Asymmetric Disulfanylbenzamides as Irreversible and Selective Inhibitors of Staphylococcus aureus Sortase A

Article abstract of DOI:10.1002/cmdc.201900687

Staphylococcus aureus is one of the most frequent causes of nosocomial and community-acquired infections, with drug-resistant strains being responsible for tens of thousands of deaths per year. S. aureus sortase A inhibitors are designed to interfere with

Full text of DOI:10.1002/cmdc.201900687

Accepted Article
Title: Asymmetric Disulfanylbenzamides as Irreversible and Selective
Inhibitors of Staphylococcus aureus Sortase A
Authors: Fabian Barthels, Gabriella Marincola, Tessa Marciniak,
Matthias Konhäuser, Stefan Hammerschmidt, Jan Bierlmeier,
Ute Distler, Peter R. Wich, Stefan Tenzer, Dirk Schwarzer,
Wilma Ziebuhr, and Tanja Schirmeister
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10.1002/cmdc.201900687
ChemMedChem
FULL PAPER
Asymmetric Disulfanylbenzamides as Irreversible and Selective
Inhibitors of Staphylococcus aureus Sortase A
Fabian Barthels[a], Gabriella Marincola[b], Tessa Marciniak [b], Matthias Konhäuser[a], Stefan
Hammerschmidt[a], Jan Bierlmeier[c], Ute Distler[d,e], Peter R. Wich[a,f], Stefan Tenzer[d], Dirk Schwarzer[c],
Wilma Ziebuhr[b], Tanja Schirmeister*[a]
[a]
F. Barthels (ORCID 0000-0001-7950-2158), M. Konhäuser, S. Hammerschmidt, Jun.-Prof. Dr. P. R. Wich (ORCID 0000-0003-1522-0340), Prof. Dr. T.
Schirmeister (ORCID 0000-0002-4587-5076)
Institute for Pharmacy and Biochemistry
Johannes-Gutenberg-University of Mainz
Staudinger Weg 5, 55128 Mainz, Germany
E-mail: schirmei@uni-mainz.de
[b]
[c]
[d]
Dr. G. Marincola (ORCID 0000-0001-9227-6554), T. Marciniak, Dr. W. Ziebuhr (ORCID 0000-0002-7059-1614)
Institute for Molecular Infection Biology
Julius-Maximilians-University of Würzburg
Josef-Schneider-Strasse 2, 97080 Würzburg, Germany
J. Bierlmeier (ORCID 0000-0002-7261-4744), Prof. Dr. D. Schwarzer (ORCID 0000-0002-7477-3319)
Interfaculty Institute of Biochemistry
Eberhard-Karls-University of Tübingen
Hoppe-Seyler-Strasse 4, 72076 Tübingen, Germany
Dr. U. Distler (ORCID 0000-0002-8031-6384), Prof. Dr. S. Tenzer (ORCID 0000-0003-3034-0017)
Institute for Immunology, University Medical Center
Johannes-Gutenberg-University of Mainz
Langenbeckstr. 1, 55131 Mainz, Germany
[e]
[f]
Dr. U. Distler (ORCID 0000-0002-8031-6384)
Focus Program Translational Neuroscience (FTN), University Medical Center
Langenbeckstr. 1, 55131 Mainz, Germany
Jun.-Prof. Dr. P. R. Wich (ORCID 0000-0003-1522-0340)
School of Chemical Engineering
University of New South Wales
Science and Engineering Building, Sydney, NSW 2052, Australia
Supporting information for this article is given via a link at the end of the document.
Abstract: Staphylococcus aureus is one of the most frequent causes
of nosocomial and community-acquired infections responsible for ten-
thousands of deaths per year by drug-resistant strains. S. aureus
addressed also in combination with classical antibiotics’ target
structures.[2] SrtA mediates the attachment of surface proteins to
the bacterial cell wall and it was shown that an S. aureus ΔSrtA
mutant is clearly attenuated in mouse infection models compared
to the wildtype.[3,4] SrtA inhibitors are likely to interfere with
adherence and intercellular communication rather than with
bacterial growth, thus imposing a lower selective pressure to
promote resistance development.[5] Since neither genetic
deletion[6] nor selective chemical inhibition[7–9] of S. aureus SrtA
was found to cause cytotoxic or growth inhibitory effects on
bacterial cells, the enzyme meets the requirements of an anti-
virulence target. Microbial surface components recognizing
adhesive matrix molecules (MSCRAMMs) are bacterial surface
proteins utilized during pathogenesis for adherence to endothelial
host cells and playing a role in immune evasion.[10] Many of these
virulence-associated proteins are secreted as precursors with C-
terminal LPXTG-tagged sorting-signals. At the bacterial cell wall,
they are recognized and cleaved between threonine and glycine
by the membrane-anchored transpeptidase SrtA.[11] Subsequent
ligation to the pentaglycine tail of the peptidoglycan layer yields
the covalent attachment to the bacterial outer surface.[12] In S.
aureus, approximately 20 surface proteins have been identified
as naturally occurring SrtA substrates, including several factors
that are involved in pathogenicity, such as protein A (SpA),
fibronectin-binding proteins (FnbpA/B), clumping factors (ClfA/B),
serine-aspartic acid repeat proteins (SdrC/D/E) and
staphylococcal surface proteins (Sas).[13,14]
sortase
A inhibitors are designed to interfere with virulence
determinants. We identified disulfanylbenzamides as a new class of
potent inhibitors against sortase A acting by covalent modification of
the active site cysteine.
