Rapid Solid-Phase Construction of Serine Hydrolase Probes Results in Selective Activity-Based Probes for Acyl Protein Thioesterases-1/2

Article abstract of DOI:10.1021/acs.jmedchem.0c01043

Serine hydrolases (SHs) are a large, diverse family of enzymes that play various biomedically important roles. Their study has been substantially advanced by activity-based protein profiling, which makes use of covalent chemical probes for labeling the active site and detection by various methodologies. However, highly selective probes for individual SHs are scarce because probe synthesis usually takes place by time-consuming solution phase chemistry. We here report a general solid-phase synthesis toward SH chemical probes, which will speed up probe library synthesis. It involves the construction of a recognition element ending in a secondary amine followed by capping with different electrophiles. We illustrate the power of this approach by the discovery of selective chemical probes for the depalmitoylating enzymes APT-1/2. Overall, this study reports new methodologies to synthesize SH probes, while providing new reagents to study protein depalmitoylation.

Full text of DOI:10.1021/acs.jmedchem.0c01043

Subscriber access provided by CARLETON UNIVERSITY
Article
Rapid solid phase construction of serine hydrolase probes results
in selective activity-based probes for acyl protein thioesterases-1/2
Roeland Vanhoutte, Merel A. T. van de Plassche, and Steven H.L. Verhelst
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c01043 • Publication Date (Web): 29 Sep 2020
Downloaded from pubs.acs.org on October 4, 2020
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the
course of their duties.

Page 1 of 35
Journal of Medicinal Chemistry
1
2
3
4
5
6
Rapid solid phase construction of serine hydrolase probes results in selective activity-
based probes for acyl protein thioesterases-1/2
7
8
9
Roeland Vanhoutte,1 Merel A. T. van de Plassche,1 and Steven H. L. Verhelst*, 1,2
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1 KU Leuven, Department of Cellular and Molecular Medicine, Laboratory of Chemical Biology,
Herestr. 49, 3000 Leuven, Belgium
2
Leibniz Institute for Analytical Sciences ISAS, e.V., Otto-Hahn-Str. 6b, 44227 Dortmund,
Germany
* Corresponding author: steven.verhelst@isas.de, steven.verhelst@kuleuven.be
1
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 35
1
2
3
4
Abstract
5
6
7
8
9
Serine hydrolases are a large, diverse family of enzymes that play various biomedically
important roles. Their study has been substantially advanced by activity-based protein
profiling, which makes use of covalent chemical probes for labeling of the active site and
detection by various methodologies. However, highly selective probes for individual serine
hydrolases are scarce, because probe synthesis usually takes place by time-consuming
solution phase chemistry. We here report a general solid phase synthesis towards serine
hydrolase chemical probes, which will speed up probe library synthesis. It involves construction
of a recognition element ending in a secondary amine followed by capping with different
electrophiles. We illustrate the power of this approach by the discovery of selective chemical
probes for the depalmitoylating enzymes APT-1/2. Overall, this study provides new
methodologies to synthesize serine hydrolase probes, while providing new reagents to study
protein depalmitoylation.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2
ACS Paragon Plus Environment

Page 3 of 35
Journal of Medicinal Chemistry
1
2
3
4
Introduction
5
6
7
8
Activity-based protein profiling (ABPP) emerged two decades ago to help discover the function
of enzymes.1 ABPP uses small molecules called activity-based probes (ABPs), which bind
covalently to the active site of the enzymes of interest, allowing a multitude of read-out
possibilities and application areas,2 including profiling in different disease states, imaging,
target identification as well as inhibitor screening by competitive ABPP (Figure 1A). ABPs
generally consist of three parts: a warhead, which binds covalently to a catalytic residue of the
target enzyme, a recognition element, which influences the selectivity of a probe, and a
reporter tag to visualize probe labeled proteins.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Fluorophosphonate (FP) probes (Figure 1B) were developed as the first serine hydrolase
reactive ABPs – either equipped with a biotin tag (FP-Biotin)3 or a fluorophore (FP-rhodamine;
FP-Rh).4,5 Serine hydrolases (SHs) are an enzyme superfamily comprising 1-2% of the human
genome. Amongst them are several pharmaceutically interesting proteins,6 such as monoacyl
glycerol lipase (MAGL) and ,-hydrolase domain containing protein 6 (ABDH6), which play a
role in the generation of endocannabinoids in the nervous system,7 acyl protein thioesterases-
1/2 (APT-1/2; previously known as lysophospholipases-1/2; LYPLA-1/2), which are involved in
depalmitoylation of Ras GTPase,8 and protein phosphatase methyl esterase 1, which plays a
role in neuroblastoma.9 For most SH members, however, the exact function remains elusive.
Conveniently, FP-Rh reacts with more than 80% of all metabolic SHs in mice.10 Hence, FP-Rh
has not only been useful for the study of well known SHs, but also for uncharacterized ones.
However, there are also some limitations of this reagent: (1) The synthesis is not trivial and
takes considerable effort. (2) It may not react with all SHs. (3) Because of its general nature, it
is problematic to implement this probe in studies for visualization of a single SH of interest.
Therefore, serine-reactive ABPs with other warheads and recognition elements have been
developed over the past two decades. These have the potential to be used for in vivo imaging
studies, as recently illustrated for MAGL.11, 12 Nevertheless, it is extremely challenging and
time-consuming to obtain highly selective ABPs, because the synthesis and optimization of
these ABPs usually takes place by solution phase chemistry. Hence, selective SH ABPs have
3
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 4 of 35
1
2
3
4
5
6
7
8
9
only been reported for a few SHs, including neutral cholesterol ester hydrolase 1 (KIAA1363),13
diacylglycerol lipase (DAGL),14, 15 and MAGL,16 although probes for the last two showed some
crossreactivity towards ABDH6. In order to produce and optimize ABPs in a fast and efficient
manner, we set out to speed up the process of SH probe development by applying solid phase
synthesis. Here, we present a mix-and-match strategy of recognition elements and serine
reactive warheads for the design and full on-resin synthesis of a SH ABP library. Triazole urea
probes in this library were potent and selective against APT-1/2, and represent new tools for
the study of protein depalmitoylation.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 1. ABPP and ABPs. (A) Left (boxed): ABPs generally consist of three elements: a
reactive electrophile as warhead that engages in a mechanism-based reaction with the active
site nucleophiles of the target, a tag that allows detection and a spacer and/or recognition
element to prevent a steric clash of the tag in the active site and to influence the selectivity of
the probe. Right: ABPP allows the tagging of active enzyme targets in a complex proteome,
such as a cell lysate, whole cell or in vivo setting, followed by various types of read-out. Inhibitor
evaluation in complex proteomes may be done by ‘competitive ABPP’, in which the proteome
is first treated with a potential inhibitor before incubation with the ABP. Disappearance of gel
bands in SDS-PAGE will then reveal inhibitor selectivity. (B) Structure of fluorophosphonate-
based probes and the mechanism of reaction with serine hydrolases: the active site serine side
chain, activated by hydrogen bonding to a histidine residue, engages in a mechanism-based
4
ACS Paragon Plus Environment

