City University, Nagoya, Aichi 467-8603, Japan

Probe-scanning with SIRT isozymes revealed that diﬀerent

combinations of probes and isozymes showed diﬀerent

responses. To illustrate the value of our probe design strategy,

we selected compound 18 for HTS application to discover

SIRT2 defatty-acylase modulators in a one-step manner. Even

with a low concentration of SIRT2, we identiﬁed compound C

as a new, small molecule SIRT2 defatty-acylase inhibitor. Our

one-step detection method has the following advantages: (i)

low background ﬂuorescence due to the probe’s stability, (ii)

longer wavelength ﬂuorescence, minimizing possible overlap,

https://pubs.acs.org/10.1021/acsmedchemlett.1c00010

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

The authors thank all the members of H.N.’s laboratory for

fruitful discussions and also thank Drs. Hirotatsu Kojima,

Takayoshi Okabe, and Tetsuo Nagano at the Drug Discovery

Initiative, the University of Tokyo for the assistance of

chemical screening. This work was supported in part by JSPS

KAKENHI Grants 26893223, 15K18899, and 18K14358

(

iii) simple manipulation for chemical screening, and (iv)

biocompatibility for cellular SIRT activity imaging. A possible

drawback is that the substrate recognition sites are diﬀerent

from those of the natural substrates (quenchers vs long-chain

fatty acids). However, in our previous paper, we established

that IC₅₀values evaluated with our one-step probe are well-

correlated with those obtained using a two-step probe,

p53(Myr)-AMC, which has a myristoyl group as a substrate

(

M.K.) and 16H05103, 19H03354, and 19KK0197 (H.N.)

as well as by the Hori Sciences and Arts Foundation (M.K.), a

Grant-in-Aid for Scientiﬁc Research on Innovative Areas from

MEXT (26111012, H.N.) and a grant from Daiichi Sankyo

Foundation of Life Science (H.N.). This research was partially

supported by the Platform Project for Supporting Drug

Discovery and Life Science Research from AMED under

Grant JP19am0101086.

recognition site. Therefore, we believe our probe set will be

useful to screen chemical libraries for modulators of not only

SIRT2 but also other SIRTs, including SIRT1, 3, and 6. Such

modulators would be useful tools to decipher the physiological

and pathological roles of the defatty-acylase activities of SIRTs

as well as to discover new SIRT inhibitors as candidate

anticancer therapeutic agents.

ABBREVIATIONS

■

SIRT, sirtuin; FRET, Fo

nicotinamide adenine dinucleotide; ADP, adenine diphos-

rster resonance energy transfer; NAD,

ASSOCIATED CONTENT

■

phate; TNF-α, tumor necrosis factor α; Dabcyl, 4-(4-

sı Supporting Information

(

dimethylamino)phenylazo)benzoic acid; SPS, solid-phase

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acsmedchemlett.1c00010.

synthesis; HPLC, high performance liquid chromatography;

HTS, high-throughput screening

ACS Med. Chem. Lett. 2021, 12, 617−624

ACS Medicinal Chemistry Letters

24) Baba, R.; Hori, Y.; Mizukami, S.; Kikuchi, K. Development of a

Letter

(

23) Yang, L.-L.; Wang, H.-L.; Yan, Y.-H.; Liu, S.; Yu, Z.-J.; Huang,

M.-Y.; Luo, Y.; Zheng, X.; Yu, Y.; Li, G.-B. Eur. J. Med. Chem. 2020,

92, 112201.

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