A broad series of derivatives were
synthesized to derive structure-activity-relationship (SAR). In vitro and
in silico methods allowed rationalizing the experimentally observed
binding affinities and selectivities. The most active compounds were
found to have single-digit micromolar Ki values and showed up to 66%
reduction of S. aureus fibrinogen attachment at an effective inhibitor
concentration of 10 µM. This new molecule class exhibited minimal
cytotoxicity, low bacterial growth inhibition and impaired sortase-
mediated adherence of S. aureus cells.
Introduction
The ongoing spread of antibiotic resistance among Gram-positive
bacteria such as Staphylococcus aureus highlights the need for
new treatment options beyond traditional antibiotics. In this
respect, exploring virulence mechanisms as drug targets might
provide novel opportunities to interfere with bacterial
pathogenicity.[1] The cysteine transpeptidase sortase A (SrtA) was
considered as a putative anti-virulence drug target, which may be
1
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10.1002/cmdc.201900687
ChemMedChem
FULL PAPER
The eight-stranded β-barrel protein SrtA possesses three
conserved residues within the sortase family: His62, Cys126, and
Arg139, each of which cannot be mutated without disrupting
enzymatic functionality.[15] Structural similarities between the
In the corresponding NMR-structure (pdb: 2MLM), the ligand
displayed a reasonable fit, mimicking substrate binding in the
active-site pocket (Fig 1B). Asymmetric disulfides are known
inhibitors of cysteine proteases[35–37], thus, we decided to
transpeptidase SrtA and the papain protease were noticed[16]
,
investigate a disulfide warhead chemotype exchange and
however, enzymatic characteristics of the S. aureus SrtA differ
significantly from most proteases: (i) The catalytic Cys126 is
“reversely protonated”, which means it does not form a thiolate-
imidazolium pair, and thus, only a small fraction (<0.1%) of SrtA
is competent for catalysis at physiological pH 7.4.[17] (ii) The active
site is predominantly defined by the intrinsic flexibility of the β6/7-
and β7/8-loops.[18] (iii) The most active form of SrtA is constituted
as a homo-dimer with a KD=55 µM.[19] (iv) The KM values for both,
the LPXTG- and Glyn-substrates are exceptionally high (KM=
5.5 mM and 0.14 mM), probably due to the fact that the enzyme
and both substrates are spatially co-localized at the outer
membrane yielding high local concentrations.[20] (v) The high
redox potential of the catalytic Cys126 (1.27 V) makes SrtA
insensitive towards oxidation stress contributing to S. aureus
phagocytotic survival.[21]
systematic scaffold optimization to generate more selective and
less cytotoxic disulfide-based SrtA inhibitors (Fig 1C).
Results and Discussion
Synthesis of the inhibitors
Disulfanylbenzamides were synthesized based on a known
procedure for asymmetric disulfides (Scheme 1).[38] Commercially
available sulfanylbenzoic acids 2a–c were activated at –50 °C
with trichloroisocyanuric acid (TCCA) to form electrophilic sulfenyl
chlorides in situ. The subsequent conversion with nucleophilic
alkyl thiols provided the disulfanylbenzoic acids 3b–g. Since
methyl mercaptan is gaseous at room temperature, for 2-
(methyldisulfanyl)benzoic acid 3a
a different strategy was
employed. S-methyl methanethiosulfonate as thiomethyl-
transferring reagent was utilized to convert thiosalicylic acid 2a to
2-(methyldisulfanyl)benzoic acid 3a.[39] Boc-protected amino
acids 4a–l were coupled to various aromatic and aliphatic amines
(R1) in the presence of 2-(1H-benzotriazole-1-yl)-1,1,3,3-
tetramethylaminium tetrafluoroborate (TBTU) to provide the
inhibitor scaffold precursors 5a–v. The deprotection of the Boc-
group was achieved by treatment with hydrochloric acid. Finally,
the disulfanylbenzoic acids 3a–g were coupled in the presence of
TBTU with the appropriate amine hydrochlorides 6a–v to provide
the desired test compounds 7a–w and 7α–ζ (Scheme 1). Parent
compound 1 was prepared in a one-pot procedure from 3a and
6a. Both reactants were coupled by means of TBTU. The
subsequent treatment with lithium hydroxide at 60 °C yielded the
elimination of methyl mercaptan and provided the
benzisothiazolinone inhibitor 1.[40]
Previous research campaigns investigated competitive-reversible
S. aureus SrtA inhibitors including promising scaffolds such as 2-
morpholinobenzoates[22], thiadiazoles[7,23], 2-phenylthiazoles[24]
,
macrocyclic peptides[25], 2-phenylbenzoxazoles[26], and various
other inhibitors.[8,27,28] However, the most active compounds were
found to be irreversible covalent inhibitors containing an
electrophilic warhead that reacts with the active-site Cys126 of
SrtA.[29–33] While having significant inhibition in the low micromolar
range, they typically exhibit poor target selectivity or are cytotoxic
such as quinones[34], rhodanines[30] or benzisothiazolinones[32]
.