Page 5 of 35
Journal of Medicinal Chemistry
1
2
3
4
5
6
reaction and results in a stable, covalent phosphonate ester probe-protein complex. (C) A
modular design of SH ABPs for construction on solid support.
7
8
9
Results and discussion
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Design & synthesis of probes. We aimed at making ABPs for SHs in a time-efficient manner
by a full on resin synthesis. Therefore, our probes comprised the following modular design
(Figure 1C): (1) a C-terminal amide which forms the anchor to the common Rink amide resin,
(2) an azide tag amenable to click chemistry-mediated detection of probe targets and
incorporated in (3) an azidolysine-aminohexanoic acid spacer that connects to (4) a recognition
element consisting of a natural amino acid, an unnatural amino acid or a peptoid (or a
combination thereof), and (5) a serine reactive electrophile. In this design, the reactive group
is introduced on resin as an N-terminal cap in the very last step prior to resin cleavage. As
warheads, we selected five types of reactive electrophiles (Scheme 1A) which have previously
been shown to react with serine hydrolases and that can potentially be installed at an amine
in
a
last step on solid support:
a
diphenylphosphoramidate,17 an N-
(nitro-)phenylcarbamate, 2-
hydroxysuccinimidylcarbamate (NHS carbamate),16
a
a
aminopyridineurea18 and a 1,2,3-triazoleurea.19 The introduction at this point of the synthesis
would minimize the incompatibilty with any used reagents and conditions except for the
coupling conditions of the reactive group and the cleavage conditions from the resin. Whereas
diphenylphosphoramidates have already been synthesized on resin,17 this possibility has not
yet been explored for the other reactive electrophiles. In a pilot experiment (Scheme 1A), we
explored coupling these reactive groups to recognition elements ending in a primary amine (a
phenyl alanine), a secondary amine (a phenylalanine peptoid) or a secondary amine in a six-
membered ring (an isonipecotic acid). We found that primary amines capped as
hydroxysuccinimidylcarbamates converted into hydantoins (Figure S1) and we could not form
triazole ureas onto primary or non-cyclic secondary amines. However, all reactive groups could
be placed onto the piperidine ring of isonipecotic acid as detected by LC-MS of crude
5
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 35
1
2
3
4
5
6
7
8
9
compound mixtures after resin cleavage (Figure S2). This inspired us to make a series of
recognition elements, consisting of a piperidine or piperazine-moiety connected to an
additional aromatic group, a combination which has previously shown to have affinity for
various serine hydrolases.19 20 21 22
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Scheme 1. Synthesis of mix-and-match SH ABP libraries.a
a (A) On resin coupling of warheads to different types of amines. Reagents and conditions: (i)
Diphenylphosphoryl chloride, DIEA, DCM. (ii) Disuccinimidylcarbonate, DIEA, DCM. (iii) 2-
aminopyridinyl phenyl carbamate, DIEA, DCM. (iv) phenyl chloroformate, DIEA, DCM. (v) 1.
Triphosgene, DIEA, DCM, 30 minutes; 2. 1H-1,2,3-triazole, DIEA, DCM. (B) Library synthesis
6
ACS Paragon Plus Environment

Page 7 of 35
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
towards compounds 4a-6f. (vi) SPPS elongation by 1. DMF/piperidine 4/1, 15 min; 2. Fmoc-
aa-OH, HBTU, DIEA, DMF. (vii) 1. DMF/piperidine 4/1, 5 min; 2. Bromoacetic acid, DIC, HOBt,
DMF. (viii) Benzylamine, DIEA, DMF. (ix) Fmoc-Inp-OH, DIC, HOBt, DMF. (x) p-
Bromomethylbenzoic acid, PyBrOP, DIEA, DMF. (xi) Piperazine, DMF. (xii) 4-nitrophenyl
chloroformate, DIEA, DCM.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Library synthesis. The three recognition elements in the probe library (Scheme 1B)
comprised of isonipecotic acid coupled to phenylalanine (1), isonipecotic acid coupled to a
phenylalanine peptoid (2), or para-piperazinylmethyl benzoic acid (3). These recognition
elements were synthesized by modified SPPS and installed onto a Lys(N3)-Ahx click-tag/linker
moiety. Specifically, elongation with Phe took place under standard conditions, whereas the
peptoid Phe was synthesized by coupling a bromoacetic acid followed by on resin nucleophilic
substitution of the bromide by benzylamine. These two recognition elements were completed
by coupling of isonipecotic acid (Inp) under influence of DIC/HOBt. The third recognition
element was formed by installation of a 4-bromomethylbenzoyl substituent followed by SN2
substitution with piperazine. All three recognition elements were then capped with six
warheads. Gratifyingly, these compounds were all compatible with Rink amide resin cleavage
conditions, giving a 3x6 library of ABPs 4a-6f.
Activity against SHs in competitive ABPP. Next, we set out to test whether these
compounds display reactivity against SHs. Mouse brain lysate was chosen as a suitable
proteome for screening, because it contains a wide variety of pharmacologically interesting
SHs. Previous studies have identified between approximately 25 and 40 different SHs in this
24 25
lysate and conveniently, the retention of several of these on SDS-PAGE is known..23
Hence, the cytosolic and membrane fractions of mouse brain lysates were pretreated with
compounds 4a-6f and residually active SHs were labeled with FP-Rh (Figure 2A). Interestingly,
this competitive ABPP experiment revealed that five library members (the three triazole ureas
4f, 5f and 6f, and two NHS-carbamates 5b and 6b) showed selective inhibition of two close-
7
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 8 of 35
1
2
3
4
5
6
7
8
9
running gel bands that have previously been identified as APT-1/2 (Figure 2A).26 A titration of
these compounds showed that the potency of the NHS carbamates 5b and 6b for these targets
was moderate (see Figure S3), whereas the triazole ureas were much more potent (low to mid
nanomolar range) (Figure 2B, see also Figure S4 for representative gels).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Probe 4f, with a Phe-Inp recognition element had an apparent IC50 of 460 ± 50 nM for APT-
1/2. For the other targets of FP-rhodamine, this probe had apparent IC50 values above 30 M
(Figure S4), resulting in a larger than 65 fold selectivity. Remarkably, the potency for APT-1/2
increased 37 fold by replacing the Phe in the recognition element by its peptoid analog in 5f
(IC50App= 11.8 ± 1.8 nM) (Figure 2B), while having an excellent selectivity (over 2500 fold, as
measured by apparent IC50 values against the other FP-Rh targets, which were above 30 M,
see Figure S4). Probe 6f, with a linear benzyl-piperazinyl recognition element also showed
high activity against APT-1/2 (IC50App = 27.9 ± 2.8 nM), although at micromolar concentrations
this probe also inhibited ABHD6 (Figure S4) with an apparent IC50 of 7.4 ± 1.7 M
(approximately 250 fold selectivity).
We next compared probe 5f with the chloro-isocoumarin-based probe JCP-174-alk27 and the
isoform selective APT-1 and APT-2 inhibitors ML-348 and ML349.26 To this end, competitive
ABPP was performed in the soluble fraction of mouse brain lysate. We found that the gel bands
that are competed with probe 5f also disappear upon treatment with JCP-174-alk (Figure 2C,
left panel). Moreover, inhibitors ML-348 and ML-349 only compete one of the two close-running
gel bands inhibited by probe 5f. Overall, this reveals that 5f has similar inhibitory capacity and
confirms APT-1/2 as target. We assessed the cell permeability of probes 5f and 6f using the
human squamous carcinoma cell line A431. In a competitive ABPP assay with FP-rhodamine
both 5f and 6f fully inhibited all active APT-1/2 at 1 M concentration (Figure 2D), confirming
their target engagement in live cells.
8
ACS Paragon Plus Environment

