University of Groningen

University of Groningen

Nature-inspired molecules containing multiple electrophilic positions Dockerty, Paul Jacques

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2018

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Dockerty, P. J. (2018). Nature-inspired molecules containing multiple electrophilic positions: Synthesis and application as activity-based probes and inhibitors. University of Groningen.

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Chapter 4

An original warhead targeting serine hydrolases

Brabantamide A is known to modify Lp-PLA

, a serine hydrolase. Antibacterial enol

carbamates based on the brabantamide A scaffold have been shown to covalently

interact with penicillin-binding proteins in Bacillus subtilis (chapter 2) and our

probes PJD2d and PJD224 based on the same scaffold modified a Cys residue of

RALDH1 in A549 (chapter 3). Intrigued by these results, we aimed in this chapter

to identify the targets of the synthesized inhibitor towards serine hydrolases on

A549 and Bacillus subtilis lysates. The activity-based probes synthesized previously

were first tested to probe recombinant Lp-PLA

and esterase from B.subtilis. After

successful initial results we were able to use PJD224 as an activity-based platform

to screen the remainder of the panel and give insight into scaffold modifications

allowed to inhibit Lp-PLA

and the esterase from Bacillus subtilis.

4.1 Introduction

Serine and cysteine residues are commonly found in the active site of enzymes, where they catalyze a large variety of reactions, often via a covalent substrate-enzyme intermediate. This adduct is rapidly hydrolysed and the enzyme reactivated. In order to inhibit the catalytic activity, electrophiles have been designed that react with these conserved serine or cysteine residues selectively in a mechanism-based fashion. β-lactams, β- lactones or activated carbamates have been designed to inhibit serine hydrolases (SHs). Upon reaction between these electrophiles and the catalytic serine, a stabilized inhibitor-enzyme adduct is formed that only slowly hydrolyzes and that is at the origin of the inhibition of SHs.

Haloketones and Michael acceptors have commonly been applied to modify nucleophilic cysteine residues.

The selectivity of these specific electrophiles for thiols can be explained by the Hard and Soft, Acids and Bases (HSAB) theory of Pearson,

which asserts that nucleophiles are more prone to form a covalent bond with an electrophile of comparable softness or hardness.

The energy levels and overlap of the frontier molecular orbitals (HOMO of the nucleophile and LUMO of the electrophile) participating in the formation of the new bond uphold the specific hard-hard and soft-soft selectivity. In contrast to the amino groups of lysine and histidine and the hydroxyl groups of serine and threonine, the thiol of a cysteine is a very reactive soft nucleophile. It therefore reacts preferably with soft electrophiles. By appending serine and cysteine-selective reactive groups onto scaffolds that bind to the protein of interest, irreversible inhibitors have been prepared for various serine and cysteine hydrolases. Even though successful, the development of novel inhibitors that bind to different subsets of protein targets and/or have an improved selectivity also relies on the simultaneous identification of unprecedented electrophile scaffolds. The synthesis and chemical profiling of compounds that feature uncommon electrophiles found in natural products led to the identification of such scaffolds.

Especially secondary metabolites contain a wide variety of rare electrophiles that have been molded to react with a specific target. By preparing derivatives of scaffolds containing a reactive diyne, a 4-chloroisoxazole motif or a cyclopropyl not only new inhibitors for RALDH1, ALDH2, ALDH4A1 and CES1 were developed but also the targets from the corresponding natural products have been identified.

With the aim to develop novel serine hydrolase inhibitors, we recently

was first found in the lipopetide Brabantamide A

and we found ourselves intrigued by this scaffold since it contains two putative electrophilic traps, an activated carbamate and an α,β-unsaturated imide. Inhibition studies with Brabantamide A and synthetic analogue 2a on lipoprotein-associated phospholipase A

(Lp-PLA

) indicated that the enol cyclocarbamate scaffold indeed might function as an electrophilic trap for serine hydrolases.

Our recent studies towards the antibacterial activity of enol carbamate further support the hypothesis that hyperreactive serine residues react with enol cyclocarbamates. Several derivatives block labeling of penicillin-binding proteins in the gram-positive bacteria Bacillus subtilis and Streptococcus pneumoniae.

Inhibition of other enzymes cannot be detected in the competitive protein profiling experiments with fluorescent penicillin analogues and this prompted us to further identify the targets using a chemoproteomics approach. We prepared chemical probes PJD120, PJD224,

enol cyclocarbamates and we applied these probes on A549 cell lysates in Chapter 3. Remarkably, we identified the aldehyde dehydrogenases RALDH1 and ALDH3A1 as the most prominent targets in this cell type.

These enzymes catalyze the oxidation of aldehydes into the corresponding carboxylic acids using a cysteine residue, which suggests that also cysteine residues react with enol cyclocarbamates. Interestingly, hardly any SHs were isolated during these chemoproteomic labeling studies.

This raised the question if SHs react with our probes and inhibitors at all.

We hypothesized that the substitution pattern of the electrophile would determine to a great extent, which proteins react with the enol carbamate and that the introduced fluorophore may affect binding to serine hydrolases. We therefore performed labeling studies with PJD120 and

carbamates synthesized in Chapter 2 in competition experiments against the broad spectrum fluorophosphonate SH probes Fp-rhodamine (Fp-rho) and Fp-biotin (Fp-bio).

These profiling studies on purified enzymes and Bacillus subtilis and A549 mammalian cell lysates revealed that the enol cyclocarbamates covalently bind to serine hydrolases, with acyl-protein thioesterase 1 (LYPLA1) and acyl-protein thioesterase 2 (LYPLA2) as important targets.

Figure 1. (A) Structures of probes PJD120 and PJD224. (B) N-Boc-Proline derived enol cyclocarbamates 2a–i containing various chains. (C) Monocyclic enol carbamates 3a–c (D) N-Boc-4-hydroxyproline derived enol cylclocarbamate 4a–e.