Zhulenkovs et al. solved the NMR-structure of SrtA in complex
with a covalent benzisothiazolinone adduct. Reaction with the
active site Cys126 occurred via ring-opening of the
isothiazolinone moiety yielding a covalent disulfide bond (Fig 1A).
O
O
O
O
S
H
N
a
b
c
N
S
OH
R
OH
OH
S
O
S
S
SH
3b-g
2a-c
3a
1
Cl
O
O
O
d
e
HN Linker
Boc
H3N Linker
HN Linker
Boc
NH
NH
R1
OH
R1
4a-l
5a-v
6a-v
O
S
H
R2
N
Linker
S
f
NH
R1
O
7a-w,
O
O
H
N
N
H
O
g
6a
7

Scheme 1. a) Trichloroisocyanuric acid, R-SH, ACN, –50 °C to rt, 15 min; 56–
69%; b) S-methyl methanethiosulfonate, MeOH, rt, 16 h, 86%; c) (i) 6a, TBTU,
DIPEA, DMF, rt, 16 h (ii) 4M LiOHaq, 60 °C, 4 h, 75%; d) R1-NH2, TBTU, DIPEA,
EtOAc, rt, 72 h, 24–96%; e) 12M HClaq/THF (1:1), rt, 1 h, 74–99%; f) R2-SS-
PhCOOH, TBTU, DIPEA, EtOAc, rt, 16 h, 23–89%; g) o-anisic acid, TBTU,
DIPEA, EtOAc, rt, 72 h, 82%.
Figure 1. A) The reaction of benzisothiazolinones with the active site Cys126 in
S. aureus SrtA. B) NMR-structure of the benzisothiazolinone inhibitor (1)
covalently bound to S. aureus SrtA (pdb: 2MLM). C) Warhead chemotype
exchange and scaffold-hopping strategy to transform the literature-known
inhibitor (1) to disulfanylbenzamides (7a–w and 12a,b).[32]
2
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For the synthesis of the aspartic acid-based inhibitors 12a,b a
different protection group strategy was used (Scheme 2). Briefly,
the Cbz/tBu-protected aspartic acid derivates 8a,b were coupled
by means of TBTU to yield the amides 9a,b. The deprotection of
the N-terminal Cbz-group was achieved by Pd-catalyzed
hydrogenolysis to yield the amines 10a,b. The disulfanylbenzoic
acids 3a or 3d were coupled to the appropriate amines in the
presence of TBTU yielding the inhibitor scaffold precursors 11a,b.
Finally, the deprotection of the tert-butyl ester with trifluoroacetic
acid yielded the compounds 12a,b.
recombinantly expressed S. aureus SrtA[41] and Abz-LPETG-
Dap(Dnp)-OH as substrate. The inhibitors 7a–w and 12a,b were
found to act as time-dependent and irreversible inhibitors.
Exemplarily, the substrate conversion plot in the presence of
inhibitor 12a is showing the time-dependency of inhibition (Fig 2).
The apparent first-order rate constant (kobs) varied hyperbolically
with the concentration of the inhibitor. A limiting value was
approached asymptotically at higher inhibitor concentrations
indicating two-step mechanism kinetics for all inhibitors except for
the fragment-like inhibitor 3a (Ki=367 µM). Benzisothiazolinone 1
was used as a reference inhibitor with a literature reported
IC50=6.11 µM.[32] For time-dependent inhibitors, reporting IC50
values is less suitable since the IC50 is strongly depending on the
incubation time of enzyme and inhibitor. Moreover, the IC50 is
depending on the substrate used for the enzyme assays, its KM
value and its concentration.[42] Therefore, we determined the
maximum inactivation rate kinact, the dissociation constant of the
reversible enzyme-inhibitor complex Ki, and the second-order rate
of inhibition k2nd. For compound 1 a kinact=0.0307 s-1 was found,
which is quite high compared to other targeted covalent
inhibitors,[43,44] but gives reason to its bactericidal effects[32] and
the unspecific cysteine labeling by benzisothiazolinones.[45]
R1
R1
O
HN
HN
O
H
N
H
N
H2N
*
*
*
Cbz
OH
O
Cbz
O
O
O
O
a
b
O
O
9a
9b
8a
8b
(R), R1 = 3-PhOPh
(S), R1 = 1-adamantane
(R)
(S)
10a,b
R1
R1
HN
H
HN
N
H
N
*
O
*
c
d
O
O
S
O
O
S
R2
S
S
R2
O
O
OH
12a,b
(R), R1 = 3-PhOPh, R2 = Et
11b (S), R1 = 1-adamantane, R2 = Me
11a
Scheme 2. a) R1-NH2, TBTU, DIPEA, EtOAc, rt, 72 h, 68–96%; b) H2 (60 psi),
Pd/C, MeOH, rt, 16 h, 92–99%; c) R2-SS-PhCOOH, TBTU, DIPEA, EtOAc, rt,
16 h, 79–83%; d) TFA/DCM, rt, 2 h, 91–99%.