Page 9 of 35
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 2. Screening of SH ABP library for inhibition by competitive ABPP. (A) Fluorescent gels
of competitive ABPP in the membrane fraction (upper) or soluble fraction (lower) of mouse
brain lysates. Pretreatment of lysate with the indicated probe was followed by measurement
of residual SH activity with FP-Rh. (B) Apparent IC50 curves were determined by competitive
ABPP of serial dilutions, followed by quantification using gel band densitometry. (C)
Competitive ABPP in mouse brain lysate (soluble fraction) with known APT1/2 inhibitor JCP-
174-alk (left panel) or APT1-selective inhibitor ML348 and APT2-selective inhibitor ML349
(right panel) in comparison with compound 5f. Residual SH activity in treated tissue lysates
was measured by incubation with FP-Rh. (D) Inhibition of APT1/2 by compounds 5f and 6f in
live A431 cells. After treatment, compounds were washed out, and residual SH activity in cell
lysates was detected with FP-Rh.
9
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 10 of 35
1
2
3
4
5
6
7
8
9
Direct profiling of probe targets. To show the utility of the synthesized molecules as novel,
highly selective ABPs against SHs, probes were applied in a labeling experiment using copper-
catalyzed azide alkyne cycloaddition (CuAAC) with an alkyne-functionalized fluorophore. At 1
µM concentration, all three probes showed strong labeling of the APT-1/2 band, and this
labeling was even retained at 100 nM concentration for probes 5f and 6f (Figure 3A-B).
Interestingly, a few other targets besides the APT-1/2 gel bands were revealed in this direct
labeling experiment (indicated with a red asterisk, Figure 3A-B), which were not detected in
the competitive ABPP experiment that made use of FP-Rh. This illustrates that not all probe
targets overlap with general SH probe FP-rhodamine. Specifically, probe 6f showed strong
labeling of a soluble protein at 30 kDa (Figure 3A) and membrane proteins at 35 and 55 kDa
(Figure 3B). Probes 4f and 5f both showed reactivity towards a different membrane protein at
33 kDa at 1 M. However SDS-PAGE analysis of 5f at 100 nM concentration did not show any
other labeling than APT-1/2 (Figure 3B, right panel)..
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Previously reported proteomics based assessments of the targets of FP-rhodamine in brain
lysates show that it labels APT-1/2 as targets at 25 kD.24 Given our competitive based ABPP
results with FP-rhodamine, we suspected these as being the targets of our NHS-carbamate
and triazole ureaprobes. To confirm that these close-running gel bands correspond to APT-
1/2, we incubated mouse brain lysates with 5f or 6f, followed by click labelling of the probe-
enzyme complexes with a trifunctional TAMRA-biotin-alkyne detection tag. This tag allowed
affinity-based enrichment of the probe hits on streptavidin-coated beads, followed by both
fluorescent visualization (Figure 3C, upper panels, full gels in Figure S5) as well as Western
blot analysis (Figure 3C; lower panels). The detected bands with anti-APT-1 and anti-APT-2
antibodies in the samples treated with probe, but not in dmso controls unequivocally reveal the
identity of the probe target.
Model of probe binding. To gain insight into the binding mode of the two most active
compounds 5f and 6f towards their targets, we performed covalent docking using the flexible
sidechain methods of Autodock 4.2.28 29 In a crystal structure of APT-1 with a bound palmitate
10
ACS Paragon Plus Environment

Page 11 of 35
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
molecule (PDB code: 6QGS), both probes overlay well with this substrate (Figure 3D-E) and
occupy a long, hydrophobic channel, which is also the binding site of the recently reported,
reversible APT-1 inhibitor ML348 (Figure S6).30 The Inp residue of the most potent probe 5f
seems locked between hydrophobic residues F73, I75, W145 and L176, whereas its peptoid
Phe moiety binds into a pocket created by the residues F73, I75, I76 and the alkyl chain of E84
(Figure 3D). Although the slightly less active probe 6f does not have the peptoid Phe, it may
compensate for this by a stacking interaction of the benzoyl group in its backbone with W145,
whereas its piperidine ring has similar hydrophobic interactions as the Inp in probe 5f with L30,
I75, W145 and L176 (Figure 3E).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 3. Covalent interaction with APT-1/2. (A) Labeling of a soluble fraction of mouse brain
lysate with probes 4f-6f, followed by click chemistry with a TAMRA-alkyne derivative shows
selective detection of the ATP-1/2 gel bands. DMSO control (-) shows click background. Other
protein targets than APT-1/2 are indicated by a *. (B) Same as (A) but in the membrane fraction
of mouse brain lysate. (C) After click chemistry with a trifunctional biotin-TAMRA-alkyne tag
11
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 12 of 35
1
2
3
4
5
6
7
8
9
and enrichment on streptavidin beads, fluorescent detection (upper panels) confirms target
engagement of the probes, while detection by anti-APT-1 or anti-APT-2 antibodies (lower
panels) confirms target identity. Note that the targets in the membrane fraction were very low
abundant and led to very weak bands. Full fluorescent gels in Figure S5. (D) Covalent docking
of 5f in a crystal structure of APT-1 (PDB code: 6QGS). Protein in cartoon format with -helices
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
in cyan, -sheets in magenta and random coil in pink. A co-crystallized palmitate is shown in
orange sticks, whereas 5f is shown in magenta. Active site residues are depicted in green,
whereas interacting residues are shown in white. Picture rendered with PyMol.31 (E) Covalent
docking of 6f (in yellow) overlayed with co-crystallized palmitate (in orange) in a crystal
structure of APT-1.
Conclusions
In conclusion, we report synthetic procedures for a full on-resin synthesis of SH chemical
probes compatible with six different types of serine-reactive electrophiles. Using this
procedure, we constructed a library of ABPs by combining the electrophiles with three different
recognition elements consisting of an aromatic unit and a piperidine or piperazine moiety.
Evaluation of these compounds in a serine-hydrolase rich mouse brain lysate revealed activity
of five of these compounds against protein depalmitoylases APT-1 and APT-2, with triazole
urea 5f showing nanomolar potency and excellent selectivity. The structure-activity relationship
from this work may provide new information on pharmacophores for the design of drug-like
APT-1/2 inhibitors and the in silico docking presented here provides a plausible model of the
binding mode of these compounds. Compared with other recently reported inhibitors and
probes for APT1/2,26 27 the compounds presented here can be readily synthesized on solid
support and allow for easy future optimization by medicinal chemistry studies.
We expect that the novel selective ABPs 5f and 6f resulting from this work will find
application in the study of APT1/2-mediated protein depalmitoylation. Furthermore, the
synthetic procedures reported in this work may be automated in future work and facilitate more
rapid development of SH inhibitors and ABPs by library synthesis on solid support. Since many
12
ACS Paragon Plus Environment