4.2 Results and discussion

4.2.1 Labeling of recombinant serine hydrolases.

We first reacted our probes PJD120 and PJD224 with recombinant Lp-PLA

to assess if they, beside aldehyde dehydrogenases, also covalently modify serine hydrolases. A mixture of purified Lp-PLA

and bovine serum albumin (BSA) was treated with increasing concentrations of the probes (from 0.01 µM to 10 µM) at 37 °C for 1 h and the labeled proteins were then analyzed by SDS-PAGE and in-gel fluorescence scanning (Figure 2A). As positive control, a similar experiment was performed with Fp-Rho (Figure 2A), a broad-spectrum SH probe. All of the enol cyclocarbamate probes preferentially labeled Lp-PLA

at sub-micromolar concentrations, even in the presence of an excess of BSA. Heat-inactivation of Lp-PLA

prior to incubating with the probe completely blocked labeling. At higher probe concentrations, PJD120, PJD224 and Fp-Rho start to label the carrier protein BSA with equal fluorescence intensities. This may be explained in part by the large excess of BSA (50 fold over Lp-PLA

). Moreover, it has been

A

N O O

HN O PJD224

N NN

NB N FF

N O

O

O H

N O PJD120 NN

N BN

N FF

N O

NH O R

2a R = (CH2)11CH3

2b R = (CH2)5CH3

2c R = CH2Ph 2d ^{R =}

2 O

2e R =

N NN (CH²)11CH3

2f R = N NN 2g R =

N NN OH

2h R =

N NN ₂

2i R =

N NN O NH 3

O (CH2)4 S

HN NH O

H H

R N O O

NH (CH2)11CH3 O

3a R = 3b R =

N NN O 2 OH

3c R = CH3

3

N O O

O

NH (CH2)11CH3 O

R

4

4b R = 4a R = CH2Ph

4c R = N NN

Ph 4d R =

N NN O 2 OH

4e R =

N NN N

B C

D

reported that BSA possesses esterase activity.

In order to confirm that probes PJD120 and PJD224 bind in an activity dependent manner to Lp- PLA

, we set up a competition experiment between Fp-Rho and our probes.

Figure 2. Labeling of recombinant Lp-PLA². (A) Initial characterization of probes PJD224 and PJD120 and control Fp-rhodamine. (B) Competition experiments between PJD224 or PJD120 (Cy2) and Fp-rhodamine (Cy3). The triangles indicate a denatured sample where the protein is boiled with 1% SDS prior to the incubation with the probe.

As expected, PJD120 and PJD224 compete with Fp-Rho for binding, which indicates that the probes based on the enol cyclocarbamate scaffold bind to the same site as Fp-Rho (Figure 2B). To evaluate if other SHs also bind to the probes, we attempted to label recombinantly expressed p-nitrobenzyl esterase from B.subtilis. This SH was also labeled in a concentration dependent fashion by probes PJD120 and PJD224 and again labeling could be blocked by heat-denaturing (Figure 3A). As for Lp-PLA

, probe PJD224 competed with FP-Rho for labeling. Pre-incubating the enzyme with 1 µM of

in the Cy3 channel. Interestingly, we were also able to label the esterase using compound PJD2d. These experiments highlight two interesting features of the enol carbamates compounds; first these compounds are indeed SHs inhibitors and secondly via adequate derivatization they can be used as chemical probes of both bacterial esterase and mammalian Lp-PLA

.

Having established that the enol cyclocarbamate probes bind to SHs, we decided to employ probe PJD224 to study the activity of our previously reported panel of lipocyclocarbamates on Lp-PLA

(Figure 4) and B.subtilis esterase (Figure 5). Direct competition experiments with probe PJD224 (at 0.1 µM) confirmed that compound 2a binds recombinant Lp-PLA

(Figure 4), which corroborates the results of Pinto and coworkers.

Decreasing the lipophilicity of the tail by replacing it with smaller substituents as in compounds 2b, 2c and 2d (respectively hexyl, benzyl and propargyl) or more hydrophilic groups as in 2g and 2i drastically reduces the inhibitory potency at the tested concentrations (1, 0.5 and 0.1 µM) (Figure 4).

0.05 0.01

0.5 0.1

PJD120 [μM] 1 ^{0.05 0.01} Fp-rho [μM] ^{1 0.5 0.1 0.05 0.01}

A Fp-rho [0.5 μM] ^{+ +} +

[μM]Px -

B

Cy3 Cy2 0.5 0.1

PJD224 [μM] 1

- +

- +

+ +

- -

PJD224 [0.1 μM]

PJD120 [0.1 μM] 120

224 0.1 Lp-PLABSA -₂ - 0.5

Figure 3. Labeling of B.subtilis esterase. (A) Initial characterization of probes PJD224, PJD120 and control Fp-rhodamine on the recombinant B.subtilis esterase. The triangles indicate a denatured sample where the protein is boiled with 1% SDS prior to the incubation with the probe. (B) Competition experiment between PJD224 (Cy2) and Fp- rhodamine (Cy3) with different conditions; lane 1 and 2: 30 min incubation; lane 3: 30 min preincubation Fp-rho followed by 30 min incubation with PJD224; lane 4: 30 min preincubation PJD224 followed by 30 min incubation with Fp-rho; lane 5: direct competition between Fp-rho and PJD224 for 30 min; lane 6 and 7: 1 h incubation. (C) Competition Fp-rho (1 µM) vs 2a.

The activity is restored when either a dodecyl or a phytyl group is introduced, as in 2e and 2h. Other lipophilic groups, such as the biphenyl in compound 2f only partly restore the activity at 1 µM, indicating a preference for long alkyl chains over more bulky lipophilic groups. Monocyclic compounds 3a-b are not active under the tested conditions, but surprisingly the very similar lipocyclocarbamate 3c blocks labeling at 1 and 0.5 µM.

Finally, all of the head-group derivatives inhibit Lp-PLA2 at 0.1 µM, except for derivate 4a, which is approximately ten-fold less potent. Similar trends were observed for the esterase from B.subtilis. All tail derivatives (2a-i) display activity at 1 µM, when competed against PJD224, but only compounds 2a, 2e and 2h inhibit labeling of this esterase at lower concentrations (Figure 5).

100 0.01

A

50 10 1 0.1

PJD224 [μM]

PJD120 [μM] Fp-rho [μM] M 5 2.5 1 0.1 0.01

-75 kDa 100 50 10 1 0.1 0.01

B

PJD224 [1 μM]

Fp-rho [1 μM]

+ + + -

+ - +

+ + + +

- + -

1 3 4 5 6

Cy3 Cy2

- +

- +

+ +

- -

PJD224 [1 μM]

PJD120 [1 μM]

Fp-rho [1 μM] M + + + + + -75 kDa 2a [μM] - 0.1 1 10 100

C

PJD2d [μM] - 5 10 25 50 50 Bodipy-N3 [50 μM] _{+ + + + + +}

2 7

Figure 4. Recombinant Lp-PLA². Competition PJD224 (0.1 µM) vs the panel of derivatives.

These three hydrophobic compounds are also the most potent inhibitors of Lp-PLA

(Figure 4). The monocyclic compounds proved to be inactive on the bacterial esterase. In contrast to Lp-PLA

, compound 3c does not inhibit this esterase (Figure 5). Finally, the head group derivatives 4a-e inhibit the bacterial esterase, as is the case for Lp-PLA

, but the potencies vary. Clearly, the inhibitory potency for Lp-PLA

and the esterase is affected by modifications on the scaffold and the substitution pattern therefore may determine the selectivity of the compound in more complex mixtures.

