Degrasyn exhibits antibiotic activity against multi-resistant: Staphylococcus aureus by modifying several essential cysteines

Article abstract of DOI:10.1039/c9cc09204h

Degrasyn inhibits deubiquitination enzymes and has anti-cancer activity. We here show that it also exhibits antimicrobial activity against multi-resistant Staphylococcus aureus. Structure activity relationship studies demonstrate an important role of the

Full text of DOI:10.1039/c9cc09204h

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Degrasyn exhibits antibiotic activity against
multi-resistant Staphylococcus aureus by
Cite this:DOI: 10.1039/c9cc09204h
modifying several essential cysteines†
Received 26th November 2019,
Accepted 20th December 2019
a
b
Kyu Myung Lee,
Philipp Le,a Stephan A. Sieber *a and Stephan M. Hacker
*
DOI: 10.1039/c9cc09204h
rsc.li/chemcomm
Degrasyn inhibits deubiquitination enzymes and has anti-cancer activity.
Given the promising antibiotic effects of degrasyn against
We here show that it also exhibits antimicrobial activity against multi- the MSSA strain NCTC8325 with a minimal inhibitory concen-
resistant Staphylococcus aureus. Structure activity relationship studies tration (MIC) of 6.25 mM, we tested its activity against various
demonstrate an important role of the electrophilic a-cyanoacrylamide methicillin-resistant S. aureus (MRSA) strains including several
moiety as a Michael acceptor. A suite of chemical proteomic techniques clinical isolates (Fig. 1B).5,11 Satisfyingly, the antibiotic activity
unraveled binding of this moiety to various cysteine residues of essential was retained in all MRSA strains (MIC r 12.5 mM) suggesting a
proteins in a reversibly covalent manner.
different mode-of-action as compared to existing drugs.
To elucidate the structure activity relationships (SAR) of
Rapid bacterial resistance development represents a major challenge degrasyn, we systematically altered its scaffold. Degrasyn (DGS)
for current antibacterial therapies.1,2 One strategy to identify novel and its enantiomer ((R)-DGS) were prepared according to estab-
antibacterials focuses on the repurposing of existing drugs originally lished synthetic procedures (Scheme 1). A change of the stereo-
developed against human proteins.3,4 In a recent drug-repurposing center resulted in a slight drop of the MIC from 6.25 mM for DGS
screen,5 we identified that the human deubiquitinating enzyme to 12.5 mM for (R)-DGS suggesting only a minor role of the
inhibitor degrasyn (also called WP1130) kills methicillin-sensitive absolute configuration (Fig. 1C). Accordingly, a racemic mixture
S. aureus (MSSA) (Fig. 1A and B). Degrasyn induces apoptosis of of (R,S)-DGS exhibited the same MIC value as DGS. Due to the
leukemia cells.6,7 In addition, anti-infective effects, i.e. the reduction minor role of the stereocenter and for the ease of synthesis, all
of intracellular replication of Listeria monocytogenes and viruses, further products were synthesized as racemic mixtures.
were previously reported. These effects were mainly attributed to the
The synthesis of DGS variants with modifications at the
inhibition of DUBs in macrophages.8–10 Direct antibiotic effects of pyridine ring (1–15, Fig. 1C) followed a modular blueprint as
degrasyn on isolated bacteria have to the best of our knowledge not outlined in Scheme 2. While an unsubstituted pyridine ring led to
been reported so far.
a loss of antibiotic activity (1, 2), varying the positions of the
Here, we synthesized 19 degrasyn derivatives and analyzed bromine substituent and the nitrogen atom was tolerated with
their effects on bacterial growth. The nitrile-substituted Michael only a slight loss in activity (3, 4). Even the replacement of the
acceptor (a-cyanoacrylamide) turned out to be a signature moiety pyridine ring with a phenyl ring retained considerable activity (5).
important for antibiotic activity through reversible covalent Therefore, due to availability of building blocks and synthetic
modification of cysteine residues in essential enzymes. Chemical accessibility, we studied substituted phenyl rings at this position
proteomics revealed several target proteins of degrasyn belonging in more detail. Bromine- and chlorine-substituted phenyl rings
to important enzyme classes involved e.g. in cell wall, lipid and were able to largely maintain antibiotic effects (5, 6, 7). In contrast,
histidine biosynthesis indicating a polypharmacological mode-of- fluoro- and iodo-substituted phenyl rings resulted in a loss of
action.
activity (8, 9, 10). While di- and tri-substitutions with electron-
donating groups (e.g. –OH, –OMe, etc.) were largely not tolerated
(11, 12, 13), the introduction of a difluoroacetal moiety (14) retained
full activity. Satisfyingly, substitution with an alkyne handle (15)
only slightly increased the MIC, which is a prerequisite for sub-
sequent target identification using conventional ABPP.