Page 13 of 35
Journal of Medicinal Chemistry
1
2
3
4
5
6
SHs are still poorly characterized, we expect that this will open the door to functional studies
of these.
7
8
9
Experimental section
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
General materials and methods
All starting materials for chemical reactions were purchased from commercial suppliers and
used without purification. All solvents were of synthesis grade or higher. Progress of solution
phase reactions was monitored by TLC on pre-coated 0.20 mm thick ALUGRAM® TLC sheets
(silicagel-60 with UV indicator) using UV light and/or staining with cerium ammonium
molybdate or ninhydrin followed by heating. Nuclear magnetic resonance spectra were
recorded on a Bruker UltrashieldTM 400 or 600 MHz NMR Spectrometer. Chemical shifts are
reported in ppm relative to the residual solvent peak. LC-MS spectra were recorded on a
Prominence Ultra-fast Liquid Chromatography system (Shimadzu) using a Waters X-bridge
2.1 mm C18 column with a gradient of 5%-80% acetonitrile in water (with 0.1% formic acid)
over 22 min. HPLC purification was performed on a Prominence Ultra-fast Liquid
Chromatography system (Shimadzu) using a Waters X-bridge 150 mm C18 prep column with
a gradient of acetonitrile in water (with 0.1% trifluoroacetic acid) over 32 min. Purity >95% of
all final compounds was assessed by integration of LC chromatograms.
Solid phase chemistry procedures
Fmoc protected Rink amide resin (1.35 g, 0.74 mmol/g) was treated with 20% piperidine in
DMF for 5 min to swell the resin and deprotect the Fmoc group. Next, the resin was washed
three times with DMF. All following Fmoc deprotections were performed according to this
procedure.
Backbone synthesis
Method 1. Fmoc-L-Lys(N3)-OH was coupled to 1 mmol of deprotected rink amide resin by
adding amino acid (1.5 eq.), HBTU (1.5 eq.) and DIPEA (3 eq.) in 4 mL of DMF to the resin
13
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 14 of 35
1
2
3
4
5
6
7
8
9
and incubating for 4 hours at room temperature. After this and each following coupling, the
resin was washed three times with DMF and three times with DCM. After deprotection, Fmoc-6-
aminohexanoic acid was coupled by adding amino acid (2 eq.), HBTU (2 eq.) and DIPEA (4
eq.) in 4 mL DMF to the resin and incubating overnight at room temperature.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Recognition element synthesis
After synthesis of the peptide backbone, the resin was divided into portions of 100 µmol on
which the different recognition elements were formed. Peptide bonds in recognition elements
were formed with the corresponding (amino)acid (3 eq.), DIC (3 eq.) and HOBt (3 eq.) in 1.5
mL DMF overnight at room temperature unless stated otherwise.
Method 2.1. Phe-peptoid was formed by coupling Br-acetic acid to the peptide backbone under
standard SPPS conditions and subsequent substitution with benzylamine (20 eq.) and DIPEA
(10 eq.) in 1.5 mL DMF per 100 µmol overnight at room temperature. Next, Fmoc-Isonipecotic
acid was coupled using standard SPPS conditions.
Method 2.2. 4-Bromomethyl phenylacetic acid (3 eq.) was coupled to the peptide backbone
with PyBrOP (3 eq.) and DIPEA (6 eq.) in 1.5 mL DMF per 100 µmol overnight at room
temperature. Subsequently, the bromide was substituted for piperazine (20 eq.) using DIPEA
(10 eq.) in 1.5 mL DMF overnight at room temperature.
Method 2.3. Fmoc-L-Phe-OH was coupled to the peptide backbone using standard SPPS
conditions, followed by coupling of Fmoc-Isonipecotic acid under standard SPPS conditions.
Warhead coupling
After synthesis of the recognition elements, the resins were divided into portions of 10 µmol
for coupling of the warheads.
Method 3.1.: Synthesis of diphenyl phosphoramidates. Resin (1 eq.) was swollen in 0.5 mL
DMF per 10 µmol of resin, to which DIEA (5 eq.) and Diphenylphosphoryl chloride (5 eq.) were
added. Reactions were shaken overnight at room temperature and checked for completion
14
ACS Paragon Plus Environment

Page 15 of 35
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
using LC/MS. After completion, the resin was washed three times with DMF and twice with
DCM. Probes were then cleaved from the resin by using a 95:2.5:2.5 mixture of TFA:TIS:water.
Method 3.2.: Synthesis of hydroxysuccinimidylcarbamates. Resin (1 eq.) was swollen in 0.5
mL DMF per 10 µmol of resin, to which DIEA (5 eq.) and Disuccinimidylcarbonate (3 eq.) were
added. Reactions were shaken overnight at room temperature and checked for completion
using LC/MS. After completion, the resin was washed three times with DMF and twice with
DCM. Probes were then cleaved from the resin by using a 95:2.5:2.5 mixture of TFA:TIS:water.
Method 3.3.: Synthesis of aminopyridine ureas. Resin (1 eq.) was swollen in 0.5 mL DMF per
10 µmol of resin, to which were added DIPEA (5 eq.) and phenyl 2-pyridinylcarbamate (3 eq.;
synthesized from 2-aminopyridine and phenyl chloroformate according to the method
described by Swanson et al.32 and isolated in 29% yield). Reactions were shaken overnight at
room temperature and checked for completion using LC/MS. After completion, the resin was
washed three times with DMF and twice with DCM. Probes were then cleaved from the resin
by using a 95:2.5:2.5 mixture of TFA:TIS:water.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Method 3.4.: Synthesis of phenylcarbamates: Resin (1 eq.) was swollen in 0.5 mL DMF per 10
µmol of resin, to which DIEA (10 eq.) and Phenyl chloroformate (10 eq.) were added. Reactions
were shaken overnight at room temperature and checked for completion using LC/MS. After
completion, the resin was washed three times with DMF and twice with DCM. Probes were
then cleaved from the resin by using a 95:2.5:2.5 mixture of TFA:TIS:water.
Method 3.5.: Synthesis of p-nitrophenylcarbamates: Resin (1 eq.) was swollen in 0.5 mL DMF
per 10 µmol of resin, to which DIEA (10 eq.) and p-nitrophenyl chloroformate (10 eq.) were
added. Reactions were shaken overnight at room temperature and checked for completion
using LC/MS. After completion, the resin was washed three times with DMF and twice with
DCM. Probes were then cleaved from the resin by using a 95:2.5:2.5 mixture of TFA:TIS:water.
Method 3.6.: Synthesis of triazole ureas: Resin (1 eq.) was swollen in 1 mL dry DCM per 10
µmol of resin, to which DIEA (15 eq.) and triphosgene (3 eq.) were added. Reactions were
shaken at room temperature for 30 min, after which the cartridge was drained into a solution
of 10% NaOH in water. The resin was washed three times with dry DCM, which was drained
15
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 16 of 35
1
2
3
4
5
6
7
8
9
into a 10%NaOH solution. Next, the resin was resuspended in 1 mL dry DCM, to which 10
equivalents of 1H-1,2,3-triazole was added. Reactions were shaken overnight at room
temperature and checked for completion using LC/MS. After completion, the resin was washed
three times with DCM. Probes were then cleaved from the resin by using a 95:2.5:2.5 mixture
of TFA:TIS:water. Note that with this on resin synthesis, the N1-isomer of the triazole ureas
was formed (Figure S7).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
N-(Diphenylphosphoryl)-isonipecotyl- L-phenylalanyl-6-aminohexanoyl-L--azidolysine
amide (4a)
Probe 4a was synthesized using subsequently methods 1, 2.3 and 3.1 and purified using
HPLC. The product was obtained as a white powder. (1.84 mg, yield = 23.7%) HRMS (MALDI-
TOF): m/z calcd. for C39H51N8O7P [M+H]+ 775.3691, found: 775.3412.
N-(Succinimidyloxycarbonyl)-isonipecotyl-L-phenylalanyl-6-aminohexanoyl-L--
azidolysine amide (4b)
Probe 4b was synthesized using subsequently methods 1, 2.3 and 3.2 and purified using
HPLC. The product was obtained as a white powder. (3.31 mg, yield = 48.4%) HRMS (MALDI-
TOF): m/z calcd. for C32H45N9O8 [M+H]+ 684.3464, found: 684.3167.
N-(2-aminopyridin-2-yl-carbonyl)-isonipecotyl-L-phenylalanyl-6-aminohexanoyl-L--
azidolysine amide (4c)
Probe 4c was synthesized using subsequently methods 1, 2.3 and 3.3 and purified using
HPLC. The product was obtained as a white powder. (2.99 mg, yield = 45.1%) HRMS (MALDI-
TOF): m/z calcd. for C34H46N10O5 [M+H]+ 663.3725, found: 663.3597.
N-(Phenyloxycarbonyl)-isonipecotyl- L-phenylalanyl-6-aminohexanoyl-L--azidolysine
amide (4d)
16
ACS Paragon Plus Environment

