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

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Nature-inspired molecules containing multiple electrophilic positions

Synthesis and application as activity-based probes and inhibitors

Paul Dockerty

The work described in this thesis was executed at the Stratingh Institute for Chemistry, University of Groningen, The Netherlands.

Cover design by Oksana Cortes Tolalpa.

Printed by Ipskamp

ISBN: 978-94-034-0310-6

eISBN: 978-94-034-0311-3

Nature-inspired molecules containing multiple electrophilic positions

Synthesis and application as activity-based probes and inhibitors

PhD thesis

to obtain the degree of PhD at the University of Groningen

on the authority of the Rector Magnificus Prof. E. Sterken

and in accordance with the decision by the College of Deans.

This thesis will be defended in public on

Friday 9 March 2018 at 14.30 hours

by

Paul Jacques Dockerty

born on 3 May 1988

Supervisor

Prof. A. J. Minnaard Co-supervisor Dr. M.D. Witte

Assessment Committee Prof. J.G. Roelfes

Prof. S. Verhelst

Prof. R. van der Hoorn

Nothing in life is to be feared, it is only to be understood.

Now is the time to understand more, so that we may fear less.

Marie Curie

Table of contents

Chapter 1

Natural products inspired scaffolds

for activity-based protein profiling 1 Chapter 2

Bicyclic enol cyclocarbamates inhibit

penicillin-binding proteins 23

Chapter 3

Enolcarbamate Probes Label the Aldehyde

Dehydrogenases RALDH1 and ALDH3A1 57 Chapter 4

An original warhead targeting serine hydrolases 79 Chapter 5

An NMR study to help the design of

more potent probes 101

Chapter 6

Summary and future perspectives 133

Chapter 1

Natural product inspired scaffolds for activity-based protein profiling

Natural products are an amazing source of inspiration for the development of drugs

and activity-based probes. Recently, impressive advances have been made in the field

of activity-based probes and its applications leading to powerful tools. We discuss

here the basis of activity-based protein profiling of natural products and the several

downstream applications helping to solve complex biological problems.

2

1.1 Natural products in the ‘omics age

There is recently an interesting inclination to revisit natural product inspired scaffolds for drug discovery and chemical proteomics.

Historically, natural products were the main (or only) source of medicines, drugs and medicinal preparations and they are still the main source for recently approved medicines. However, for a while, other techniques like high-throughput screenings for specific targets and combinatorial chemistry were the central interest of pharmaceutical companies and research groups. Natural compounds were considered not compatible with these high-throughput methodologies, since their scaffolds are often challenging to synthesize, characterize or even to isolate. Sample preparation thus hampered the use of natural products in these campaigns and as a consequence natural products were disregarded as leads for several decades.

Figure 1. Structure of natural products today on the market and their corresponding activities and origins.

Despite the challenges in sample preparation, it is remarkable that natural products were ignored as leads, in particular, since the results of high- throughput screenings were to some extend disappointing. Moreover secondary metabolites are sculpted to interact with biological targets, often display high bioavailability and have obtained great results reaching the market. An abundant number of secondary metabolites and their associated activities remain undiscovered and it is captivating to contemplate that

NSO3H HN

O N O O O OH

N S NH₂

Aztreonam monobactam antibiotic Chromobacterium violaceum

Fumagillin antimicrosporidial Aspergillus fumigatus

N S

O H

O OH HN

O

Penicillin G antibiotic Penicillium chrysogenum

O O

O H

H H

O O

Artemisin antimalarial Artemisia annua O

O

O O

O

OH O

these metabolites can be used to fine-tune biological responses.

This reasoning, in combination with the failures in high-throughput screenings led to a renewed interest of the drug discovery field in natural product inspired scaffolds.

Key step in designing drugs and chemical probes based on natural product scaffolds is the identification of scaffolds that have interesting biological properties (Figure 1). The identification of such scaffolds can be divided in two steps, a chemical and a biological step. The chemical identification is based on a thorough study of literature of the scaffold’s structure elucidation. The synthetic accessibility of the scaffold should then be evaluated together with the possible scaffold modifications for structure- activity relationship (SAR) study or incorporation of tags. The scaffold should preferably be structurally different from known inhibitors and drugs to increase the chance of finding unprecedented targets and leads. As for the biological identification, phenotypic assays or protein-based assays are commonly used to assess the biological properties. Results from these experiments can help to gain information into the potential target, off targets or pathways leading to the observed activity. The information collected from these two steps should help the design and characterization of efficient drugs and chemical probes.

After a novel scaffold has been selected, the mechanism of action has to be

elucidated before the natural product can be converted into successful drugs

and research tools. This requires a full characterization of the biological

activity of the compound, including identification of its targets and off-

targets. The recent advances in genomics, proteomics and metabolomics and

the maturing of the chemical biology field in the past decade led to

development of a variety of techniques that enable to do so. These

techniques range from assays that enable determining the activity of natural

products on specific proteins in a high-throughput manner (activity assays),

to correlation of biological activities with known phenotypes, to protein

profiling with chemical probes (ABPP and AfBP).

Especially, the latter

technique has become increasingly popular. Depending on the type of tag

that has been incorporated into the probe molecule, protein profiling with

chemical probes can be used to identify the targets and potential off-targets,

to provide insight in the selectivity profile and to improve the

understanding of the role of the target(s) in biologically relevant setting

(Figure 2).

4

Figure 2. Structure of activity-based probes inspired by natural products. An azide, an alkyne, a Bodipy and a biotin are here used as tags for visualisation and/or enrichment.