Substrate-based diacyl hydrazide inhibitors (16a,b) were
synthesized starting from proline benzyl ester 13 and Boc-leucine
to yield the dipeptide ester 14. Hydrazinolysis of the benzyl ester
gave the hydrazide 15, which was either TBTU-coupled with 3a to
yield the disulfanylbenzamide 16a or converted with maleic
anhydride to the mono-maleamide 16b (Scheme 3).
H
O
O
OBn
N
Boc
HN
O
O
Boc
HN
O
OBn
NH2
a
b
N
N
HN
15
14
13
O
H
S
O
S
N
Boc
HN
O
N
H
c
N
16a
O
O
H
N
OH
O
Figure 2. A) Fluorometric assay with compound 12a showing time-dependent
enzyme inhibition with hyperbolic substrate conversion plots in the presence of
12a. The fluorescence was recorded for 30 min every 30 s. For clarity, only
every fourth data point is shown. Lines represent non-linear fits for t<10 min. A
magnification plot of the crucial initial phase can be found in SI figure 8. B) kobs
vs. [I] for the determination of inhibition constants (Ki, kinact).
Boc
HN
O
d
N
H
15
N
16b
Scheme 3. a) Boc-Leu-OH, TBTU, DIPEA, EtOAc, rt, 72 h, 56%; b) Hydrazine
hydrate, MeOH, rt, 16 h, 74%; c) 3a, TBTU, DIPEA, EtOAc, rt, 16 h, 16%; d)
Maleic anhydride, AcOH, rt, 16 h, 79%.
To date, only a few studies characterized irreversible SrtA
inhibitors by their inactivation kinetics.[31,46,47] Most of these
inhibitors contained the LPAT sorting-signals but utilized different
electrophilic warheads (diazoketone, chloroketone or vinyl
sulfone). Compared to the parent compound 1, these warheads
Irreversible inhibition of S. aureus sortase A
To evaluate the inhibition potency of the compounds, these were
tested by means of
a fluorometric enzyme assay with
3
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10.1002/cmdc.201900687
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FULL PAPER
were 150–5000-fold less reactive (kinact=0.0002 s-1–6.6⋅10-6 s-1),
however, the vinyl sulfone was shown to gain significant reactivity
above pH 8.00 due to the deprotonation of Cys126 (pKa=9.4[48]).
Since the peptidoglycan is slightly acidic[49] and several Gram-
positive bacteria have trained themselves by evolution for survival
in low-pH environment,[50] we raised the hypothesis that inhibitors
with an optimum effect at pH 8.00 or above are unsuitable to
target SrtA in cellulo. To investigate the pH-dependence of the
novel disulfanylbenzamide warhead in comparison to a common
Michael-acceptor warhead, we designed sorting-signal derived
leucine-proline dipeptide inhibitors, with disulfanylbenzamide
(16a) and mono-maleamide (16b) warheads and characterized
their inhibition kinetics and pH-dependence as presented in table
1 and figure 3.
completely reversible by the addition of 1 mM reducing agents
such as DTT or TCEP. Thus, we postulate that covalent targeting
of the SrtA Cys126 under physiological conditions was much
more effective by disulfanylbenzamides and should be optimized
for potency and selectivity (see next chapter).
Disulfanylbenzamide inhibitor 16a showed irreversible inhibition,
with
a twofold increased affinity compared to the parent
compound 1 (Ki: 34.2 vs. 16.6 µM), but its overall inhibition
potency (k2nd) was threefold lower, mainly due to its reduced
inactivation rate constant (kinact=0.0044 s-1). The dipeptide mono-
maleamide 16b did not show significant inhibition in the standard
fluorometric assay, but a two-hour pre-incubation of the enzyme
and inhibitor prior to substrate addition led to an IC50=20.4 µM.
We hypothesized a covalent inhibition mode of 16b with very slow
inactivation kinetics due to the poor nucleophilicity of Cys126. We
measured the pH-dependence of SrtA inactivation by 16a and
16b at eight different pH-values (6.75–8.50). As shown in figure
3, the general enzymatic activity increased with higher pH-values,
which is in coherence with the reported enzyme optimum at
pH 8.80.[51] The inhibition potency of the mono-maleamide 16b
was overall low but increased significantly above pH 8.00 either
due to the deprotonation of the Cys126 or in situ conversion of
16b to a more electrophilic maleic isoimide, maleimide or
pyridazinedione.[52–54]
Figure 3. pH-dependency on the inhibition potency of the disulfide inhibitor 16a
and the mono-maleamide inhibitor 16b at a final inhibitor concentration of 20 µM.
The residual enzymatic activity was assessed from the initial slope of the
substrate conversion plots. For the inhibitors, no preincubation was used. All
results include the mean value and standard deviations from triplicate
measurements.