Page 17 of 35
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
Probe 4d was synthesized using subsequently methods 1, 2.3 and 3.4 and purified using
HPLC. The product was obtained as a white powder. (3.37 mg, yield = 50.8%) HRMS (MALDI-
TOF): m/z calcd. for C34H46N8O6 [M+H]+ 663.3613, found: 663.3211.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
N-(4-Nitrophenyloxycarbonyl)-isonipecotyl-
L-phenylalanyl-6-aminohexanoyl-L--
azidolysine amide (4e)
Probe 4e was synthesized using subsequently methods 1, 2.3 and 3.5 and purified using
HPLC. The product was obtained as a white powder. (1.93 mg, yield = 27.3%) HRMS (MALDI-
TOF): m/z calcd. for C34H45N9O8 [M+H]+ 708.3464, found: 708.3064.
N-(1H-1,2,3-triazol-1-yl-carbonyl)-isonipecotyl-L-phenylalanyl-6-aminohexanoyl-L--
azidolysine amide (4f)
Probe 4f was synthesized using subsequently methods 1, 2.3 and 3.6 and purified using HPLC.
The product was obtained as a white powder. (0.36 mg, yield = 5.6%) HRMS (MALDI-TOF):
m/z calcd. for C30H43N11O5 [M+H]+ 638.3521, found: 638.3790.
N-(Diphenylphosphoryl)-isonipecotyl-(N-benzyl)glycyl-6-aminohexanoyl-L--
azidolysine amide (5a)
Probe 5a was synthesized using subsequently methods 1, 2.1 and 3.1 and purified using
HPLC. The product was obtained as a white powder. (0.33 mg, yield = 4.3%) HRMS (MALDI-
TOF): m/z calcd. for C39H51N8O7P [M+H]+ 775.3691, found: 775.3742.
N-(Succinimidyloxycarbonyl)-isonipecotyl-(N-benzyl)glycyl-6-aminohexanoyl-L--
azidolysine amide (5b)
Probe 5b was synthesized using subsequently methods 1, 2.1 and 3.2 and purified using
HPLC. The product was obtained as a white powder. (1.29 mg, yield = 18.9%) HRMS (MALDI-
TOF): m/z calcd. for C32H45N9O8 [M+H]+ 684.3391, found: 684.3342.
17
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 18 of 35
1
2
3
4
5
6
N-(2-aminopyridin-2-yl-carbonyl)-isonipecotyl-(N-benzyl)glycyl-6-aminohexanoyl-L--
azidolysine amide (5c)
7
8
9
Probe 5c was synthesized using subsequently methods 1, 2.1 and 3.3 and purified using
HPLC. The product was obtained as a white powder. (2.82 mg, yield = 42.5%) HRMS (MALDI-
TOF): m/z calcd. for C34H46N8O6 [M+H]+ 663.37301, found: 663.3648.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
N-(Phenyloxycarbonyl)-isonipecotyl-(N-benzyl)glycyl-6-aminohexanoyl-L--azidolysine
amide (5d)
Probe 5d was synthesized using subsequently methods 1, 2.1 and 3.4 and purified using
HPLC. The product was obtained as a white powder. (2.80 mg, yield = 42.2%) HRMS (MALDI-
TOF): m/z calcd. for C33H46N10O5 [M+H]+ 663.3619, found: 663.3772.
N-(4-Nitrophenyloxycarbonyl)-isonipecotyl-(N-benzyl)glycyl-6-aminohexanoyl-L--
azidolysine amide (5e)
Probe 5e was synthesized using subsequently methods 1, 2.1 and 3.5 and purified using
HPLC. The product was obtained as a white powder. (2.97 mg, yield = 41.9%) HRMS (MALDI-
TOF): m/z calcd. for C34H45N9O8 [M+H]+ 708.3469, found: 708.3151.
N-(1H-1,2,3-triazol-1-yl-carbonyl)-isonipecotyl-(N-benzyl)glycyl-6-aminohexanoyl-L--
azidolysine amide (5f)
Probe 5f was synthesized using subsequently methods 1, 2.1 and 3.6 and purified using HPLC.
The product was obtained as a white powder. (1.06 mg, yield = 16.6%) HRMS (MALDI-TOF):
m/z calcd. for C30H43N11O5 [M+Na]+ 660.3346, found: 660.2908.
N-(Diphenylphosphoryl)-4-(piperazinyl-methyl)-benzoyl-6-aminohexanoyl-L--
azidolysine amide (6a)
18
ACS Paragon Plus Environment

Page 19 of 35
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
Probe 6a was synthesized using subsequently methods 1, 2.2 and 3.1 and purified using
HPLC. The product was obtained as a white powder. (0.93 mg, yield = 12.9%) HRMS (MALDI-
TOF): m/z calcd. for C36H47N8O6P [M+H]+ 719.3435, found: 719.3851.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
N-(Succinimidyloxycarbonyl)-4-(piperazinyl-methyl)-benzoyl-6-aminohexanoyl-L--
azidolysine amide (6b)
Probe 6b was synthesized using subsequently methods 1, 2.2 and 3.2 and purified using
HPLC. The product was obtained as a white powder. (0.93 mg, yield = 12.9%) HRMS (MALDI-
TOF): m/z calcd. for C29H41N9O7 [M+H]+ 628.3201, found: 628.3192.
N-(2-aminopyridin-2-yl-carbonyl)-4-(piperazinyl-methyl)-benzoyl-6-aminohexanoyl-L--
azidolysine amide (6c)
Probe 6c was synthesized using subsequently methods 1, 2.2 and 3.3 and purified using
HPLC. The product was obtained as a white powder. (0.69 mg, yield = 11.4%) HRMS (MALDI-
TOF): m/z calcd. for C31H42N8O5 [M+H]+ 607.3351, found: 607.3222.
N-(Phenyloxycarbonyl)-4-(piperazinyl-methyl)-benzoyl-6-aminohexanoyl-L--
azidolysine amide (6d)
Probe 6d was synthesized using subsequently methods 1, 2.2 and 3.4 and purified using
HPLC. The product was obtained as a white powder. (0.49 mg, yield = 8.1%) HRMS (MALDI-
TOF): m/z calcd. for C30H42N10O4 [M+H]+ 607.3463, found: 607.3647.
N-(4-Nitrophenyloxycarbonyl)-4-(piperazinyl-methyl)-benzoyl-6-aminohexanoyl-L--
azidolysine amide (6e)
Probe 6e was synthesized using subsequently methods 1, 2.2 and 3.5 and purified using
HPLC. The product was obtained as a white powder. (0.63 mg, yield = 9.7%) HRMS (MALDI-
TOF): m/z calcd. for C31H41N9O7 [M+H]+ 652.3202, found: 652.2670.
19
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 20 of 35
1
2
3
4
5
6
7
8
N-(1H-1,2,3-triazol-1-yl-carbonyl)-4-(piperazinyl-methyl)-benzoyl-6-aminohexanoyl-L--
azidolysine amide (6f)
9
Probe 6f was synthesized using subsequently methods 1, 2.2 and 3.6 and purified using HPLC.
The product was obtained as a white powder. (0.34 mg, yield = 5.8%) HRMS (MALDI-TOF):
m/z calcd. for C27H39N11O4 [M+H]+ 582.3259, found: 582.3079.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Preparation of tissue lysates
Mouse brains were thawed on ice, after which the hemispheres were separated using a razor
blade and cut into small pieces. The pieces were collected in a Dounce homogenizer and
homogenized on ice using 1.5 mL cold (4 °C) lysis buffer (20 mM HEPES, 2 mM DTT, 1 mM
MgCl2, 25 U/mL DNAse I, pH 7.2) until thorough lysis of the organ was visually assessed. After
homogenization, the suspension was collected into Eppendorf tubes and the Dounce
homogenizer was rinsed twice with small aliquots of lysis buffer, which were added to these
tubes. The suspension was centrifuged (2500 g, 3 min, 4°C) to remove cell debris. The
supernatant was carefully pipetted and transferred to an ultracentrifuge tube. The debris was
resuspended in 0.25 mL lysis buffer and centirfuged again using the same procedure. The
supernatant was added to the ultracentrifuge tubes, which were then spun in an ultracentrifuge
(100,000 g, 45 min, 4 °C, Beckman OptimaTM TLX, TLA 120.2 rotor). The supernatant (=
cytosolic protein fraction) was collected and the pellet (= membrane protein fraction) was
suspended in 1.5 mL storage buffer (20 mM HEPES, 2 mM DTT, pH 7.2). Protein
concentrations were determined using BCA assay on a Spectramax ID3 platereader. Aliquots
were snap frozen in liquid nitrogen and stored at -80 °C until use.
Competition of probes with FP-rhodamine
30 L of cytosolic or membrane fraction of mouse brain lysate (1 mg/mL) was incubated for 1
h at room temperature with 0.3 L of a stock solution of probe (10 mM, DMSO) or 0.3 L
20
ACS Paragon Plus Environment