1.2 Protein profiling to characterize the targets of natural products

Direct and competitive protein profiling methodologies have been established to identify the targets of natural products. In the direct approach, the natural product serves as lead for the synthesis of a chemical probe that can be used to visualize and enrich the targets in an unbiased fashion. Incorporating a tag at a position that is not essential for the biological activity is often sufficient to obtain probe molecules of natural products containing a putative electrophilic trap (Figure 3A). The binding partners of the natural product become covalently modified upon reaction with the electrophile and the resulting protein-probe adducts can therefore be detected using for example a fluorescent tag (Figure 3C). Furthermore, when an affinity handle has been introduced, the tag also facilitates enrichment of the modified proteins by means of pull-down purification.

Biotin has been commonly employed for this purpose, since the strong streptavidin-biotin interaction allows for efficient enrichment and stringent washing conditions to be used. Streptavidin pull-down thereby simplifies subsequent identification of the protein targets by mass spectrometry (Figure 3C). In many cases, the tag affects the biological activity of the

NSO₃H HN

O N O O O NH

N S NH₂

Aztreonam probe Fumagillol-biotin probe

O O

O O

O NH

HN HN O

O O

5 5

S NH HN O H

H

N S

O H

O O^- HN

O

Bocillin-FL

Na+

HN O N⁺ NB^-F

F

O O

H

H H

O O

Artemisin probe NH

O

N₃

chemical probe. To overcome this issue, two-step approaches have been developed in which the tag is introduced onto the probe in a post-labeling step via a bio-orthogonal reaction, like the Staudinger ligation, copper catalyzed click chemistry and inverse electron-demand Diels-Alder reactions.

The relatively small bio-orthogonal handles are less invasive and the probes therefore mimic the natural product more closely.

Incorporating an affinity tag on natural products that lack a putative

electrophile also enables enrichment of the interaction partners. However,

this often results in a poor signal-to-noise ratio. Due to the transient nature

of the interactions, weakly binding targets are lost during the washing steps

and target identification therefore has met with less success. To improve the

signal-to-noise ratio, the natural product can be equipped either with an

electrophilic trap - a so-called warhead - that reacts with a nucleophilic

amino acid in the proximity of the active site or with a photocrosslinker that

gets activated upon radiation with light. The resulting probes will covalent

modify their targets in a similar fashion as probes that exploit electrophiles

present in natural products (Figure 3B). There are numerous examples that

illustrate how converting natural products into probes has helped to

pinpoint the targets and the mechanism by which the targets are inhibited

(by mass-spectrometry identification of the modified residues). This method

was applied to identify the protein targets of Vancomycin, a well-known

non-covalent antibiotic.

By incorporating benzophenone as a

photocrosslinker and a small alkyne handle as a tag into the Vancomycin

scaffold (Figure 4), Eirich et al. were able to confirm that the vancomycin

photoprobe interact with the autolytic machinery by inhibiting Atl (a

bifunctional autolysin) in E.faecalis. An ABC transporter protein was also

labeled and identified. The inhibition of this transporter by Vancomycin

probably reduces drastically the uptake of necessary nutrients for the

bacteria. Applying this strategy on natural products that display

antibacterial activity such as Vancomycin aids to the most important fight

against bacterial resistance.

6

Figure 3. Design of a direct probe from an electrophilic (A) and a non-electrophilic natural product (B); Example of tags and their specific applications (B).

m/z SDS-PAGE

tag

Direct probe

reactive group

“warhead”

Electrophilic Natural products

1. Electrophiles found in nature

O

present within the natural scaffold provide selectivity by modifying nucleophilic residues

Tag or label 1. Affinity tag 2. Fluorescent tag

S NH

HN O

H

H NB^-N⁺

F

biotin bodipyF

pull-down with streptavidin followed by

mass spectrometry to identify target protein in-gel detection

O S

O O

tag

Direct probe Non-electrophilic

Natural products reactive group

reactive group

“warhead”

incorporated by synthesis

1. Electrophile 2. Photocrosslinker

N O

H O

O O

N N

choice of an electrophile usually based on the nucleophile to modify

provides a carbene or radical that allows covalent interaction with the protein

A

B

incorporated by synthesis to serve downstream applications

O O

C

Some natural products already contain an electrophilic trap and are therefore readily available for protein profiling methodologies. Excellent examples that reflect this are the studies towards the antibacterial agents Showdomycin and Rugulactone by Sieber and coworkers. The group of Sieber realized that both compounds contain an α,β-unsaturated carbonyl motif that can function as a Michael acceptor and that these natural products therefore likely react with hyper-reactive amino acid residues, in particular cysteine. To identify the targets and to elucidate the mechanism of action of Showdomycin, they prepared a probe of this nucleoside antibiotic that was first isolated from Streptomyces showdoensis.

Since the maleimide was considered to be essential for the activity, Sieber and coworkers incorporated an alkyne on the other side of the scaffold to avoid impairing the interaction with nucleophilic residues (Figure 4). The resulting probe displayed equal antibacterial activity against Staphylococcus aureus as the parent compound. Labeling experiments revealed that the probe binds 13 different enzymes that are related to oxidative stress resistance in S. aureus, with the main hit being UDP-N-acetylglucosamine 1-carboxyvinyl transferase 1 (murA1). This enzyme catalyzes the first step of the cell wall biosynthesis in S. aureus and inhibition of this enzyme forms a likely explanation for the observed antibiotic activity. By profiling the targets of the Showdomycin probe in the methicillin-resistant S. aureus (MRSA) strain Mu50 and comparing the labeling profiles with nonresistant S. aureus strains, Sieber and coworkers demonstrated that murA2 was labeled in Mu50 strain but not in the nonresistant strains, while the expression levels for murA2 were similar in both strains. Such a tool is therefore also interesting to distinguish between closely related strains that display different pathogenicity and understand the mechanism of antibiotic resistance.