Structure-activity relationship
A broad series of disulfanylbenzamides was synthesized to derive
structure-activity-relationship (SAR). In fact, 26 out of 32 analogs
(7a–w, 12a,b and 16a) did inhibit SrtA in the fluorometric enzyme
assay, but with varying potency. Based on the k2nd values, eight
compounds (12a, 7a–g) appeared to be more potent than parent
compound 1, while all investigated disulfanylbenzamides
exhibited at least twofold reduced kinact values ranging from
0.0013 s-1 to 0.0174 s-1 (Table 2). Strikingly, we observed a drop
in SrtA inhibition for most modifications on the glycine amino acid
linker. This finding suggested that substitution at this position is
not well tolerated, except for (R)-aspartic acid in inhibitor 12a,
which we hypothesized to interact with Arg139 (Fig 6).
Considering the k2nd value, 12a was the most potent irreversible
inhibitor (82,136 M-1min-1). Compounds 7α–ζ were found to be
non-binders displaying <30% inhibition at 50 µM. Interestingly,
the (R)-phenylalanine derivate 7p which showed one of the
highest binding affinities (Ki=2.72 µM) had very low inactivation
kinetics (kinact=0.0013 s-1). The (S)-phenylalanine enantiomer 7γ,
however, did not show any inhibition. No significant loss in activity
was observed upon amide-methylation of glycine (7f) to sarcosine
(7h), demonstrating that the activity is not mediated by an in situ
activation to benzisothiazolinones. More pronounced effects
could be associated with the replacement of the amide substituent
(R1), but no clear structural trend was identified. It should be noted
that the two most potent compounds identified here (12a, 7a)
incorporated a 3-phenoxyaniline substituent (R1). Intriguingly, the
change of the disulfide warhead (R2) affected both, affinity (Ki) and
reactivity (kinact). The ortho-configuration (7f) seemed to be
strongly preferred over meta (7u) and para (7α). The alkyl group
(R2) followed the trend: Me~Et>iPr~tBu>EtPh. A methyldisulfanyl-
(7e) to methoxy- (7β) exchange led to a complete loss of inhibition.
We concluded from these findings that both, the warhead’s
positioning and the steric demand were likely to influence the
inhibitor’s potency.
Table 1. Inhibition constants (Ki, kinact, k2nd) of the compounds 1, 3a and 16a,b
for S. aureus SrtA.
Ki
[µM]
kinact
[s-1]
k2nd
Cpd.
1
structure
[M-1min-1]
34.2
±5.87
0.0307
±0.0016
53,860
±6,665
367
±24.4
0.0072
±0.0012
1,177
±119
3a
16.6
±2.88
0.0044
±0.0003
15,904
±1,735
16a
16b
IC50=20.4±1.30 µM[a]
[a] Two-hour preincubation of the enzyme with inhibitor at pH 7.50. All results
include the mean value and standard deviations from triplicate measurements.
In contrast, the disulfide inhibitor 16a showed strong and pH-
independent inhibition, indicating that either a negatively charged
thiolate as a nucleophile is not required for the reaction with
disulfanylbenzamides or that an inhibitor binding-induced
zwitterion formation might occur.[55] Besides the classical SN2-
mechanism involving a thiolate,[56] thiol-disulfide conversion was
reported to proceed via oxidative and radical-mediated pathways,
which might be relevant for the action of disulfanylbenzamides on
SrtA.[57–60] We could also show the inhibition by 16a was
4
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10.1002/cmdc.201900687
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FULL PAPER
Table 2. Inhibition constant values (Ki, kinact, k2nd) of the compounds 7a–ζ and 12a,b for S. aureus SrtA.
Cpd.
R1
HN-linker-C(=O)
R2
Ki [µM]
kinact [s-1]
k2nd [M-1min-1]
12a
7a
7b
7c
7d
7e
7f
3-PhOPh
3-PhOPh
(R)-aspartic acid
glycine
Et (ortho)
Et (ortho)
Et (ortho)
Et (ortho)
Et (ortho)
Me (ortho)
Et (ortho)
Me (ortho)
Et (ortho)
Et (ortho)
Et (ortho)
Et (ortho)
Et (ortho)
tBu (ortho)
Et (ortho)
iPr (ortho)
Et (ortho)
Et (ortho)
Et (ortho)
EtPh (ortho)
Et (ortho)
Me (ortho)
Et (meta)
Et (ortho)
Et (ortho)
Et (para)
10.3±2.30
11.1±2.15
12.6±2.34
8.32±1.31
12.7±2.73
16.2±2.87
14.3±2.87
17.0±3.04
20.5±3.53
1.88±0.32
10.0±1.87
10.9±1.91
14.4±2.31
6.40±0.84
13.5±3.47
8.24±1.12
2.72±0.43
9.60±1.93
5.37±0.83
21.6±3.75
11.0±2.05
40.2±8.73
24.1±5.94
25.3±5.81
13.8±3.68
n.d.
0.0141±0.0007
0.0134±0.0009
0.0151±0.0010
0.0094±0.0005
0.0130±0.0010
0.0160±0.0012
0.0138±0.0011
0.0163±0.0012
0.0174±0.0013
0.0013±0.0001
0.0066±0.0004
0.0068±0.0004
0.0089±0.0005
0.0039±0.0002
0.0082±0.0011
0.0044±0.0002
0.0013±0.0001
0.0028±0.0002
0.0015±0.0001
0.0059±0.0005
0.0026±0.0001
0.0092±0.0011
0.0031±0.0003
0.0029±0.0003
0.0014±0.0001
n.d.