Page 21 of 35
Journal of Medicinal Chemistry
1
2
3
4
DMSO. Then 0.3 L FP-rhodamine (100 µM, DMSO) was added to each sample and the
5
6
7
8
samples were incubated for another hour. Next, 10 L 4x sample buffer was added to the
samples, which were subsequently boiled at 95 °C for 2 minutes. Samples were resolved by
SDS-PAGE on 12% acrylamide gels and visualized by measuring in-gel fluorescence on a
Typhoon FLA 9500 scanner with excitation at 532 nm and detection at 568 nm with the
photomultiplier set at 900 V. Patterns of probe-treated samples were compared with an FP-
rhodamine treated control sample. Compounds that led to full disappearance of gel bands were
considered hits.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Comparison of 5f with JCP-174 alk
30 L of cytosolic or membrane fraction of mouse brain lysate (1 mg/mL) was incubated for 1
h at room temperature with either 0.3 L of a stock solution of probe 5f (10 µM, DMSO), 0.3
L of a stock solution of JCP-174 alk (1 mM, DMSO) or 0.3 L DMSO. Then 0.3 L FP-
rhodamine (100 µM, DMSO) was added to each sample and the samples were incubated for
1 hour at room temperature. Next, 10 L 4x sample buffer was added to the samples, which
were subsequently boiled at 95 °C for 2 minutes. Samples were resolved by SDS-PAGE on
12% acrylamide gels and visualized by measuring in-gel fluorescence on a Typhoon FLA 9500
scanner with settings as described above.
Comparison of 5f with ML-348 and ML-349
30 L of cytosolic fraction of mouse brain lysate (1 mg/mL) was incubated for 1 h at room
temperature with either 0.3 L of a stock solution of probe 5f (10 µM, DMSO), 0.3 L of a stock
solution of ML-348 or ML-349 (50 mM, DMSO) or 0.3 L DMSO. Then 0.3 L FP-rhodamine
(100 µM, DMSO) was added to each sample and the samples were incubated for 5 minutes at
room temperature. Next, 10 L 4x sample buffer was added to the samples, which were
subsequently boiled at 95 °C for 2 minutes. Samples were resolved by SDS-PAGE on 12%
21
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 22 of 35
1
2
3
4
5
6
acrylamide gels and visualized by measuring in-gel fluorescence on a Typhoon FLA 9500
scanner with settings as described above.
7
8
9
Cell permeability assay in A431 cells
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
A431 cells were seeded at 300,000 cells per well into a 6-well plate and allowed to adhere
overnight. The media was refreshed with 1 mL DMEM (with 10% FBS and pen/strep), spiked
with either 1 µL 5f or 6f (1 mM, DMSO) or 1 µL DMSO for 2h at 37 °C. Next, the medium was
aspirated and the cells were washed three times with PBS (5 min). Lysates were prepared by
incubating cells in 150 µL lysis buffer (20 mM HEPES, 150 mM NaCl, 2 mM DTT, 25 U/mL
Benzonase, 0.5% NP-40, pH 7.2) before scraping cells. Lysates were centrifuged (13000 g, 5
min, room temperature) to pellet debris, after which the supernatant was pipetted off. Protein
concentrations of all samples were measured using a BCA assay, and normalized to 2.39
mg/mL with storage buffer (20 mM HEPES, 2 mM DTT, pH 7.2). 90 µL of each sample was
incubated with either 0.9 µL FP-rhodamine (DMSO, 100 µM) or 0.9 µL DMSO at room
temperature for 1 hour. Next, 30 L 4x sample buffer was added to the samples, which were
subsequently boiled at 95 °C for 2 minutes. Samples were resolved by SDS-PAGE on 12%
acrylamide gels and visualized by measuring in-gel fluorescence on a Typhoon FLA 9500
scanner with settings as described above.
IC50 determination
Apparent IC50 values were measured by using six different compound concentrations in a 1:3
serial dilution, starting at a highest concentration of 30 M or 370 nM, depending on the
potency of the compound.. To this end 30 L of cytosolic or membrane fraction of mouse brain
lysate (1 mg/mL) was incubated for 1 h at room temperature with 0.3 L of a 100x stock solution
of probe in DMSO. Then, 0.3 L FP-rhodamine (100 M, DMSO) was added to each sample
and the samples were incubated for another hour. Next 10 L 4x sample buffer was added to
the samples, which were subsequently boiled at 95 °C for 2 minutes. Samples were resolved
22
ACS Paragon Plus Environment