The group of Sieber used a similar approach to identify the targets and

elucidated the mechanism of action of Rugulactone,

an antibacterial agent

that was first isolated from Cryptocarya rugulosa.

An alkyne was introduced

to enrich proteins that react with α,β-unsaturated ketone in Rugulactone

(Figure 4). Several targets were identified by the Rugulactone probe with

mass spectrometry after pull-down experiment. MurA2 was one of the main

hits and ThiD, a kinase involved in the biosynthesis of thiamine was for the

first time inhibited by a small molecule. Culturing bacteria in thiamine-

deficient medium in the presence of Rugulactone led to more potent

inhibition of bacterial growth, thereby linking the activity to the isolated

target.

8

Figure 4. Structure of the probes based on Showdomycin, Rugulactone and Vancomycin.

Highlighted in blue is the Micheal acceptor present in Rugulactone and Showdomycin and the benzophenone photocrosslinker incorporated in the Vancomycin scaffold.

Highlighted in red is the alkyne tag.

Converting natural products into chemical probes can clearly be used to further the biological understanding of natural products in general and it resulted in novel lead compounds and potential novel drug targets, but this strategy also has some disadvantages. The most important limitation of the direct protein profiling approach is that the introduction of a tag and a warhead has a great influence on the physical and biochemical properties.

For example, these modifications can alter the hydrophobicity of the resulting probe and consequently the cell-permeability. As such, the tag and warhead may affect the biological activity and they thus determine the outcome of target identification studies. Varying the tag often leads to notable differences in the labeling profile of the probe. Even though the effect of tag can be minimized by employing two-step labeling strategies (vide supra), ideally the targets of the non-modified parent compound are identified. Competitive protein profiling with broad-spectrum probe molecules that react with one family or super-family of proteins allows this for natural products containing putative electrophilic traps. This method capitalizes on the fact that the electrophile in essence determines which hyper reactive amino acid residues are modified (usually a conserved nucleophilic residue in the active site), while the rest of the scaffold via non- covalent interaction generates selectivity for one or another target within the class. By carefully selecting electrophiles that react with a specific amino acid residue and equipping these with a bio-orthogonal handle, broad- spectrum probes for the majority of the hyper reactive amino acid residues

Showdomycin probe O

OH OH HN

O O O

O O O

O

Rugulactone probe O

O

NH HN

Cl

NH O O

HN NH HO

O HN O O

O NH

Cl

O O NH₂

OH

OH O O HOOH

OH O

OH HN

O NH

HO OH

NH O

O

O

Vancomycin probe

have been obtained.

Michael acceptors, epoxides, chloro- and iodoacetamides and sulfonate ester based probes have been developed to react predominantly with hyper reactive cysteine (Cys) residues.

Iodoacetamide alkyne (IAA) is mostly used as a proteomic platform to target Cys residues. Activated Ser residues, which are found in the active site of several enzyme families including the serine hydrolases (SHs), a superfamily of proteins that comprises 1% of the human proteome, have been profiled with carbamate, diphenyl phosphonate, β-lactam, β-lactone and fluorophosphonate (Fp) probes.

Especially probes containing the latter type of electrophile have become popular tools to study SHs (Fp-rhodamine and Fp-biotin).

Recently, broad-spectrum probes targeting nucleophilic lysine (Lys) residues have also been added to the profiling toolbox. An alkyne-functionalized N-hydroxysuccinimide-ester probe (NHS-ester) was developed by Ward et al.

as a versatile reactivity-based probe. This small electrophile mainly labels Lys residues (around 50%) but also serines, threonines, and tyrosines in mouse liver proteomes. Furthermore, the group of Cravatt used a sulfotetrafluorophenyl (STP) based probe that showed a better selectivity towards Lys residues (> 80%) (Figure 5).

Figure 5. Structure of broad-spectrum probes targeting Ser (A), Cys (B) and Lys (C) residues.

The available Cys, Ser and Lys targeted probes provide a proteomic platform to screen inhibitors and identify the proteins that react with an electrophilic natural product by comparing the labeling profiling of these broad-spectrum probes in the presence and the absence of the natural product. Selecting the appropriate broad-spectrum probe is key in these profiling experiments. Hits within the serine hydrolase families will be missed, when a natural product is screened against a broad-spectrum

S NH HN O

H P H

NH EtO O

F

O

NH R

O O

NMe2 NMe2+

CO2- R= R=

Fp-rhodamine Fp-biotin

I N

H O

iodoacetamide alkyne

B A

O O

O N O

O NHS-ester probe

C

O O

SO3Na F F

F F

STP probe

10

electrophilic traps can help to predicted which protein classes/residues will likely react with the putative electrophile within the natural product of interest and thus against which type of probe the product has to be screened (Figure 6). When a natural product is used as an inspiration to develop a specific activity-based probe, it is recommended to choose a tag that is different from the one used by the broad-spectrum probe to be able to screen them in a competitive experiment (Figure 6A and 6B, BODIPY and rhodamine can for example be screened simultaneously). The various profiles obtained from the competitive and direct activity based protein profiling can be compared in order to identify the targets of both the natural product and the newly developed natural product based probe simultaneously (Figure 6B). The main purpose of such studies is to be as thorough as possible to identify the protein targeted by a natural product. If the natural product screened interacts with proteins via non-covalent interactions or if the selected broad-spectrum probe is inadequate (or limited), the number of non-identified target proteins will be high and important hits might be overlooked.