82,136±15,136
72,432±9,581
71,339±8,765
63,873±6,238
61,417±8,957
59,259±6,283
57,902±7,348
57,529±6,285
50,927±5,414
41,489±4,005
39,600±5,217
37,431±4,518
37,083±3,985
36,563±2,984
36,444±4,848
32,039±2,962
28,676±2,397
17,500±2,380
16,760±1,515
16,389±1,509
14,182±2,186
13,731±1,418
7,718±1,242
6,877±924
6,087±1,294
n.d.
1-cyclohexanemethyl
N,N-dicyclohexyl
4-fluorophenyl
1-adamantyl
1-adamantyl
1-adamantyl
1-adamantyl
4-cyclohexanephenyl
1-napthyl
glycine
glycine
glycine
glycine
glycine
7g
7h
7i
(S)-proline
sarcosine
glycine
7j
glycine
7k
7l
1-(thiophene-2-methyl)
2-adamantyl
1-adamantyl
isobutyl
glycine
glycine
7m
7n
7o
7p
7q
7r
glycine
glycine
1-adamantyl
1-adamantyl
1-adamantyl
1-adamantyl
1-adamantyl
1-adamantyl
1-adamantyl
1-adamantyl
1-adamantyl
1-adamantyl
1-adamantyl
1-adamantyl
1-adamantyl
2-phenylethyl
1-adamantyl
1-adamantyl
glycine
(R)-phenylalanine
β-alanine
(R)-proline
glycine
7s
7t
isonipecotic acid
(S)-aspartic acid
glycine
12b
7u
7v
7w
7α
7β
7γ
7δ
7ε
(S)-asparagine
(S)-alanine
glycine
glycine
see Scheme 1
Et (ortho)
Et (ortho)
Et (ortho)
Et (ortho)
n.d.
n.d.
n.d.
(S)-phenylalanine
glycine
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
(R)-leucine
(S)-terleucine
n.d.
n.d.
n.d.
7ζ
n.d.
n.d.
n.d.
n.d.= <30% inhibition after 30 min at 50 µM of final compound concentration. All results include the mean value and standard deviations from triplicate
measurements.
5
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10.1002/cmdc.201900687
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Characterization of covalent protein adducts
protein was destabilized to a lower melting point (47.1 °C). This
agreed with the previously solved NMR-structure (pdb: 2MLM)
showing a higher degree of disorder upon covalent complex
formation (RMSDmean: 1.57 Å vs 3.12 Å; Fig 5B). From a
thermodynamic point of view, the loss of the most energetically
favorable fold might be compensated here by the released
enthalpy of the warhead reaction.
For asymmetric disulfanylbenzamides, two covalent protein
adducts are principally possible: the transfer of a thioethyl
fragment (+60.0 Da) or the transfer of the thiosalicylamide subunit
(+356.2 Da). By using mass spectrometry, we aimed to determine
the mode of inhibitor action (Fig 4). The site-specific modification
of Cys126 was investigated using trypsin digestion and followed
by LC-MS/MS analysis of the tryptic peptides. Three samples
were analyzed: the native SrtA and the inhibitor-labeled protein
either with 7h or with 16b.
Treatment of sortases with S-alkyl methanethiosulfonates led to
the quantitative formation of thioalkylated sortase proteins.[64,65]
Labeling of SrtA with S-ethyl methanethiosulfonate (EMTS) was
performed to generate a purely thioethylated SrtA protein, which
was found to melt slightly higher than the native SrtA protein
(52.7 °C). The protein melt analysis of 7h-labeled SrtA showed a
similar melting point (52.4 °C), thus, these results are
strengthening the hypothesis of a thioethyl-fragment transfer.
For labeling with disulfanylbenzamide 7h, the measurement was
in agreement with the predicted tryptic peptide QLTLITC(SS-
Et)DDYNEK (m/z 808.41), encompassing a thioethylated Cys126
(Fig 4A). MS/MS fragmentation confirmed the correct peptide
sequence (SI Fig 3). In contrast, the untreated protein sample was
lacking this type of modification (Fig 4B). The transfer of a
thioethyl fragment (+60.0 Da) seems to be the predominant mode
of action, but minor modifications with the thiosalicylamide subunit
(+356.2 Da) cannot be completely excluded since corresponding
adducts may be below the detection limit or show poor ionization.
Thiosalicylamide-protein adducts from benzisothiazolinones were
previously linked to haptenization and allergic contact
dermatitis[61,62], thus, we evaluate the absence of this adduct form
as potentially beneficial. The MS/MS analysis for the mono-
maleamide inhibitor 16b (SI Fig 2) indicated that the inhibition
became irreversible due to slow covalent modification of the
Cys126 even at physiological pH. However, from labeling with
400 µM inhibitor, we observed only 27% modified peptide masses,
supporting the fluorometric assay results (Fig 3), which showed
that this reaction is not very efficient at pH 7.50.
Figure 5. Differential scanning fluorimetry to characterize labeled SrtA proteins.