We next turned our synthetic efforts towards the reactive
a-cyanoacrylamide moiety. Acrylamides are irreversible covalent
inhibitors, which readily react with cysteine residues.12 Further
a Center for Integrated Protein Science, Department of Chemistry and Chair of
¨
¨
¨
Organic Chemistry II, Technische Universitat Munchen, Garching bei Munchen,
Germany. E-mail: stephan.sieber@tum.de
b
¨
¨
Department of Chemistry, Technische Universitat Munchen, Garching bei
¨
Munchen, Germany. E-mail: stephan.m.hacker@tum.de
† Electronic supplementary information (ESI) available: Supporting scheme,
figures and tables, experimental procedures and compound characterization.
See DOI: 10.1039/c9cc09204h
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Fig. 1 (A) Structure of degrasyn (DGS). (B) MIC values for DGS in various strains of S. aureus. *: Clinical isolates of S. aureus . (C) Substitution patterns and
MIC values in S. aureus NCTC8325 for various degrasyn analogues synthesized in this study in order to investigate the SAR of DGS.
Scheme 1 Synthesis of DGS. (R)-DGS was synthesized using the same pro-
Scheme 2 Synthesis of degrasyn derivatives (see Fig. 1 for specific R
tocol starting from (R)-1-phenylbutan-1-amine. (a) 1-Cyanoacetyl-3,5-dimethyl-
groups). (a) (i) NH2OHÁHCl, NaOH, EtOH/H2O, 3 h (ii) H2, Pd/C, MeOH,
1H-pyrazole, toluene, reflux, 2 h, 75%; (b) 6-bromo-2-pyridinecarboxaldehyde,
24 h (iii) 1-cyanoacetyl-3,5-dimethyl-1H-pyrazole, toluene, reflux, 2 h, 69%;
piperidine, EtOH, reflux, 3 h, 65%.
(b) aryl aldehyde, piperidine, EtOH, reflux, 3 h, 33–75%.
fine-tuning with electron-withdrawing substituents such as nitriles antibiotic activity highlighting that both the Michael acceptor and
yields reversibly covalent inhibitors, which have received great the electron-withdrawing substituent are mandatory (Fig. 1C).
attention in drug development.13,14 As degrasyn bears this signature
In order to better understand the mechanism-of-action, we
moiety, we investigated, if the ability to act as a Michael acceptor performed target identification studies in S. aureus. We first
and the fine-tuning as a-cyanoacrylamide are both relevant for the applied conventional ABPP with 15 (Fig. S1, ESI†).15,16 Live
antibiotic effect. The synthesis of 16, without the double bond of the bacterial cells were incubated with the probe, lysed, labeled
Michael acceptor, was achieved by reduction of DGS using NaBH4
proteins modified with biotin azide using copper-catalyzed
(Scheme S1, ESI†). The synthesis of 17, without the nitrile moiety, azide–alkyne cycloaddition (CuAAC)17 and subsequently analyzed
was performed using a Horner–Wadsworth–Emmons reaction via liquid chromatography coupled to tandem mass spectrometry
(Scheme S1, ESI†). Interestingly, neither 16 nor 17 showed any (LC-MS/MS). The labeling was performed at three different
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concentrations (6.25 mM, 12.5 mM and 25 mM) and data analysis
Besides the ability to obtain residue-specific information of
was performed with label-free quantification (LFQ).18 While we binding with an unmodified covalent protein ligand, another
saw some labeling in a gel-based analysis, no prominent enrich- important advantage of this approach is that also cysteines,
ment of protein targets was observed by mass spectrometry which are reversibly engaged by DGS, are identified.14 Satisfyingly,
(Fig. S2 and Table S1, ESI†). In addition, competition studies treatment of S. aureus lysate with various concentrations of DGS
with various concentrations of parent DGS did not lead to an ranging from 100 mM to 10 mM led to the identification of 151
obvious target profile (Fig. S3 and Table S1, ESI†). Thus, the cysteines in 114 proteins targeted by DGS (Fig. 2B, Fig. S4 and
reversibly covalent binding mechanism is not suitable for Table S1, ESI†). Interestingly, 96 of these cysteines were not
conventional ABPP studies as the transient target binding is liganded by any of 19 covalent a-chloro-acetamides that were
incompatible with the enrichment procedure for MS.