The advantages as well as the weaknesses of competitive profiling approaches over converting a natural product into a chemical probe are exemplified in a study by Abegg et al.

They used a combination of direct profiling and two broad-spectrum hyper-reactive cysteine probes to characterize the targets of curcumin (Figure 7). To assure that the majority of the Cys residues could be labeled, they developed a novel alkylating agent based on benziodoxolone (EBX) that is complementary to the widely used iodoacetamide alkyne (IAA). Prior studies had shown that EBX selectively and rapidly modifies thiols in high-yield.

Labeling of the resulting probe JW-RF-010 in HeLa cells was compared with the IAA. At pH 7.4 without additives at room temperature, both probes reacted with proteins. Using 10 µ M of probe, 2257 peptides were enriched by JW-RF-010 and 2184 by IAA.

Among these peptides, 793 were unique to IAA and 866 unique to JW-RF-

010, which indicates that the two probes indeed address different subsets of

hyper-reactive Cys residues and thus complement each other.

Figure 6. Broad-spectrum probes modifying cysteine, serine or lysine hyperreactive residues; direct and two-step natural product based probes (A); competitive activity- based profiling can be used to identify the proteins targeted by a natural product by comparing the obtained profile with the adequate controls, a reprensentation of a gel is used to illustrate the competitive activity-based protein profiling assays (B); as an example, the protein indicated by number 1 in bold and italic targeted by the NP, the NP probe and the broad-spectrum probe is highlighted on the gel representation.

Furthermore, JW-RF-010 revealed to be more specific probe towards Cys than IAA (97 versus 91.4%), which also targets to some extend Asp, Glu, Ser

A

B

P EtOO

F

I N

H O OH SH

Ser Cys

CuSO₄

N₃

direct probe two-step probe N

N N

natural product based probes

broad-spectrum probes

NaO₃S F

F

F

F O

O

H2NLys

broad-spectrum probe

pre-incubated with a NP

then with broad-spectrum probe pre-incubated with a NP probe then with broad-spectrum probe

NP probe

pre-incubated with a NP then with a NP probe

broad-spectrum probe targets NP probe targets

NP targets

5 2 1

1

1 2

non-identified targets

competitive activity-based protein profiling activity-based protein profiling

1 2

3 4 5

1 2 3 4 5

1

12

complementarity of these Cys-specific probes. In a competitive experiment between curcumin and the Cys-specific probes, 57 proteins were identified while a direct curcumin-alkyne probe led to the identification of only 42 targets. Among the identified proteins, 19 were specific to IAA and 16 to JW-RF-010. This unambiguously demonstrates that, besides labels of direct probes, also the choice of the broad-spectrum probe can affect the outcome of the experiment. Determining the targets both with competitive profiling and direct probes derived from the natural product of interest aids to define the real targets.

Figure 7. Structures the broad-spectrum probes JW-RF-001 and IAA targeting Cys

residues and of the curcumin probe.

Even with these direct and competitive profiling methods, it is sometimes difficult to distinguish between hits that are related to the biological activity of natural products, the probe specific off-targets and non-specific background. Highly expressed proteins can impair the detection of less abundant protein targets by fluorescence in-gel scanning. To avoid this intrinsic bias of working at the protein level, mass spectrometry based- techniques that enable quantification on a peptide level have been explored.

Development of these quantitative proteomics approaches was assisted by the increased sensitivity and resolution of mass spectrometry equipment and the improvements in the analysis of large proteomic datasets. Using quantitative mass spectrometry approaches enabled the identification of targets that are overlooked by in-gel detection methods. The main quantitative techniques include SILAC (stable isotope labelling by amino acids in culture), iTRAQ (isobaric tag for relative and absolute quantitation), dimethyl labelling (DiMe), and label-free based methods (such as spectral counting) (these methods are critically compared in a review by Bantscheff et al.

). For the labeling approaches, the incorporation of isotopes into peptides is necessary. The mass spectrometer can distinguish between samples containing isotopes and non-treated samples to facilitate quantification. One example of this is the study towards the mode of action of hypothemycin in the parasite Trypanosoma brucei by Nishino et al.

This polyketide natural product inhibits the growth of the parasite. In mammalian cells, hypothemycin has been shown to bind to a diverse subset

I O O

Me

JW-RF-001

I N

H O

iodoacetamide alkyne

MeO

HO

OMe

O

O O

Curcumin probe

of kinases, including MEK, ERK, PDGFR, VEGFR2, and FLT3. These kinases have in common that a cysteine residue precedes the catalytic DXG motif and Nishino et al. therefore hypothesized that CDXG kinases also were the targets in the T.brucei parasite. After synthesis of the hypothemycin alkyne probe (Figure 8), they first confirmed that its potency was similar to the natural product in parasite growth inhibition assays. The Michael acceptor present in hypothemycin indeed also reacted with CDXG kinases in T.brucei and TbGSK3 was identified as the main hit based on the abundance and protein coverage. Gel-free quantitative labeling experiments of four samples pre-treated by hypothemycin (0, 20, 200, or 1000 nM) and labeled with unique iTRAQ reagents at the peptide level revealed the extent of CDXG kinase labeling by the probe. From an analysis of the quantity of kinase recovery as a function of hypothemycin pretreatment (0, 20, 200, or 1000 nM) it appeared that the kinase TbCLK1/2 rather than TbGSK3 was most sensitive to hypothemycin and silencing TbCLK1 by RNAi confirmed that TbCLK1 inhibition is important for parasite growth inhibition.