A) Derivative –d(RFU)/dt of the protein denaturing curves to determine the
melting temperature (native SrtA: 50.5 °C, +cpd 1: 47.1 °C, +cpd 7h: 52.4 °C,
+EMTS: 52.7 °C). B) Structural super-positioning of the NMR-structures pdb:
1IJA (apo SrtA) and pdb: 2MLM (+cpd 1) showing differences in overall disorder.
Molecular modeling of the ligand-binding
Figure 4. Mass spectrometric analysis of SrtA labeled with compound 7h
revealed the mode of inhibition for disulfanylbenzamides. A) TIC chromatogram
of the labeled SrtA showed the distinct modification of the active site Cys126.
The peak at m/z=808.41 in the TIC chromatogram (Rt=41.7 min) corresponds
to the thioethylated peptide QLTLITC(SS-Et)DDYNEK. B) Native SrtA did not
show the modification. C) The predominant mode of SrtA inhibition was the
transfer of the thioethyl-fragment to Cys126.
Molecular docking studies were performed by FlexX docking
within the LeadIT worksuite. The docking of the non-covalently
bound inhibitors resulted in a conformation that aligned well with
the benzisothiazolinone inhibitor of the NMR-structure pdb: 2MLM
(Fig 6). When docked into the active site of SrtA 12a inserted its
space-filling 3-phenoxyaniline moiety into the lipophilic sub-
pocket generated by the side chains of Thr122, Ile124 and the
hydrophobic stretch of the β6/7-loop (Val108–Leu111). This might
explain why altering the 3-phenoxyaniline fragment to smaller or
less hydrophobic moieties, such as isobutylamine at the R1
position, reducing the potency up to threefold. However, the
flexible β6/7-loop was previously shown to adapt to
In addition to the mass spectrometric analysis, differential
scanning fluorimetry (DSF) was used to characterize covalently
labeled SrtA proteins (Fig 5A).[63] The native SrtA unfolding
temperature was determined to be 50.5 °C in absence of any
ligand. By reaction with the benzisothiazolinone compound 1, the
equilibrium was pulled toward the unfolded complex and the
6
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10.1002/cmdc.201900687
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substrate/inhibitor binding, and hence, it was difficult to predict the
optimal R1 substituent ab inito by molecular modeling.[66]
On the amino acid linker, both the carbonyl oxygen and the
aspartic acid side chain group were positioned towards the highly
conserved side chain of Arg139, suggesting a potential hydrogen-
bonding network. This could explain the observed reduction in
activity when non-polar functional groups were introduced to the
amino acid position of the inhibitor scaffold. The
disulfanylbenzamide aromatic system was enclosed by π-π
interactions in a sub-pocket comprising His62, Tyr129 and Trp136
positioning the disulfide warhead towards the targeted Cys126.
With meta or para-substituted inhibitors (7u and 7α), no
reasonable docking pose could be generated explaining their low
inhibitory activity. Furthermore, we found the alkyl sulfur atom at
a distance of 3.53 Å to the Cys126, whereas the aromatic sulfur
atom had a distance of 3.90 Å. The closer proximity of the alkyl
sulfur atom supported the findings that most likely the thioethyl
fragment was transferred. The docking calculations suggested
the dithioethyl group to be the largest tolerated R2 substituent due
to the gatekeeping Leu39 residue. Correspondingly, larger alkyl
substituents such as iPr, tBu, EtPh led to a substantial decrease
in inhibition.
Figure 7. Analysis of fibrinogen-mediated adherence inhibition of S. aureus
SA113. Different concentration (5 and 10 µM) of the various inhibitors were
added to the bacterial inoculum and the biofilm was allowed to form overnight
by static grow at 30 °C. The total biofilm was determined. Untreated bacteria
served as control. Graphs represent the results of four independent biological
replicates. Error bars indicate the mean value with the standard deviation.
Inhibition of synthetic substrate incorporation in S. aureus
The activity of SrtA on the surface of S. aureus was determined
by employing a fluorescein-conjugate of the LPXTG-substrate
(FAM-GSLPETGGS-NH2). When added to the cell culture, the
fluorescence label is incorporated into the cell wall, and thus, SrtA
activity can be measured by fluorescence quantification.[25,68] 7f
was selected as the model compound because it showed one of
the best potencies in both the fluorometric assay and the
adherence assays. 7f inhibited the incorporation of the substrate
in a concentration-dependent manner in SA113 cells (Fig 8). For
the USA300 strain, however, only weak inhibition (17% at 100 µM
of 7f) was detected, which is in agreement with the results of the
fibrinogen adherence assays.
The combined data suggest interesting strain-specific effects of
disulfanylbenzamides. Of note, the USA300 strain was described
before as less susceptible to SrtA-targeting compounds. Thus,
rhodanines (which are known as covalent modifiers of SrtA[30]
)
were found to be 40-fold weaker biofilm inhibitors in USA300
compared to SA113.[69] Our data suggest that this insusceptibility
of USA300 might also hold true for the disulfanylbenzamides
tested in this study. The reasons for these differences remain
unknown, but it is conceivable that the two strains might differ
regarding their overall cell wall composition, and that the inhibitors
are therefore unable to reach the SrtA protein in the cell wall of
USA300. However, at the present stage the issue needs further
investigation.