previously used to profile cysteines in S. aureus.19 This indicates
We therefore performed competitive residue-specific proteomics that the reversibly covalent a-cyano-acrylamide is able to interact
using the recently developed isoDTB-ABPP method19 that is based with a unique set of cysteines. Many cysteines respond to DGS
on the isoTOP-ABPP (isotopic tandem orthogonal proteolysis-ABPP) treatment in a clearly concentration-dependent manner (Fig. 2C
platform12,20 as an alternative strategy. In this approach, S. aureus and Tables S1, S2, ESI†). For the 98 cysteines that are liganded by
cells are lysed and the lysate is split into two samples. One sample is DGS and quantified at all concentrations, the data could be fitted
treated with DGS and the other one with DMSO as a control (Fig. 2A). with a dose–response curve with a median R2 value of 0.92. This
Afterwards, the samples are both alkynylated with iodoacetamide- demonstrates that residue-specific proteomics with the isoDTB
alkyne (IA-alkyne). Competitive cysteine binders such as DGS block tags using a few different concentrations of a covalent protein
this labeling at their specific binding sites in the proteome. In order ligand is able to deliver robust information on the binding affinity
to read out these differences in alkynylation, isotopically labeled for many cysteines in parallel.
desthiobiotin azide (isoDTB) tags19 are appended using CuAAC.17
31 of the targeted cysteines belong to proteins with an
The samples are combined, enriched on streptavidin, and digested. essential function for the viability of S. aureus. DGS e.g. binds
The resulting peptides are identified and quantified relative to each cysteine C100 of the bifunctional protein GlmU (EC50 = 22.3 mM,
other using LC-MS/MS. The ratio R between the heavy-labeled UniProt Code Q2G0S3), which is located close to the magnesium
(DMSO-treated) and light-labeled (DGS-treated) samples is a measure ion in the UDP-GlcNAc binding site of this protein.21 GlmU is an
for the degree of modification of the specific cysteine with DGS.
essential protein in the synthesis of UDP–GlcNAc and therefore
Fig. 2 (A) Workflow for competitive, residue-specific proteomics with the isoDTB-ABPP technology. Competition of IA-alkyne labeling by reversibly
binding DGS is preserved throughout the workflow by the irreversibly binding probe. DTB: desthiobiotin. (B) Volcano plot for the isoDTB-ABPP experiment
with 100 mM DGS used as the competitor. Significantly competed cysteines that are discussed in the text are highlighted in red. Other liganded cysteines that
are included in the concentration-dependent analysis (C) are highlighted in blue. The grey lines indicate cut-offs at Àlog10(p) = 1.3 and log2(R) = Æ2 that
were used as a criterion for hit selection. (C) Dependence of the degree of competition in isoDTB-ABPP experiments on the DGS concentration. Data points
are measured values from isoDTB-ABPP experiments and lines are non-linear dose–response curve fits. The values in parentheses are the EC50 values of the
competition for the respective cysteine. EC50 values with confidence intervals are given in Table S2 (ESI†). In (B) and (C), for each indicated cysteine, the
name or the UniProt code of the respective protein and the residue number of the competed cysteine are given. All data results from duplicates.
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to the alteration of multiple essential pathways, which is reflected
by complex changes in the proteome.
In summary, we identified degrasyn as a novel antibiotically
active compound against S. aureus including clinically isolated MRSA
strains. In line with the essential role of the a-cyanoacrylamide as
Michael acceptor covalent capture of target proteins failed due to
limited stability of the linkage throughout the chemoproteomic
protocol. While reversible covalent inhibitors are very promising,
this is one technological drawback. Competitive residue-specific
proteomics using the isoDTB-ABPP method turned out as an
excellent complementary strategy to identify the targeted cysteines
of degrasyn in vitro. 31 of these cysteines were labelled in proteins
essential for viability of S. aureus highlighting a polypharmacolo-
gical mode-of-action which was further corroborated by the up- or
down-regulation of several proteins in a whole proteome study.
While for degrasyn significant human toxicity is a challenge
(IC50 = 3.4 Æ 0.3 mM in an MTT assay in A549 cells), the high
antibacterial potency obtained by screening a small compound
library encourages the repurposing of other human inhibitors or
drugs for exploiting their antibacterial potential.
SAS acknowledges funding by the Center for Integrated
Protein Science (CIPSM) and the European Research Council
(grant agreement No. 725085, CHEMMINE, ERC consolidator grant),
SMH by the Fonds der Chemischen Industrie (Liebig Fellowship)
and the TUM Junior Fellow Fund and KML by the Korea Research
Institute of Chemical Technology (KRICT).
Fig. 3 (A) Volcano plot for a whole proteome analysis of protein expression
comparing S. aureus NCTC8325 treated with 21MIC concentration of DGS to
treatment with DMSO as a control. Upregulated proteins involved in the
histidine biosynthetic process are highlighted in red. (B) Enrichment analysis
of Gene Ontology terms of the category ‘‘biological process’’ comparing
the upregulated proteins to all proteins detected in the whole proteome
analysis. The five terms with the lowest corrected p-value are shown. All
data results from four independent biological replicates. The grey lines
indicate cut-offs at Àlog10(p) = 1.3 and log2(R) = Æ1 that were used as a
criterion for selection of up- and down-regulated proteins.
Conflicts of interest
There are no conflicts to declare.
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