Page 23 of 35
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
by SDS-PAGE on 12% acrylamide gels and visualized by in-gel fluorescence measuring on a
Typhoon scanner as for the initial screening. Remaining enzyme activity was determined by
measuring the integrated optical gel band intensity using ImageJ software. Values were
normalized to the FP-rhodamine labeled control sample without inhibitor, which was set at
100% activity. IC50 values were determined from a dose-response curve generated in
GraphPad Prism.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Direct labelling of probe targets
30 L of cytosolic or membrane fraction of mouse brain lysate (1 mg/mL) was incubated for 1
h with 0.3 L of a 100x stock solution of the appropriate concentration. Next, 0.5 L TAMRA-
alkyne (1.5 mM in DMSO), 0.3 L THPTA (5 mM in 4:1 t-BuOH: DMSO), 0.6 L sodium
ascorbate (50 mM in water) and 0.6 L CuSO4 (50 mM in water) were added to each sample
and the samples were incubated 1 h at room temperature. Afterwards, 10 L of 4x sample
buffer was added to the samples, which were subsequently boiled at 95 °C for 2 minutes.
Samples were resolved on an SDS-PAGE 12% acrylamide gel and visualized by in-gel
fluorescence measuring on a Typhoon scanner.
Western blot against APT-1/2
Mouse brain lysates (90 L, 1 mg/mL of either cytosolic or membrane fraction) were treated
for 1 h with 1 M probe (from a 100x dmso stock) or a corresponding amount of dmso (control
sample). All samples were subsequently treated with 1,5 L tritag (TAMRA-Lys(biotin)-NH-
propyne; 1.5 mM in DMSO), 0.9 L THPTA (5 mM in water), 1.8 L CuSO4 (50 mM in water)
and 1.8 L sodium ascorbate (50 mM in water), after which they were incubated for another
hour at room temperature. Next, 360 L of cold acetone (-80 °C) was added to each sample
to precipitate proteins and eliminate residual click reagents. The samples were incubated at -
20 °C for 30 minutes to ensure efficient protein precipitation, after which they were spun down
23
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 24 of 35
1
2
3
4
5
6
7
8
using a microcentrifuge at max speed. The supernatant was discarded, another 360 L cold
acetone was added to the pellet of the samples and the procedure was repeated once more.
Protein pellets were then redissolved in 90 L PBS with 2% SDS. Next, SDS was diluted with
9
steps of 90 L to a final concentration of 0.4% SDS. Then, 30 L of Ultralink Streptavidin resin
slurry was added to each sample to pull down all biotin-labeled proteins. To this end, all
samples were incubated on a thermomixer (1h, 1200 rpm, 25 °C). Afterwards, the samples
were spun down using a microcentrifuge at full speed.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
The supernatant was carefully pippeted from the beads, after which the beads were washed
with 120 L of 0.1% SDS in PBS, subsequently with 120 L of 1M NaCl, then with 120 L 1M
urea, then with 120 L PBS and finally with 120 L MilliQ water. After each washing step, the
samples were spun down using a microcentrifuge at full speed and the supernatant was
discarded. The beads were resuspended in 120 L sample buffer and boiled for 10 minutes to
release the proteins from the beads. Samples were then resolved on a 12% acrylamide gel
using SDS-PAGE.
A fluorescent image of the gels was made (Typhoon scanner), after which the proteins were
transferred onto nitrocellulose membranes. Both membranes were incubated for 1h with 3%
milk/PBST. The milk was discarded and one of the gels was incubated overnight with 15 mL
anti-APT-1 antibody Ab 91606 (0.5 µg/mL in PBST), whereas the other membrane was
incubated overnight with 15 mL anti-APT-2 antibody Ab 151578 (0.5 g/mL in PBST). The
antibody-solutions were then decanted and the membranes washed four times with PBST.
Next, both membranes were incubated with anti-rabbit HRP antibody (0.2 g/mL in PBST) for
1h. Afterwards, the membranes were washed four times with PBST and incubated with
SuperSignal West Pico Plus chemiluminescent substrate (Life Technologies) just prior to
development using a BioRad ChemiDoc imaging system.
Covalent docking
24
ACS Paragon Plus Environment

Page 25 of 35
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
The different probes were drawn in Chemdraw professional 15.1, converted to a pdb file using
Chem3D 15.1 and converted into a PDBQT by AutoDockTools 1.5.6. Probes were attached
with their carbonyl function (with elimination of the leaving group) to the side chain of residue
S119 of APT-1 (PDB code: 6QGS, from which water molecules and the palmitate ligand were
removed), using the provided script at the autodock website (see for download:
http://autodock.scripps.edu/resources/covalentdocking) with a slight modification. Molecules
were then docked according to the flexible side chain method of AutoDock 4.2, as described
by Bianco et al.29 The connected molecules were treated as a fully flexible side chain. The
binding site was covered by preparing a 60ꢀ×ꢀ76ꢀ×ꢀ70 size grid box with grid spacing of 0.375
Å and the center at 7.837, 127.491, 18.586.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
25
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 26 of 35
1
2
3
4
Associated content
5
6
Supporting information
7
8
9
The Supporting Information is available free of charge at https://pubs.acs.org/
Additional synthetic procedures, Supporting Figures S1-S7, copies of NMR spectra and HPLC
purity data, coomassie stains of gels
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Coordinates of docked probes
Molecular formula strings
Funding
We acknowledge financial support from Research Foundation Flanders FWO (project number
G0E3617N), the Ministerium für Kultur und Wissenschaft des Landes Nordrhein-Westfalen,
the Regierende Bürgermeister von Berlin–inkl. Wissenschaft und Forschung, and the
Bundesministerium für Bildung und Forschung.
Acknowledgments
The authors thank Luc Baudemprez for recording NMR spectra and Rita Derua for HRMS.
Abbreviations
ABDH6, ,-hydrolose domain containing protein 6; ABP, activity-based probe; ABPP, activity-
based protein profiling; APT-1/2, acyl protein thioesterase-1/2; CuAAC, copper(I)-catalyzed
azide alkyne cycloaddition; DAGL, diacylglycerol lipase; FP, fluorophosphonate; MAGL,
monoacylglycerol lipase; NHS, N-hydroxysuccinimide; SH, serine hydrolase
References
1.
enzyme chemistry to proteomic chemistry. Annu. Rev. Biochem. 2008, 77, 383-414.
2. Sadaghiani, A. M.; Verhelst, S. H. L.; Bogyo, M., Tagging and detection strategies for
Cravatt, B. F.; Wright, A. T.; Kozarich, J. W., Activity-based protein profiling: from
activity-based proteomics. Curr. Opin. Chem. Biol. 2007, 11, (1), 20-8.
26
ACS Paragon Plus Environment