Figure 5. Recombinant esterase. Competition PJD224 (1 µM) vs the panel of derivatives.

3a 3b

1 0.5 0.12d 1 0.5 0.12e

1 0.5 0.12f -

1 0.5 0.12g 1 0.5 0.12h

1 0.5 0.12i -

1 0.5 0.1 1 0.5 0.1

3a 3b

1 0.5 0.1 3c -

Inhibitor [μM]

3a 3b

4a 4b 4c

-

1 0.5 0.14d 1 0.5 0.14e

- M

Inhibitor [μM] 1 0.5 0.1 1 0.5 0.1 1 0.5 0.1

Inhibitor [μM]

BSA - Lp-PLA2 -

1 0.5 0.1 1 0.5 0.1

2b 2c

0.5 0.1 2a

- M

-75 kDa Inhibitor [μM]

5 1 0.2

2d 5 1 0.2

2c 5 1 0.2

2b Inhibitor [μM]

Inhibitor [μM]

- 0.2 1 5

2g 5 1 0.2

2f 5 1 0.2

2e

-

5 1 0.2

3a 5 1 0.2

2i 5 1 0.2

2h

- 0.2 1 5

4a 5 1 0.2

3c 5 1 0.2

3b

-

5 1 0.2

4e 5 1 0.2

4d 5 1 0.2

4c

- 5

1 0.2

4b Inhibitor [μM]

1 0.1 -

2a

Inhibitor [μM] 10 100

4.2.2 Competitive protein profiling on A549 and B.subtilis lysates.

We then decided to perform competitive reactive serine profiling experiments in Bacillus subtilis 168 lysates and A549 cell line lysates using Fp-rhodamine as a read-out. In order to identify the targeted proteins we used Fp-biotin to perform pull-down experiments using streptavidin beads.

After separation of the proteins on a SDS-PAGE gel and coomassie stain, we excised the most prominent bands and performed mass spectrometry analysis and label free quantification in order to identify the targeted proteins. Comparisons with reported proteins identified by Fp-biotin from these two biological sources were used to confirm the targets.

Competition experiments on lysates of A549 cells and label free quantification of the proteins that are retrieved by Fp-biotin in the presence and absence of inhibitor revealed that monocyclic analogs 3a-c, compounds

inhibit LyPLA1 and LyPLA2. All the other compounds did block labeling of these two enzymes. LyPLA1 hydrolyzes a variety of lysophospholipids and shows a thioesterase/esterase activity on several peptide substrates. LyPLA2 has 60% homology with LyPLA1 and displays the same type of activities. In particular, compounds 4b and 4d displayed interesting specificity towards LyPLA2 at 10 µM.

Figure 6. A549 cell line. Competition experiment Fp-rho (1 µM) vs the panel of derivatives (10 and 100 µM).

-+ + 2b 100 +

2b 10 +

2c 100 +

2c 10 +

2e 100 +

2e 10 +

2f 100 +

2f 10

- + +

3b 100 +

3b 10 +

3c 100 +

3c 10 +

4a 100 +

4a

10 +

4c 100 +

4c 10 Inhibitor

[μM]

Fp-rho (1 μM) - + 2g 100 +

2g 10 +

2h 100 +

2h 10 +

2i 100 +

2i 10 +

3a

100 +

3a 10

LyPLA1LyPLA2 FASN

-+ + 4d 100 +

4d 10

kDa 75

25 37 50

kDa 75

25 kDa

75

25

M

M M Inhibitor

[μM] - Fp-rho (1 μM) + +

2a 100

+ 2a 10

+ 2a 1

+ 4b 100

+ + 4b 1

+ 2d 100

+ 2d 10

kDa 75

25

+ LyPLA1 LyPLA2 FASN

4b 10

Furthermore, Fp-biotin pull down and subsequent label free quantification revealed that Lp-PLA

was targeted by compound 2a which corroborates the results of Pinto and Thirkettle.

Identification of the Fp-biotin labeled proteins in the 100-250 kDa region in combination with label free quantification indicated that compounds 2b, 2c, 2f and 2g target fatty acid synthase (FASN). The main function of this protein is the synthesis of palmitate from acetyl-CoA and malonyl-CoA and has been reported as a potential chemotherapeutic target. Its inhibition apparently leads to NADPH accumulation and consequent cell death.

Analog 2f targets LyPLA1, LyPLA2 and FASN at 100 µM but displayed a remarkable selectivity towards FASN at 10 µM.

On B.subtilis lysates, three main protein bands were visible in the 25-35 kDa region after Fp-rho labeling that are targeted by the majority of the analogs.

The less electrophilic monocyclic analogs 3a-b and the hydrophobic derivative 4c do not inhibit labeling of these proteins. The pull-down and label free quantification identified several proteins (10 proteins, see appendix) of 25-35 kDa and these results correspond well with the previously identified proteins by Meier et al.

Based on in-gel fluorescence scanning, the majority of these proteins are also inhibited by derivatives that do not display antibacterial activity and it is therefore not very likely that these proteins are involved in the reported growth inhibition of B.subtilis by compounds 2a, 2e, 4d and 4e.

The three different bands visible by in-gel fluorescence scanning seem to contain several proteins based on the pull- down results. Surfactin synthase subunit 3 (srfAC), protein of 143 kDa, was also identified as a target of several compounds. Compound 2g especially displayed high selectivity towards this protein involved in the surfactin biosynthesis.

Figure 7. Bacillus subtilis 168. Competition experiment Fp-rho (1 µM) vs the panel of derivatives (100 µM).

kDa 75

25

Inhibitor [μM] - Fp-rho (1 μM) + +

100 2a + 100 2d

+ 100 4b

+ 100 2b

+ 100 2c

+ 100 2e

+ 100 2f

+ 100 2g

srfAC

- + +

100 2h + 100 2i

+ 100 3a

+ 100 3b

+ 100 3c

+ 100 4a

+

100 4c -

+ + 100 4e

+ PJD224 100

+ 120PJD 100 +

100 4d

Surfactin is a powerful surfactant that is mainly used for its ability to stabilize monovalent and divalent cations (in between the claw formed by aspartate and glutamate).