Figure 8. Structure of hypothemycin and 4-hydroxy-2-nonenal based probes (A) and isotopically-tagged UV-cleavable reagent Az-UV-biotin (B).

Studying the site of modification rather than the common protein based enrichment-identification sequence in combination with quantitative mass spectrometry techniques also provided insight in dynamics of modification of proteins with electrophiles in live cells. Yang et al.

identified modification of peptides by the electrophilic lipid 4-hydroxy-2-nonenal metabolite (HNE) using their novel isotopically-tagged, UV-cleavable

A

B

hypothemycin alkyne probe O

OH O O

O OH OH O

alkyne HNE probe O

OH H

S HN NH

O H H HN

O O NH O

O O

O O O N3

light/heavy Az-UV-biotin

O N3

O N3

12C

13C

14

CuAAC reagent (“Az-UV-biotin”) (Figure 8). After incubation of HNE- probe with RKO colon cancer cells, cell lysis and protein digestion; the Az- UV-biotin is reacted with the peptides followed by enrichment-release by UV irradiation. This method then greatly facilitated the identification of modified peptides and their quantification. No less than 398 alkylation events were identified on intact cells. They also exploited this approach to follow the dynamics of HNE in live cells and follow adduct turnover rates.

The results obtained confirmed that the repair or reversion processes are only effective on intact cells (the same adducts were stable on lysates). The use of such quantitative platform is interesting as it provides a versatile method to follow cellular dynamics.

1.3 Downstream applications of natural product derived chemical probes

The use of natural product derived probes is not limited to target identification studies. These probes can also be employed to gain a deeper insight into the role of the targets in living cells. Probes equipped with fluorophores are particularly suitable for these studies, as they enable efficient and rapid in-gel detection of labeled proteins and fluorescent microscopy studies.

The fluorescent probes that specifically and potently label one or a small

sub-set of proteins can be used for live-cell imaging of the target proteins in

their native environment. By virtue of fluorescence imaging microscopy

insight can be obtained about the localization of the protein of interest under

physiologically relevant conditions. This is elegantly illustrated for

penicillin-binding proteins (PBPs) in bacteria. These enzymes are involved

in the final steps in the synthesis of the peptidoglycan cell wall that

surrounds most bacteria. The growing peptidoglycan chain is elongated by

Class A high molecular weight (HMW) PBPs, which display

glycosyltransferase activity. Furthermore, all PBPs catalyze crosslinking of

the peptidoglycan and trimming of the stem-peptide. These latter activities

are inhibited by natural product based antibiotics, such as penicillin or

cephalosporin. The β-lactam motif within these antibiotics covalently

modifies the active site serine residue required for the

transpeptidase/carboxypeptidase activity. To study the role of PBPs,

fluorescent derivatives of penicillin and cephalosporin have been

prepared.

In a recent comparative protein profiling experiment,

Kocaoglu et al. showed that the in vivo labeling pattern for Bocillin-FL and Cephalosporin C-F and C-T differ in Bacillus subtilis (Figure 9).

While Bocillin-FL labels PBPs 1a/b, 2b, 2c, 3, 4 and 5, Ceph C-T only labels PBPs 1a/b, 2b, 2c, and 4. The authors exploited these differences to study the localization of PBP3 and PBP5 with fluorescence imaging. By treating live B.

subtilis cells first with Ceph C-T and subsequently with Bocillin-FL, they revealed that both probes were not co-localized within the same region of a cell, which suggests that PBP3 and PBP5 may have specific function during cell division. Similar results were obtained for Streptococcus pneumonia. Also in this cell line Ceph C-T labeled a different subset of PBPs than Bocillin-FL (PBPs 1b and 3 for Ceph C-T and PBPs 1a/b, 2x/a/b and 3 for Bocillin-FL).

Super-resolution images were then captured after dual labeling and only little co-localization was observed indicating again an evident difference in distribution. As such profiling of the PBPs with probes for the first time showed that the endogenously expressed active PBPs localize to different sites within the cell and that these PBPs may have different functions in the synthesis and homeostasis of peptidoglycan.

Figure 9. Cephalosporin and Penicillin based probes (A) and schematic representation of the gel and the microscopy picture on B.subtilis demonstrating the absence of co- localization indicating that the PBPs targeted by the probes are found in different areas of the cell (B).

A

B

NH O NH

R

O O

OH H

N

O N

S

O HO O

O H O

O O

HO

COO^-

O N⁺

N

COO^-

Ceph C-F Ceph C-T

N S O

H

O O^- HN O

Bocillin-FL

Na+

HN O N⁺ NB^-F R= F

Ceph C-T Boc-FL PBP

1a/1b

2a 2b2c 3 4

5

16

Furthermore, specific natural product-derived probes in combination with broad-spectrum ABPs have helped to screen in an almost high-througput manner (focused) compound libraries for enzyme specific inhibitors.

Competition experiments with the broad-spectrum probe give a good indication for the selectivity of a compound within an enzyme family, as was shown for the fluorophosphonates probes Fp-rhodamine and Fp-biotin.

The large serine hydrolase enzyme family consists of more than 200 enzymes in humans and the enzymes are involved in the cleavage of ester, amide or thioester bonds in a wide range of pathways.