Page 27 of 35
Journal of Medicinal Chemistry
1
2
3
4
3.
Liu, Y. S.; Patricelli, M. P.; Cravatt, B. F., Activity-based protein profiling: The serine
5
6
7
8
hydrolases. Proc. Natl. Acad. Sci. USA 1999, 96, (26), 14694-14699.
4. Patricelli, M. P.; Giang, D. K.; Stamp, L. M.; Burbaum, J. J., Direct visualization of serine
9
hydrolase activities in complex proteomes using fluorescent active site-directed probes.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Proteomics 2001, 1, (9), 1067-71.
5.
Jessani, N.; Liu, Y. S.; Humphrey, M.; Cravatt, B. F., Enzyme activity profiles of the
secreted and membrane proteome that depict cancer cell invasiveness. Proc. Natl. Acad. Sci.
USA 2002, 99, (16), 10335-10340.
6.
potential of serine hydrolases. Nat. Rev. Drug. Discov. 2012, 11, (1), 52-68.
7. Savinainen, J. R.; Saario, S. M.; Laitinen, J. T., The serine hydrolases MAGL, ABHD6
Bachovchin, D. A.; Cravatt, B. F., The pharmacological landscape and therapeutic
and ABHD12 as guardians of 2-arachidonoylglycerol signalling through cannabinoid receptors.
Acta Physiol. 2012, 204, (2), 267-76.
8.
palmitoylation. Med. Chem. Comm. 2014, 5, (3), 268-276.
9. Sontag, J. M.; Nunbhakdi-Craig, V.; Mitterhuber, M.; Ogris, E.; Sontag, E., Regulation
Davda, D.; Martin, B. R., Acyl protein thioesterase inhibitors as probes of dynamic S-
of protein phosphatase 2A methylation by LCMT1 and PME-1 plays a critical role in
differentiation of neuroblastoma cells. J. Neurochem. 2010, 115, (6), 1455-65.
10.
Bachovchin, D. A.; Ji, T.; Li, W.; Simon, G. M.; Blankman, J. L.; Adibekian, A.; Hoover,
H.; Niessen, S.; Cravatt, B. F., Superfamily-wide portrait of serine hydrolase inhibition achieved
by library-versus-library screening. Proc. Natl. Acad. Sci. USA 2010, 107, (49), 20941-20946.
11.
Cheng, R.; Mori, W.; Ma, L.; Alhouayek, M.; Hatori, A.; Zhang, Y.; Ogasawara, D.;
Yuan, G.; Chen, Z.; Zhang, X.; Shi, H.; Yamasaki, T.; Xie, L.; Kumata, K.; Fujinaga, M.; Nagai,
Y.; Minamimoto, T.; Svensson, M.; Wang, L.; Du, Y.; Ondrechen, M. J.; Vasdev, N.; Cravatt,
B. F.; Fowler, C.; Zhang, M. R.; Liang, S. H., In Vitro and in Vivo Evaluation of (11)C-Labeled
Azetidinecarboxylates for Imaging Monoacylglycerol Lipase by PET Imaging Studies. J. Med.
Chem. 2018, 61, (6), 2278-2291.
27
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 28 of 35
1
2
3
4
12.
Chen, Z.; Mori, W.; Fu, H.; Schafroth, M. A.; Hatori, A.; Shao, T.; Zhang, G.; Van, R.
5
6
7
8
S.; Zhang, Y.; Hu, K.; Fujinaga, M.; Wang, L.; Belov, V.; Ogasawara, D.; Giffenig, P.; Deng,
X.; Rong, J.; Yu, Q.; Zhang, X.; Papisov, M. I.; Shao, Y.; Collier, T. L.; Ma, J. A.; Cravatt, B. F.;
Josephson, L.; Zhang, M. R.; Liang, S. H., Design, Synthesis, and Evaluation of (18)F-Labeled
Monoacylglycerol Lipase Inhibitors as Novel Positron Emission Tomography Probes. J. Med.
Chem. 2019, 62, (19), 8866-8872.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
13.
integral membrane hydrolase KIAA1363. Angew. Chem. Int. Ed. Engl. 2012, 51, (4), 966-70.
14. van Rooden, E. J.; Kohsiek, M.; Kreekel, R.; van Esbroeck, A. C. M.; van den
Chang, J. W.; Moellering, R. E.; Cravatt, B. F., An activity-based imaging probe for the
Nieuwendijk, A.; Janssen, A. P. A.; van den Berg, R.; Overkleeft, H. S.; van der Stelt, M.,
Design and Synthesis of Quenched Activity-based Probes for Diacylglycerol Lipase and
alpha,beta-Hydrolase Domain Containing Protein 6. Chem. Asian J. 2018, 13, (22), 3491-
3500.
15.
van Rooden, E. J.; Kreekel, R.; Hansen, T.; Janssen, A. P. A.; van Esbroeck, A. C. M.;
den Dulk, H.; van den Berg, R.; Codee, J. D. C.; van der Stelt, M., Two-step activity-based
protein profiling of diacylglycerol lipase. Org. Biomol. Chem. 2018, 16, (29), 5250-5253.
16.
Chang, J. W.; Cognetta, A. B., 3rd; Niphakis, M. J.; Cravatt, B. F., Proteome-wide
reactivity profiling identifies diverse carbamate chemotypes tuned for serine hydrolase
inhibition. ACS Chem. Biol. 2013, 8, (7), 1590-9.
17.
Haedke, U. R.; Frommel, S. C.; Hansen, F.; Hahne, H.; Kuster, B.; Bogyo, M.; Verhelst,
S. H., Phosphoramidates as novel activity-based probes for serine proteases. ChemBioChem
2014, 15, (8), 1106-10.
18.
enzyme classes: the serine hydrolases. Bioorg. Med. Chem. Lett. 2014, 24, (16), 3807-13.
19. Adibekian, A.; Martin, B. R.; Wang, C.; Hsu, K. L.; Bachovchin, D. A.; Niessen, S.;
Otrubova, K.; Srinivasan, V.; Boger, D. L., Discovery libraries targeting the major
Hoover, H.; Cravatt, B. F., Click-generated triazole ureas as ultrapotent in vivo-active serine
hydrolase inhibitors. Nat. Chem. Biol. 2011, 7, (7), 469-78.
28
ACS Paragon Plus Environment

Page 29 of 35
Journal of Medicinal Chemistry
1
2
3
4
20.
Long, J. Z.; Jin, X.; Adibekian, A.; Li, W.; Cravatt, B. F., Characterization of tunable
5
6
7
8
piperidine and piperazine carbamates as inhibitors of endocannabinoid hydrolases. J. Med.
Chem. 2010, 53, (4), 1830-42.
9
21.
Aaltonen, N.; Savinainen, J. R.; Ribas, C. R.; Ronkko, J.; Kuusisto, A.; Korhonen, J.;
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Navia-Paldanius, D.; Hayrinen, J.; Takabe, P.; Kasnanen, H.; Pantsar, T.; Laitinen, T.;
Lehtonen, M.; Pasonen-Seppanen, S.; Poso, A.; Nevalainen, T.; Laitinen, J. T., Piperazine and
piperidine triazole ureas as ultrapotent and highly selective inhibitors of monoacylglycerol
lipase. Chem. Biol. 2013, 20, (3), 379-90.
22.
Lamani, M.; Malamas, M. S.; Farah, S. I.; Shukla, V. G.; Almeida, M. F.; Weerts, C. M.;
Anderson, J.; Wood, J. T.; Farizatto, K. L. G.; Bahr, B. A.; Makriyannis, A., Piperidine and
piperazine inhibitors of fatty acid amide hydrolase targeting excitotoxic pathology. Bioorg. Med.
Chem. 2019, 27, (23), 115096.
23.
Baggelaar, M. P.; van Esbroeck, A. C.; van Rooden, E. J.; Florea, B. I.; Overkleeft, H.
S.; Marsicano, G.; Chaouloff, F.; van der Stelt, M., Chemical Proteomics Maps Brain Region
Specific Activity of Endocannabinoid Hydrolases. ACS Chem. Biol. 2017, 12, (3), 852-861.
24.
Blankman, J. L.; Simon, G. M.; Cravatt, B. F., A comprehensive profile of brain enzymes
that hydrolyze the endocannabinoid 2-arachidonoylglycerol. Chem. Biol. 2007, 14, (12), 1347-
56.
25.
van Rooden, E. J.; van Esbroeck, A. C. M.; Baggelaar, M. P.; Deng, H.; Florea, B. I.;
Marques, A. R. A.; Ottenhoff, R.; Boot, R. G.; Overkleeft, H. S.; Aerts, J.; van der Stelt, M.,
Chemical Proteomic Analysis of Serine Hydrolase Activity in Niemann-Pick Type C Mouse
Brain. Front. Neurosci. 2018, 12, 440.
26.
Adibekian, A.; Martin, B. R.; Chang, J. W.; Hsu, K. L.; Tsuboi, K.; Bachovchin, D. A.;
Speers, A. E.; Brown, S. J.; Spicer, T.; Fernandez-Vega, V.; Ferguson, J.; Hodder, P. S.;
Rosen, H.; Cravatt, B. F., Confirming target engagement for reversible inhibitors in vivo by
kinetically tuned activity-based probes. J. Am. Chem. Soc. 2012, 134, (25), 10345-10348.
29
ACS Paragon Plus Environment