The results from the pull down experiments with PJD2d described in Chapter 3 indicate that the enol cyclocarbamate scaffold modifies cysteine residues. We were therefore interested in screening our panel of compounds also against a broad-spectrum cysteine probe, such as iodoacetamide alkyne (IAA). To this end, we performed competitive protein profiling experiments using IAA. No noticeable differences were observed for the samples that were preincubated with the enolcarbamates, presumably because the used concentration of IAA did not completely saturate the Cys residues. As reported before,

IAA is a difficult tool to use for such gel based competitive experiments and in-depth in vivo quantitative proteomics should therefore be employed to map these residues.

4.3 Conclusion

This chapter reveals that the enol cyclocarbamate probes PJD120 and

in an activity-based manner. We were able to use PJD224 in a competitive labeling profiling experiment to screen our panel of derivatives. This profiling study provided insight into the scaffold derivatizations that are required for efficient Lp-PLA

inhibition. To complete our understanding of this scaffold, we used Fp-rho and Fp-bio to identify the targets of these compounds in more complex biological settings (A549 lysates and B.subtilis lysates). Several compounds showed interesting selectivity towards important proteins in A549 cells lysates. These results may have an impact into the future design of potent inhibitors or probes based on the enol cyclocarbamate scaffold and participate into our effort to fully characterize this scaffold. The combined results of Chapter 3 and 4 confirm that these compounds target serine and cysteine residues, but proper tuning may accentuate the specificity for one or another residue and thus will result in more selective probes and inhibitors.

4.4 Experimental

4.4.1. General biochemical procedures

Proteins. Recombinant Lp-PLA2 (PAF-AH human, SRP3136 SIGMA) was purchased from Sigma-Aldrich. The protein was reconstituted in buffer (50 mM Tris HCl pH 8.0, 150 mM NaCl)

at 20 ng/µL in the presence of BSA (Bovin Serum Albumin) at 1 mg/mL and stored in 50 µL working aliquots at -20 °C. Recombinant Esterase from Bacillus subtilis (96667-10MG SIGMA) was purchased from Sigma-Aldrich. The protein was reconstituted in PBS (NaCl 137 mM, KCl 2.7 mM, Na²HPO⁴ 10 mM, KH²PO⁴ 1.8 mM, pH 7.4) at 100 ng/µL and stored in 100 µL working aliquots at -20 °C.

Probes, reagents and material. Fp-rhodamine (ActivX™ TAMRA-FP Serine Hydrolase Probe, 88318) was purchased from Thermo Fisher Scientific and 10 µL working aliquots at 50 µM were prepared in DMSO and then stored at -20 °C. Fp-biotin (sc-215056A) was purchased from Santa Cruz biotechnology and 10 µL working aliquots at 10 µM were prepared in DMSO and then stored at -20 °C. TABTA was prepared as described and stock solutions of TABTA ligand was prepared in water and stored at -20 °C. Stock solutions of CuSO4 ⋅5H2O and sodium ascorbate were prepared fresh in water before each experiment. Sample buffer (SB) 4× contains 200 mM Tris-HCl (pH 6.8), 400 mM dithiothreitol (DTT), 8% sodium dodecyl sulfate (SDS), 0.4%

bromophenol blue and 40% glycerol. Probes PJD120 and PJD224 and the panel of derivatives, were dissolved in DMSO and the stock solutions were stored at -20 °C.

Cell Culture conditions. A549 cells were grown in t75 culture flasks in DMEM supplemented with 10% FBS (Fetal Bovine Serum), 1% L-Glutamine, and 1% Pen/Strep into an incubator at 37

°C and 5% CO2 humidified air. At about 70-90% confluency cells were detached from the flask by Trypsin/EDTA treatment, pelleted and reseeded in 4 ml complete cell culture medium per T75 flask or washed two times with PBS and conserved at -80°C. The pellets were lysed using a NP40 lysis buffer [0.5 % NP40, Tris·HCl (10 mM), NaCl (150 mM), MgCl2 (5 mM), pH 7.4]

during 10 min over ice, the lysates were then spin down during 10 min at 10 000 rpm and the supernatant was collected and submitted to a Bradford protein assay to assess the protein concentration before subsequent dilution to 1 mg/mL. One t75 flask usually provided 200-250 µL at 1 mg/mL of protein.

Bacillus subtilis 168 cells were cultured in LB broth overnight. The cells were then diluted in LB to OD=0.1 and cultured 2-3 hours up to OD=0.4-0.5. The cells were then washed three times with PBS and lyzed in PBS using lysozyme (1 mg/mL) together with sonication over ice. The lysates were then spin down during 10 min at 10 000 rpm and the supernatant was collected and submitted to a Bradford protein assay to assess the protein concentration. Lysates were then aliquoted, snap-frozen with liquid nitrogen and stored at -80 °C. Before each experiment the lysates were diluted to a concentration of 1 or 2 mg/mL of protein.

SDS-PAGE and pull-down analysis. Laemmli type SDS‐PAGE was performed according to standard literature procedures.¹⁹ Gels were prepared using acrylamide-bis ready-to-use solution 40% (37.5:1) (Merck Millipore) and separated on a Mini-PROTEAN Tetra cell (Bio‐

Rad). Fluorescence scanning of SDS-PAGE gels was performed on a Typhoon 9500 FLA model (GE Healthcare) using the CY2 settings for BODIPY (blue laser excitation at 488 nm and emission filter at 520 nm). Coomassie staining was carried out with colloidal CBB G250 staining according to the manufactures protocol (Roti-Blue, Carl Roth). BioRad precision plus protein standards dual color was used as molecular weight marker. Pull-down was performed using Dynabeads® MyOne™ Streptavidin C1 and DynaMag™-2 Magnet (ThermoFisher Scientific).

4.4.2. Labeling experiments with recombinant proteins

SDS-PAGE labeling with recombinant Lp-PLA2.

Initial characterization: 1 µL of the probe PJD120 and PJD224 (0.01 µM to 1 µM) or Fp-rhodamine (0.01 µM to 5 µM) was incubated with 9 µL of the Lp-PLA2 solution (20 ng/µL stock solution) for 1 hour at 37 °C. SB (with DTT) was added and the proteins were resolved on a 15% SDS-PAGE and fluorescence was visualized using a Typhoon scanner by in-gel fluorescence scanning. (SB

= sample buffer, DTT = dithiothreitol)

Competition experiment: 1 µL of either DMSO or enol cyclocarbamates 2a-i, 3a-c and 4a-e (1, 0.5 and 0.1 µM) were incubated with 9 µL of the Lp-PLA2 solution (20 ng/µL stock solution) for 1 hour at 37 °C. Then 1 µL of the probe PJD224 (0.1 µM) was added and the solution was incubated for one more hour. SB (with DTT) was added and the proteins were resolved on a 15% SDS-PAGE and fluorescence was visualized using a Typhoon scanner by in-gel fluorescence scanning.