By screening carbamates versus Fp-rhodamine and Fp-biotin,

the selectivity of carbamate containing inhibitors could be determined and these leads were successfully developed further into a handful of carbamate based ABPs.

However some enzymes within the serine hydrolase family do not react with Fp-rhodamine and Fp-biotin, such as diacyl glycerol lipase-α (DAGL- α), or are less abundant. Screening for inhibitors of these proteins can be achieved by using a combination of specific and broad-spectrum probes, as demonstrated by Baggelaar et al.

To develop novel reversible inhibitors of DAGL-α, the authors used the drug tetrahydrolipstatin (THL), which is derived from the natural product lipstatin, as a starting point. This natural product derivative covalently inhibits DAGL-α and introduction of a BODIPY-dye on THL yielded the probe MB064 (Figure 10), which reacts with tubulin, glyceraldehyde-3-phosphate dehydrogenase, ABHD16 and DAGL-α in mouse brain membrane proteome. The labeling profile of MB064 is much more specific than Fp-rhodamine in the same biological settings.

Competitive activity based protein-profiling assays with MB064 facilitates sensitive detection of DAGL-α inhibition, while Fp-rhodamine enabled determining the general selectivity of the compound within the SH family.

These screen campaigns resulted in the identification of specific inhibitors

for FAAH and DAGL-α and demonstrate the effectiveness of screening

simultaneously against selective and broad-spectrum probes.

Figure 10. MB064 (A) and schematic outcome of the competitive activity-based profiling of THL versus Fp-rho or MB064 (B).

1.4 Conclusion

In conclusion, chemical probes are completing the traditional methods -such as biochemical and genetic modifications- to gain insight in the connections between proteins, their specificities for substrates and their importance in diseases. Natural products are a great inspiration for the development of probes that can be employed to characterize the targets of the natural product and to study the function of the protein by using the probe in downstream application.

Several interesting guidelines emerge from the selected examples. Firstly, for the development of novel probe molecules, it is essential to recognize interesting and original natural products both containing and not containing electrophilic traps. These scaffolds should be accessible in an effective way to allow modification of the original scaffold with the necessary tags and/or reactive group. For the target identification it is essential to combine broad- spectrum and natural product derived probes. It is also preferable to identify the targets using quantitative methods to assure the relevance of the hit and overcome the limitation of gel-based assays. Thirdly, the field of ABPP encloses a plethora of techniques such as fluorescence microscopy that should be implemented to gain insight into biological processes or diseases. Finally, it is necessary to develop and characterize new broad- spectrum ABPs and to carefully take advantage of the ones already available to screen scaffolds and obtain specific and potent inhibitors and ABPs.

Inspired by nature (or not), it is safe to believe that chemical probes can be designed for almost any protein of interest containing a nucleophilic residue within the active site by combining an adequate scaffold and electrophile.

A B

Fp-Rho MB-064

DAGL-α -

O O

NN N

O

NF FBN OH

O O

HN O O

MB064

THL - THL

-

18

These probes will help comprehending nature by pursuing the goal to understand every single protein function.

1.5 Brabantamide as an inspiration and outline of the thesis

The research in this thesis focused at employing the above described methodologies to characterize the targets of the enol cyclocarbamate scaffold found in brabantamides. These lipopeptides have been reported to have variety of interesting bioactivities. Brabantamide A has antibacterial, antifungal, antioomycete activity and inhibits the mammalian phospholipase A

(Lp-PLA

) (a SH).

Also from a structural point of view, Brabantamides are interesting. These lipopeptides consist of a rhamnosylated beta-hydroxy fatty acid that is attached via an imide to a rare 5,5 (brabantamide A, B and C) or a 5,7-fused bicyclic enol carbamate ring system (SB-31502) (Figure 11).

Simplified(semi-) synthetic analogues that lack the rhamnose, as in deglycosylated brabantamide A,

and the imide, as in the derivative synthesized by Pinto et al.

(Figure 12) maintained activity towards Lp- PLA

. Even changing the stereochemistry from R to S ring or the chain length did not greatly affect activity, which suggested that the bicyclic enol carbamate ring system is at the origin of the observed activity.

Figure 11. Brabantamides A,B and C natural products first isolated from Pseudomonas

N O

O HN O

O

O O

OH OH

OH SB-253514 brabantamide A

N O

O HN O

O

ORha

R R=

R=

SB-253517 brabantamide B

SB-253518 brabantamide C

SB-315021 HN

O

O O

OH OH

N OH O

O O

H

1

14

14 1

16

16 1

The two putative electrophilic traps within the scaffold, the activated carbamate and the Michael acceptor, advert potential covalent interaction with Ser and Cys residues. Mass spectrometry by Thirklette et al. confirmed that phospholipase-brabantamide A adducts are formed.

Figure 12. Brabantamide A bears two putative electrophiles potentially reacting with Ser, Cys or Thr ; several approaches can be envisioned to identify the targets of the synthesized enol carbamates.

Based on this, we decided to study the mechanism of action of this natural product scaffold using chemical proteomics. In this thesis, a series of probe molecules based on the Brabantamide A scaffold is developed. A set of inhibitors, two-steps probes and direct probes were synthesized and biological activity was characterized. The aim of the thesis is to explain the observed phenotype in bacteria by identifying the molecular targets using the competitive and direct profiling approaches, to gain insight into proteins that react with enol-cyclocarbamate in mammalian cells and to improve the understanding of the nuances of reactivity with the aim to develop

brabantamide A (1)

Approach 2:

Global identification

N O O

O HN

N O O

O HNC12H25 O

Nu^-

O O NH

O HO OH HO N O O O

NH N O

O O

Nu N

H HN O

N Nu H N O

O O

Nu

addition to the Michael acceptor?