The same protocol was applied for the competition between PJD120 (0.5 µM) or PJD224 (0.1 µM) and Fp-rhodamine (0.5 µM).

SDS-PAGE labeling with recombinant esterase from B.subtilis.

Initial characterization: 1 µL of the probe PJD120 or PJD224 (0.01 µM to 100 µM) or Fp-rhodamine (0.01 µM to 5 µM, incubated for 30 min) were incubated with 9 µL of the esterase (10 ng/µL stock solution) for 1 hour (enol carbamate) or 30 min (Fp-rho) at 37 °C. SB (with DTT) was added and the proteins were resolved on a 12% SDS-PAGE and fluorescence was visualized using a Typhoon scanner by in-gel fluorescence scanning.

Competition experiment: 1 µL of compound 2a (0.1 µM to 10 µM) or DMSO was incubated with 9 µL of the esterase (100 ng/µL stock solution) for 1 hour at 37 °C. Then 1 µL of probe PJD224 (1 µM) or Fp-rhodamine (1 µM) was added and incubated for 1 more hour. SB (with DTT) was added and the proteins were resolved on a 12% SDS-PAGE and fluorescence was visualized using a Typhoon scanner by in-gel fluorescence scanning.

1 µL of the compound, 2a-i, 3a-c and 4a-e (0.2 µM, 1µM and 5µM) or DMSO was incubated with 9 µL of the esterase (100 ng/µL stock solution) for 1 hour at 37 °C. Then 1 µL of probe PJD224 (1 µM) was added and incubated for 1 more hour. SB (with DTT) was added and the proteins were resolved on a 12% SDS-PAGE and fluorescence was visualized using a Typhoon scanner by in-gel fluorescence scanning.

Click conditions: 1 µL of compound PJD2d was incubated with 9 µL of the esterase (100 ng/µL stock solution) for 1 hour at 37 °C. Bodipy-N3 was then clicked using the following conditions:

Bodipy-N3 50 µM, CuSO4 50 µM, TABTA 250 µM and sodium ascorbate 1.25 mM for 2 hours before adding SB and resolving the proteins on a 12% SDS-PAGE followed by in-gel fluorescence scanning.

4.4.3. Labeling experiments and pull-down with lysates

SDS-PAGE labeling with A549 and B.subtilis lysates.

1 µL of enol cyclocarbamate or DMSO was incubated one hour at 37 °C with 19 µL of lysates (1 mg/mL) and then 2 µL Fp-rhodamine (10 µM) was added and incubated during 30 minutes. SB (with DTT) was added and the proteins were resolved on a 12% SDS-PAGE and fluorescence was visualized using a Typhoon scanner by in-gel fluorescence scanning.

Pull-down experiment with A549 and B.subtilis lysates. – protocol adapted from Baggelaar et al.²⁰

(i) The lysates (500 µL, 2.0 mg/mL) were incubated with vehicle (DMSO) or 2a (100 µM) for 30 minutes at 37 °C.

excess of probe was then removed by chloroform/methanol precipitation. 750 µL of methanol was added to the 250 µL protein solution (protein samples were usually splitted in two and combined in the next step). The samples were vortexed vigorously. 200 µL of chloroform were added and the samples were vortexed vigorously. Add 600 µL of H²O (to get phase separation), vortex the tube vigorously and centrifuge it (2 min at room temperature, 9,000 g). We discarded the upper layer and then add 600 µL of methanol, vortex the mixture at low speed, thoroughly, and centrifuge it (2 min at room temperature, 9,000 g) and finally removed the supernatant by pipetting and air-dried the pellet for exactly 5 min the tube upside-down.

(iii) The precipitated proteome was suspended in 500 µL PBS and allowed to incubate for 15 minutes.

(iv) Denaturation and alkylation. We then added 5 µL (1 M DTT) and the mixture was heated to 65 °C for 15 minutes. The sample was allowed to cool during 10 min and we added 40 µL (0.5 M) iodoacetamide and the sample was alkylated for 30 minutes in the dark. We then added 140 µL 10% (wt/vol) SDS and the proteome was heated for 5 minutes at 65 °C.

(v) Pull-down with paramagnetic beads. 100 µL of streptavidin magnetic beads (prewashed with PBS three times) were added and incubated for 3 h at room temperature under constant shaking.

(vi) The tube were placed on the Dynamag for 3 min and the supernatant was removed.

(xi) The beads were then washed three times with PBS (250 µL) using the Dynamag.

(xii) Elution from the beads. The beads were boiled with 100 µL of 1× sample buffer containing 10 µM biotin for 5 min at 100 °C.

Samples were run on a 12% SDS-Page and a MS compatible coomassie stain was performed and the main bands excised. The bands or section containing bands excised are highlighted in the appendix 1 (the Fp-rho gel is used as a reprensentation). After coomassie the bands were immediately excised to avoid dust accumulation and subjected to mass spectrometry sample preparation and measurement.

Sample preparation for LC-MS. The gel pieces were completely de-stained with 50%

acetonitrile (ACN), 50 mM ammonium bicarbonate (ABC), dehydrated with 150 µL acetonitrile, reduced with 10 mM dithiothreitol (30 min at 55°C) and alkylated with 40 mM iodoacetamide (45 min at RT, in the dark) and overnight digested with 10 µL of a 10 ng/µL trypsin (V5111;

Promega) at 37°C. Peptides were extracted from the gel pieces by adding, sonicating and collecting sequentially in the same tube: 40 µL 2% trifluoroacetic acid (TFA), 40 µL 33% ACN;

1.7% TFA and 40 µL 67% ACN, 0.7% TFA. The samples were dried under vacuum and reconstituted with 100 µL, 2% ACN. 20 µL, 5% trifluoroacitic acid (TFA) was added to the sample to reach pH < 3. Solid phase extraction was performed with Pierce® C18 tips (87784;

Thermo) according to the suppliers manual. The eluate fraction was dried under vacuum and reconstituted with 20 µL 2% ACN, 0.1% formic acid (FA).

Mass spectrometry. Peptide separation was performed with 2 µL peptide sample using a nano- flow chromatography system (EASY nLC II; Thermo) equipped with a reversed phase HPLC column (75 µm, 15 cm) packed in-house with C18 resin (ReproSil-Pur C18–AQ, 3 µm resin; Dr.

Maisch) using a linear gradient from 95% solvent A (0.1% FA, 2% acetonitrile) and 5% solvent B (99.9% acetonitrile, 0.1% FA) to 28% solvent B over 45 min at a flow rate of 200 nL/min. The peptide and peptide fragment masses were determined by an electrospray ionization mass spectrometer (LTQ-Orbi-trap XL ; Thermo).

Data processing. Raw files were imported into the Peaks Studio software (Bioinformatics Solutions) analyzed against forward and reverse peptide sequences of either Uniprot/Swiss-

Prot database; B. subtilis (strain 168), 4185 proteins or Uniprot/Swiss-Prot database; Human, 20171 proteins. Carbamidomethylation (C) was set as fixed modification; oxidation (M) and deamination (NQ) as variable modification. The mass tolerance was set to 10 ppm for precursor ions and 0.5 Da for fragment ions.

4.5 References

(1) Lodola, A.; Capoferri, L.; Rivara, S.; Tarzia, G.; Piomelli, D.; Mulholland, A.; Mor, M. J. Med.

Chem. 2013, 56 (6), 2500.

(2) Shannon, D. A.; Weerapana, E. Curr Opin Chem Biol 2015, 24, 18.

(3) Pearson, R. G. J. Am. Chem. Soc. 1963, 85 (22), 3533.

(4) LoPachin, R. M.; Gavin, T. Free Radic. Res. 2016, 50 (2), 195.

(5) Kreuzer, J.; Bach, N. C.; Forler, D.; Sieber, S. A. Chemical Science 2014, 6, 237.

(6) Heydenreuter, W.; Kunold, E.; Sieber, S. A. Chem. Commun. 2015, 51, 15784.

(7) Wirth, T.; Schmuck, K.; Tietze, L. F.; Sieber, S. A. Angew. Chem. Int. Ed. 2012, 51 (12), 2874.

(8) Thirkettle, J.; Alvarez, E.; Boyd, H.; Brown, M.; Diez, E.; Hueso, J.; Elson, S.; Fulston, M.;

Gershater, C.; Morata, M. L.; Perez, P.; Ready, S.; Sanchez-Puelles, J. M.; Sheridan, R.; Stefanska, A.; Warr, S. The Journal of Antibiotics 2000, 53 (7), 664.

(9) Thirkettle, J. The Journal of Antibiotics 2000, 53 (7), 733.

(10) Pinto, I. L.; Boyd, H. F.; Hickey, D. M. Bioorganic & Medicinal Chemistry Letters 2000, 10 (17), 2015.

(11) Dockerty, P.; Edens, J. G.; Tol, M. B.; Morales Angeles, D.; Domenech, A.; Liu, Y.; Hirsch, A.

K. H.; Veening, J. W.; Scheffers, D.-J.; Witte, M. D. Org. Biomol. Chem. 2017, 15 (4), 894.

(12) Liu, Y.; Patricelli, M. P.; Cravatt, B. F. Proceedings of the National Academy of Sciences 1999, 96 (26), 14694.

(13) Patricelli, M. P.; Giang, D. K.; Stamp, L. M.; Burbaum, J. J. Proteomics 2001, 1 (9), 1067.

(14) Watanabe, H.; Tanase, S.; Nakajou, K.; Maruyama, T.; Kragh-Hansen, U.; Otagiri, M.

Biochemical Journal 2000, 349 (3), 813.

(15) Tuin, A. W.; Mol, M. A. E.; van den Berg, R. M.; Fidder, A.; van der Marel, G. A.;

Overkleeft, H. S.; Noort, D. Chem. Res. Toxicol. 2009, 22 (4), 683.

(16) Meier, J. L.; Niessen, S.; Hoover, H. S.; Foley, T. L.; Cravatt, B. F.; Burkart, M. D. ACS Chem.

Biol. 2009, 4 (11), 948.

(17) Cui, Y.; Xing, P.; Wang, Y.; Liu, M.; Qiu, L.; Ying, G.; Li, B. Oncotarget 2017, 8 (20), 32576.

(18) Abegg, D.; Frei, R.; Cerato, L.; Hari, D. P.; Wang, C.; Waser, J.; Adibekian, A. Angew. Chem.

Int. Ed. 2015, 54 (37), 10852.

(19) Green, M. R.; Sambrook, J. Molecular cloning: a laboratory manual 4 edition Cold Spring Harbor Laboratory Press; New York, 2012.

(20) Baggelaar, M. P.; Chameau, P. J. P.; Kantae, V.; Hummel, J.; Hsu, K.-L.; Janssen, F.; van der Wel, T.; Soethoudt, M.; Deng, H.; Dulk, den, H.; Allarà, M.; Florea, B. I.; Di Marzo, V.; Wadman, W. J.; Kruse, C. G.; Overkleeft, H. S.; Hankemeier, T.; Werkman, T. R.; Cravatt, B. F.; van der Stelt, M. J. Am. Chem. Soc. 2015, 137 (27), 8851.

4.6 Appendix

Appendix 1. Work-flow for pull-down experiments using Fp-biotin. The bands that were excised and submitted to mass spectrometry are highlighted by letters (A-E for A549 and A-B for B.subtilis).

SDS-PAGE +coomassie pull down

cell lysate inhibitor cell lysate +

++ +-

Inhibitor

[μM] -

Fp-rho (1 μM) + + 100 2a

A B C

E kDa

75

25

A B

D

Inhibitor

[μM] -

Fp-rho (1 μM) + + 100 2a

kDa 75

25

A549 B.sub.

12 3

HN POEt

O F Fp-rhodamine O

O CO2-

N

N⁺

HN POEt

O O

F HN

O S HN O NH H

H

Fp-biotin Fp-bio

Fp-bio

Sample B - A549 - Protein List

Protein Group Protein

ID Accession -10lgP Coverage(%) #Peptides #Unique PTM Avg.

Mass Description

2 2 sp|P04264|K2C1_HUMAN 156.81 16 8 8 Y 66039 Keratin, type II cytoskeletal 1 OS=Homo sapiens GN=KRT1 PE=1 SV=6 3 4 sp|P35527|K1C9_HUMAN 145.90 16 7 7 Y 62064 Keratin, type I cytoskeletal 9 OS=Homo sapiens GN=KRT9 PE=1 SV=3 1 29 sp|Q15102|PA1B3_HUMAN 112.49 35 10 10 Y 25734 PlatelePlatelet-actiactivating factor aceating factor acetylylhydrolase IB subunit gamma OS=Homo sapiens drolase IB subunit gamma OS=Homo sapiens