Nu^- addition to the carbamate?

Nu = Ser, Thr

TAG

TAG

Approach 1:

Competitive profiling for reactive serines

F POEt O

TAG TAG: biotin, TAMRA

Nu = Cys

20

improved probes and inhibitors. Chapter 2 describes the design and synthesis of a panel of derivatives as well as their activity versus Bacillus subtilis 168 and Streptococcus pneumoniae. We used a combination of fluorescence microscopy and competitive ABPP to study the mechanism action and we identified penicillin-binding proteins as one of the targets of these compounds. In Chapter 3, the reactivity of four enol cyclocarbamate probes (2 two-steps probes and 2 fluorescent probes) is studied in the mammalian A549 cells. Chemoproteomics studies identified ALDH3A1 and RALDH1 and labeling experiments on recombinantly expressed confirmed that the novel probes label the important cancer markers RALDH1 and ALDH3A1. Chapter 4 describes the use of Fp-rhodamine and Fp-biotin to study the reactivity of the panel of compounds towards serine hydrolases first on recombinant Lp-PLA

and esterase from Bacillus subtilis and then on both bacteria and mammalian cells (respectively Bacillus subtilis and A549).

In Chapter 5, we studied the reactivity of a model enol carbamate to gain insight in how different nucleophiles react with the scaffold. We used this insight to tune the electrophilicity of both the carbamate and the Michael acceptor present in the brabantamide scaffold by synthesizing vinyl sulfone, vinyl ester and monocyclic derivatives. In Chapter 6, the research is summarized and future directions towards the applications of enol carbamate electrophiles as well as improvement of the probes are discussed.

1.5 References

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

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Taunton, J. J. Am. Chem. Soc. 2017, 139 (2), 680.

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(25) Bantscheff, M.; Schirle, M.; Sweetman, G.; Rick, J.; Kuster, B. Anal Bioanal Chem 2007, 389 (4), 1017.

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L.; Renslo, A. R.; McKerrow, J. H.; Taunton, J. eLife 2013, 2, 2139.

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E.; Kearns, D. B.; Carlson, E. E. ACS Chem. Biol. 2012, 7 (10), 1746.

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B.; Saghatelian, A.; Cravatt, B. F. Nature Chemical Biology 2016.

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Niessen, S.; Cravatt, B. F. Proc. Natl. Acad. Sci. U.S.A. 2010, 107 (49), 20941.

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Hoogendoorn, S.; McGuire, R.; Florea, B. I.; Meeuwenoord, N.; van den Elst, H.; van der Marel, G. A.; Brouwer, J.; Di Marzo, V.; Overkleeft, H. S.; van der Stelt, M. Angew. Chem. Int. Ed. 2013, 52 (46), 12081.

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Raaijmakers, J. M.; Gross, H. ChemBioChem 2014, 15 (2), 259.

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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.

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22

Chapter 2

Bicyclic enol cyclocarbamates inhibit penicillin-binding proteins

Natural products form attractive leads for the development of chemical probes and drugs. The antibacterial lipopeptide Brabantamide A contains an unusual enol cyclocarbamate and we used this scaffold as inspiration for the synthesis of a panel of enol cyclocarbamate containing compounds. By equipping the scaffold with different groups, we identified structural features that are essential for antibacterial activity. Some of the derivatives block incorporation of hydroxycoumarin carboxylic acid-amino

-alanine into the newly synthesized peptidoglycan. Activity-based protein-profiling experiments revealed that the enol carbamates inhibit a specific subset of penicillin-binding proteins in B. subtilis and S. pneumoniae.

This chapter is adapted from the original publication: Dockerty, P. et al. Bicyclic enol cyclocarbamates inhibit penicillin-binding proteins. Org. Biomol. Chem. 2017, (15) 4, 894.

24

2.1 Introduction

Microorganisms produce a large variety of secondary metabolites for communication, nutrient uptake and self-defense. These structurally diverse compounds often have interesting properties, like antimicrobial and cytotoxic activity. Because of these properties, natural products and parts of their scaffold have served as starting point for the development of drugs

and chemical probes.

Examples of natural product based drugs include antibiotics like beta-lactams, which inhibit cell wall synthesis, and tetracyclines and aminoglycosides, which block protein synthesis. The natural products vibrolactone, epoxomicin and cyclophellitol have been converted into tools to study serine hydrolases,

the proteasome and glycosidases, respectively.

Further examples of probe molecules derived from metabolites are fluorescent penicillin and moenomycin A analogues, which have been prepared to study the transpeptidation and transglycosylation step during peptidoglycan synthesis, respectively.

The vast majority of the isolated natural products have not yet been exploited for probe and drug development, while derivatization of many of these scaffolds may also lead to interesting research tools or lead compounds. Of particular interest are natural products that display antibacterial activity.

The rising resistance to commonly applied antibiotics necessitates the identification of leads that inhibit bacterial growth. A scaffold that triggered our interest was the enol cyclocarbamate. These proline-derived 5,5-fused and 5,7-fused bicyclic ring systems are found in lipopeptides that have been isolated from Pseudomonas extracts.

Compounds containing this scaffold, including natural product Brabantamide A (1a) (Figure 1A) and (semi-) synthetic derivatives 1b

and 2a

potently inhibit lipoprotein-associated phospolipase A

(Lp-PLA

), a mammalian group VII phospholipase A

that belongs to the serine hydrolase superfamily.