GN=

GN=PAFAH1B3 PE=1 SV=1AH1B3 PE=1 SV=1

Sample A - A549 - Protein List

Protein Group Protein

ID Accession -10lgP Coverage(%) #Peptides #Unique PTM Avg.Mass Description

3 2 sp|P04264|K2C1_HUMAN 139.28 50 40 35 Y 66039 Keratin, type II cytoskeletal 1 OS=Homo sapiens GN=KRT1 PE=1 SV=6 4 1 sp|P13645|K1C10_HUMAN 134.86 52 30 24 Y 58827 Keratin, type I cytoskeletal 10 OS=Homo sapiens GN=KRT10 PE=1 SV=6 6 3 sp|P35908|K22E_HUMAN 129.87 62 32 22 Y 65433 Keratin, type II cytoskeletal 2 epidermal OS=Homo sapiens GN=KRT2 PE=1 SV=2 5 4 sp|P35527|K1C9_HUMAN 116.81 46 23 22 Y 62064 Keratin, type I cytoskeletal 9 OS=Homo sapiens GN=KRT9 PE=1 SV=3 1 10 sp|O75608|LYPA1_HUMAN 116.52 82 17 17 Y 24670 Acyl-protein thioesteAcyl-protein thioesterase 1 OS=Homo sapiens GN=ase 1 OS=Homo sapiens GN=LYPLA1 PE=1 SV=1YPLA1 PE=1 SV=1 7 5 sp|P08779|K1C16_HUMAN 115.56 49 23 9 Y 51268 Keratin, type I cytoskeletal 16 OS=Homo sapiens GN=KRT16 PE=1 SV=4 8 6 sp|P02533|K1C14_HUMAN 113.93 43 22 6 Y 51562 Keratin, type I cytoskeletal 14 OS=Homo sapiens GN=KRT14 PE=1 SV=4 11 8 sp|P48668|K2C6C_HUMAN 110.03 36 24 0 Y 60025 Keratin, type II cytoskeletal 6C OS=Homo sapiens GN=KRT6C PE=1 SV=3 2 11 sp|O95372|LYPA2_HUMAN 109.67 70 19 19 Y 24737 Acyl-protein thioesteAcyl-protein thioesterase 2 OS=Homo sapiens GN=ase 2 OS=Homo sapiens GN=LYPLA2 PE=1 SV=1YPLA2 PE=1 SV=1 10 7 sp|P02538|K2C6A_HUMAN 109.54 34 23 1 Y 60045 Keratin, type II cytoskeletal 6A OS=Homo sapiens GN=KRT6A PE=1 SV=3

9 9 sp|P04259|K2C6B_HUMAN 109.37 36 24 1 Y 60067 Keratin, type II cytoskeletal 6B OS=Homo sapiens GN=KRT6B PE=1 SV=5 12 13 sp|Q04695|K1C17_HUMAN 87.37 21 12 2 Y 48106 Keratin, type I cytoskeletal 17 OS=Homo sapiens GN=KRT17 PE=1 SV=2 23 19 sp|P09211|GSTP1_HUMAN 86.64 57 7 7 Y 23356 Glutathione S-transferase P OS=Homo sapiens GN=GSTP1 PE=1 SV=2 13 14 sp|P13647|K2C5_HUMAN 86.05 23 14 4 Y 62378 Keratin, type II cytoskeletal 5 OS=Homo sapiens GN=KRT5 PE=1 SV=3 20 15 sp|Q9NUJ1|ABHDA_HUMAN 85.52 30 8 8 Y 33933 Mycophenolic acid acyl-glucuronide esterase, mitochondrial OS=Homo sapiens GN=ABHD10 PE=1 SV=1 26 12 sp|P28074|PSB5_HUMAN 81.61 33 7 7 Y 28480 Proteasome subunit beta type-5 OS=Homo sapiens GN=PSMB5 PE=1 SV=3 28 18 sp|Q5VWZ2|LYPL1_HUMAN 80.17 41 8 8 Y 26316 Lysophospholipase-like protein 1 OS=Homo sapiens GN=LYPLAL1 PE=1 SV=3 17 22 sp|O95678|K2C75_HUMAN 77.12 10 8 0 Y 59560 Keratin, type II cytoskeletal 75 OS=Homo sapiens GN=KRT75 PE=1 SV=2 16 16 sp|Q01546|K22O_HUMAN 76.03 10 9 0 Y 65841 Keratin, type II cytoskeletal 2 oral OS=Homo sapiens GN=KRT76 PE=1 SV=2 14 20 sp|P19012|K1C15_HUMAN 74.49 11 7 0 Y 49212 Keratin, type I cytoskeletal 15 OS=Homo sapiens GN=KRT15 PE=1 SV=3

14 3 sp|P35908|K22E_HUMAN 109.87 8 3 3 Y 65433 Keratin, type II cytoskeletal 2 epidermal OS=Homo sapiens GN=KRT2 PE=1 SV=

2

4 439 sp|Q8N2K0|ABD12_HUMAN 92.38 6 4 4 Y 45097 MonoacylglMonoacylglycerol lipase ABHD12 OS=Homo sapiens GN=ABHD12 PE=1 SV=2cerol lipase ABHD12 OS=Homo sapiens GN=ABHD12 PE=1 SV=2 5 18 sp|Q5VWZ2|LYPL1_HUMAN 91.29 16 4 4 Y 26316 LLysophospholipase-liysophospholipase-like protein 1 OS=Homo sapiens GN=e protein 1 OS=Homo sapiens GN=LYPLAL1 PE=1 SV=3YPLAL1 PE=1 SV=3 9 887 sp|P10619|PPGB_HUMAN 91.17 7 3 3 Y 54466 LLysosomal protectiysosomal protective protein OS=Homo sapiens GN=Ce protein OS=Homo sapiens GN=CTSA PE=1 SV=2A PE=1 SV=2

7 31 sp|P02768|ALBU_HUMAN 87.69 7 4 4 Y 69367 Serum albumin OS=Homo sapiens GN=ALB PE=1 SV=2