Besides being Lp-PLA

inhibitors, enol cyclocarbamate containing lipopeptides, like 1a, have also been shown to affect bacterial, fungal and oomycete growth by targeting specific pathways in these microorganisms.

13

Brabantamide A (1a) increases phospholipase D activity in oomycetes.

Mode of action studies with reporter strains that carry a firefly luciferase

gene fused to a promoter that is induced by antibiotics that block the most

important biosynthetic pathways in bacteria indicated that the bactericidal

activity of lipopeptide 1a is caused by inhibition of the peptidoglycan

synthesis pathway and/or cell membrane stress.

Although 1a has

surfactant-like properties, non-specific disruption of the cell membrane has been excluded as the sole mode of action, since lipopeptide 1a did not permeabilize model membranes at biologically relevant concentrations.

Despite the fact that it has been shown that the enol cyclocarbamate is essential to potently inhibit the mammalian Lp-PLA

, it has not yet been established if the enol cyclocarbamate is required for the observed activity in bacteria. Neither have the molecular targets of molecules containing this scaffold been identified in bacteria. We aimed to investigate this by preparing a series of enol cyclocarbamate derivatives 2–4 (Figure 1B-D) that vary in the substitution pattern and by studying the antibacterial activity of the resulting compounds. To determine the role of the 5,5-fused ring system on the biological activity of the scaffold and to probe the impact of the substitution pattern, we synthesized monocyclic analogues 3a–c (Figure 1C) and substituted derivatives 2a–i and 4a–e (Figure 1B–D), respectively.

Figure 1. (A) Structure of the natural product Brabantamide A 1a and its deglycosylated form 1b. The enol cyclocarbamate scaffold is depicted in grey. (B) N-Boc-Proline derived enol cyclocarbamates 2a–i containing various chains. (C) Monocyclic enol carbamates 3a–c (E and Z isomers). (D) N-Boc-4-hydroxyproline derived enol cylclocarbamate.

To circumvent possible decomposition of the acid and base-labile enol cyclocarbamate during synthesis,

we developed a novel route in which the enol cyclocarbamate is introduced at a late stage in the synthesis and that

N O

NHR O

2a R = (CH2)11CH3

2b R = (CH2)5CH3

2c R = CH2Ph

2d ^{R =}

2 O

2e^{R =} _{N N}^{N (CH2)11CH3}

2f ^{R = N NN} 2g ^{R =}

N NN OH

2h ^{R =}

N NN ₂

2i ^{R =}

N NN O N

H 3

O (CH2)4 S

HN NH O

H H

R N O O

NH (CH2)11CH3 O

3a ^{R =} 3b ^{R =}

N NN O₂ OH 3c ^{R = CH3} 3

N O O

O

NH (CH2)11CH3 O

R

4

4b ^{R =} 4a ^{R = CH}2Ph

4c ^{R =} N NN

Ph

4d ^{R =} _{N N}^{N O}2 ^OH

4e ^{R =}

N NN N

B C

D A

O O

NH O

SB-253514 Brabantamide A O

R

HOHO OH

O

1a^R= 1b^R=OH Deglycosylated

Brabantamide A O

26

allows straightforward functionalization of this labile scaffold in the final step with copper-catalyzed click chemistry. With the panel of molecules synthesized, we identified the structural features that are required for antibacterial activity. Using metabolic labeling and activity-based protein profiling, we reveal that these enol cyclocarbamates interfere with peptidoglycan synthesis and that they inhibit Class A high molecular weight penicillin-binding proteins.

2.2 Results and discussion

2.2.1. Chemical synthesis

The synthesis of the panel of enol carbamate derivatives commenced with condensing Meldrum’s acid to Boc-protected building blocks 5, 8 and 11 (Scheme 1A-D) using N,N’-dicyclohexylcarbodiimide (DCC) and 4- (dimethylamino)pyridine (DMAP). For the hydroxy proline (Hyp) derived enol carbamates, Boc-protected Hyp 14 was first alkylated with either benzyl bromide or propargyl bromide to obtain respectively 15a and 15b and subsequently condensed with Meldrum’s acid as mentioned before.

Filtration and subsequent acid-base extraction gave the crude Meldrum’s

acid derivative intermediates. Attempts to directly convert these into β-keto

amides using five equivalents of amine in refluxing toluene resulted in

complex mixtures and the β-keto amide was therefore installed using a two-

step procedure. First, the Meldrum’s acid intermediates were smoothly

converted into the corresponding β-keto esters 6, 9, 12 and 16a–b by

refluxing the intermediates in anhydrous methanol.

In a second step, the β-

keto esters were reacted with the amine of interest to form β-keto amides

7a–d, 10, 13 and 17a–b. Refluxing β-keto ester 6 with hexylamine or

benzylamine in THF did not result in the desired β-keto amide products 7b

and 7c, but afforded the corresponding enamine products instead. The

same, undesired products were obtained when the β-keto esters were

reacted with the amine in the presence of sodium methoxide

or titanium

isopropoxide.

Traces of the desired β-keto amides could be obtained by

treating the amine with trimethylaluminum to generate the

dimethylaluminum amide in situ.

We therefore turned our attention to

DABAL-Me

, the adduct of 1,4-diazabicyclo[2.2.2]octane (DABCO) and

trimethylaluminum.

Activating the amine in this fashion improved the

yield of the β-keto amide formation reaction and products 7a–d, 10, 13 and

17a–b were obtained in moderate to reasonable yields. The final steps of the