10 889 sp|P10768|ESTD_HUMAN 82.52 9 3 3 Y 31463 S-forS-formylglutathione ylglutathione hydrolase OS=Homo sapiens GN=ESD PE=1 SV=2drolase OS=Homo sapiens GN=ESD PE=1 SV=2 6 15 sp|Q9NUJ1|ABHDA_HUMAN 78.80 7 3 3 Y 33933 Mycophenolic acid acyl-glucuronide esterase, mitochondrial OS=Homo sapiens GN=ABHD10 PE=1 SV=1 19 10 sp|O75608|LYPA1_HUMAN 77.03 14 3 3 N 24670 Acyl-protein thioesterase 1 OS=Homo sapiens GN=LYPLA1 PE=1 SV=1 30 94 sp|Q8NFV4|ABHDB_HUMAN 59.30 7 2 2 Y 34690 Protein ABHD11 OS=Homo sapiens GN=ABHD11 PE=1 SV=1Protein ABHD11 OS=Homo sapiens GN=ABHD11 PE=1 SV=1 29 24 sp|Q9HB40|RISC_HUMAN 58.64 2 1 1 N 50831 Retinoid-inducible serine carboxypeptidase OS=Homo sapiens GN=SCPEP1 PE=1SV=1 15 85 sp|P49327|FAS_HUMAN 57.14 1 2 2 Y 273424 Fatty acid synthase OS=Homo sapiens GN=FASN PE=1 SV=3 83 888 sp|Q8WZ82|OVCA2_HUMAN 53.53 5 1 1 Y 24418 Esterase OVCA2 OS=Homo sapiens GN=OVCA2 PE=1 SV=1 84 460 sp|Q99685|MGLL_HUMAN 44.44 5 1 1 Y 33261 Monoglyceride lipase OS=Homo sapiens GN=MGLL PE=1 SV=2 85 1 sp|P13645|K1C10_HUMAN 39.38 2 1 1 N 58827 Keratin, type I cytoskeletal 10 OS=Homo sapiens GN=KRT10 PE=1 SV=6 86 467 sp|P11498|PYC_HUMAN 35.35 1 1 1 N 129634 Pyruvate carboxylase, mitochondrial OS=Homo sapiens GN=PC PE=1 SV=2 32 188 sp|Q02388|CO7A1_HUMAN 25.33 1 2 2 N 295216 Collagen alpha-1(VII) chain OS=Homo sapiens GN=COL7A1 PE=1 SV=2

Protein Group Protein

ID Accession -10lgP Coverage(%) #Peptides #Unique PTM Avg.

Mass Description

6 4 sp|P35527|K1C9_HUMAN 271.06 43 17 17 Y 62064 Keratin, type I cytoskeletal 9 OS=Homo sapiens GN=KRT9 PE=1 SV=3 1 2 sp|P04264|K2C1_HUMAN 269.93 42 24 20 Y 66039 Keratin, type II cytoskeletal 1 OS=Homo sapiens GN=KRT1 PE=1 SV=6 8 3 sp|P35908|K22E_HUMAN 228.44 31 15 9 Y 65433 Keratin, type II cytoskeletal 2 epidermal OS=Homo sapiens GN=KRT2 PE=1 SV=2 2 437 sp|Q86TX2|ACOT1_HUMAN 225.36 31 15 2 Y 46277 Acyl-coenzyme A thioesteAcyl-coenzyme A thioesterase 1 OS=Homo sapiens GN=ACase 1 OS=Homo sapiens GN=ACOT1 PE=1 SV=1T1 PE=1 SV=1

Sample C - A549 - Protein List

4 438 sp|P49753|ACOT2_HUMAN 220.68 31 15 2 Y 53219 Acyl-coenzyme A thioesteAcyl-coenzyme A thioesterase 2, mitochondrial OS=Homo sapiens GN=ACase 2, mitochondrial OS=Homo sapiens GN=ACOT2 T2 PE=1 SV=6

PE=1 SV=6

7 1 sp|P13645|K1C10_HUMAN 216.35 26 15 13 Y 58827 Keratin, type I cytoskeletal 10 OS=Homo sapiens GN=KRT10 PE=1 SV=6 3 101 sp|Q6PIU2|NCEH1_HUMAN 203.40 34 11 11 Y 45808 NeutNeutral cholesterol ester al cholesterol ester hydrolase 1 OS=Homo sapiens GN=NCEH1 PE=1 SV=drolase 1 OS=Homo sapiens GN=NCEH1 PE=1 SV=

3

15 6 sp|P02533|K1C14_HUMAN 187.15 18 7 2 Y 51562 Keratin, type I cytoskeletal 14 OS=Homo sapiens GN=KRT14 PE=1 SV=4 12 441 sp|P63261|ACTG_HUMAN 182.70 29 11 9 Y 41793 Actin, cytoplasmic 2 OS=Homo sapiens GN=ACTG1 PE=1 SV=1 11 9 sp|P04259|K2C6B_HUMAN 177.95 28 14 1 Y 60067 Keratin, type II cytoskeletal 6B OS=Homo sapiens GN=KRT6B PE=1 SV=5 9 8 sp|P48668|K2C6C_HUMAN 175.50 28 14 0 Y 60025 Keratin, type II cytoskeletal 6C OS=Homo sapiens GN=KRT6C PE=1 SV=3 10 7 sp|P02538|K2C6A_HUMAN 172.67 28 14 1 Y 60045 Keratin, type II cytoskeletal 6A OS=Homo sapiens GN=KRT6A PE=1 SV=3 5 439 sp|Q8N2K0|ABD12_HUMAN 162.14 22 11 11 Y 45097 MonoacylglMonoacylglycerol lipase ABHD12 OS=Homo sapiens GN=ABHD12 PE=1 SV=2cerol lipase ABHD12 OS=Homo sapiens GN=ABHD12 PE=1 SV=2 13 5 sp|P08779|K1C16_HUMAN 158.15 18 7 2 Y 51268 Keratin, type I cytoskeletal 16 OS=Homo sapiens GN=KRT16 PE=1 SV=4 16 85 sp|P49327|FAS_HUMAN 155.81 3 7 7 Y 273424 Fatty acid synthase OS=Homo sapiens GN=FASN PE=1 SV=3 14 14 sp|P13647|K2C5_HUMAN 123.72 15 9 3 Y 62378 Keratin, type II cytoskeletal 5 OS=Homo sapiens GN=KRT5 PE=1 SV=3 17 446 sp|Q99487|PAFA2_HUMAN 121.03 14 6 6 Y 44036 PlatelePlatelet-actiactivating factor aceating factor acetylylhydrolase 2, cytoplasmic OS=Homo sapiens GNdrolase 2, cytoplasmic OS=Homo sapiens GN

=PAFAH2 PE=1 SV=1AH2 PE=1 SV=1

27 450 sp|Q9Y570|PPME1_HUMAN 114.87 16 4 4 Y 42315 Protein phosphatase metProtein phosphatase methylesteylesterase 1 OS=Homo sapiens GN=PPME1 PE=1 SVase 1 OS=Homo sapiens GN=PPME1 PE=1 SV

=3

=3

24 27 sp|P05787|K2C8_HUMAN 99.51 6 3 1 N 53704 Keratin, type II cytoskeletal 8 OS=Homo sapiens GN=KRT8 PE=1 SV=7 22 449 sp|O60218|AK1BA_HUMAN 99.38 20 6 6 Y 36020Aldo-Aldo-keto reductase family 1 member B10 OS=Homo sapiens GN=AKR1B10 PEeto reductase family 1 member B10 OS=Homo sapiens GN=AKR1B10 PE

=1 SV=2

=1 SV=2