Figure 6. Docking pose of inhibitor 12a in complex with the NMR structure (pdb:
2MLM) highlighting the proposed interaction features upon non-covalent binding.
Effect on fibrinogen-mediated adherence of S. aureus
To determine the effect of SrtA inhibitors on living bacterial cells
we studied the ability of various S. aureus strains to adhere to
fibrinogen-coated surfaces, a prerequisite mechanism for biofilm
formation and the pathogenesis of bloodstream infections.[67] The
treatment of the S. aureus SA113 strain with a set of selected
disulfanylbenzamides efficiently reduced staphylococcal binding
to fibrinogen (Fig 7). Here, we identified compound 7g as the most
potent adherence inhibitor with 66% adherence reduction at a
concentration of 10 µM. The mono-maleamide 16b did not show
a significant reduction of adherence. In contrast to the efficient
reduction of adherence in SA113 cells, we did not observe
significant effects in S. aureus USA300 cells at final inhibitor
concentrations of 10 µM (data not shown).
7
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10.1002/cmdc.201900687
ChemMedChem
FULL PAPER
200 mg/L, indicating that these compounds only affect Gram-
positive bacteria. These results indicate that most
disulfanylbenzamides selectively inhibit SrtA activity and do not
function as antibiotics for S. aureus strains.
Protease inhibition selectivity
Mammalian cathepsin B, L and SrtA are all structurally related to
papain-like proteases, thus, we used their relatedness to study
the selectivity of our inhibitors.[70] In fact, disulfides and
isothiazolinones are known inhibitors of the cathepsin family.[71–73]
In line with previous cytotoxicity studies,[74,75] parent compound 1
and the fragment-based inhibitor 3a showed the weakest
selectivity and up to 100% inhibition at 20 µM on both cathepsins
(Table 4). However, most disulfanylbenzamides displayed no
inhibition of cathepsin L and only moderate inhibition of cathepsin
B, indicating a favorable shift of selectivity.
Figure 8. Inhibition of SrtA-mediated incorporation of a synthetic fluorescence
substrate into the cell wall of S. aureus SA113 and USA300. Different
concentrations of compound 7f (5, 10 and 100 µM) were added to the bacteria
inoculum containing 0.3 mM FAM-GSLPETGGS-NH2 and cells were grown
overnight. After washing and removal of non-covalently bound FAM-substrate,
the fluorescence was measured. Graphs represent the results of three
independent biological replicates and error bars indicate the mean with the
standard deviation.
Table 4. Protease inhibition selectivity of a representative compound set.
Compounds were tested at a final concentration of 20 µM inhibitor.
inhibition [%] at 20 µM
Cpd.
structure
cathepsin
B
cathepsin
L
NS2B/NS3
(ZIKV)
Growth inhibition of bacterial cells
To test the bacterial growth inhibition by the compounds, we
determined the minimum inhibitory concentration (MIC) by a
microbroth dilution assay. The inhibitors which showed effective
adherence reduction (7e, 7f, 7g, 12b, and 16a) were tested for
growth inhibition in two strains of S. aureus (SA113 and USA300)
and one E. coli strain (Table 3). Unlike parent compound 1, which
rapidly killed Staphylococci (MIC=2.92 µM[32]), the addition of
most disulfanylbenzamides to staphylococcal cultures had no
measurable effect on the growth of S. aureus strains at effective
adherence inhibition concentrations (5–10 µM).
98%
±0.4%
79%
±2.2%
1
n.i.
n.i.
100%
±0.5%
93%
±5.6%
3a
98%
±0.1%
14%
±8.9%
18%
±1.1%
7a
60%
±0.5%
12%
±3.1%
7f
7g
7h
n.i.
n.i.
18%
±3.9%
17%
±7.7%
Table 3. The minimal inhibitory concentration of representative compounds on
two strains of S. aureus and one E. coli strain.
69%
±1.7%
23%
±11%
13%
±8.0%
MIC [µM]
Cpd.
Structure
SA113
16.0
USA300
16.0
E. coli
90%
±0.5%
15%
±5.7%
12a
n.i.
7e
7f
>500
n.i.= no inhibition at 20 µM compound concentration. All results include the
mean value and standard deviations from triplicate measurements.
7.74
116
15.4
464
>500
>500
As endopeptidases, both cathepsins prefer large hydrophobic
amino acids in the P2 site.[76] This might explain why the 3-
phenoxyaniline inhibitors (7a and 12a) showed the highest
cathepsin inhibition (90–98%) among all disulfanylbenzamides.
The exopeptidase activity makes cathepsin B unique among
cysteine cathepsins, thus, we hypothesized this could cause the
selectivity of our inhibitors within the cathepsin family and might
favor the binding of the carboxylic acid 12a to the histidine-rich
occluding loop of cathepsin B.[77] The ZIKV NS2B/NS3 protease
is a serine protease and contains only two non-catalytic cysteine
residues, thus as expected, only minimal inhibition by all of our
thiol-reactive compounds was observed.[78] Compound 7g
showed neither relevant inhibition of cathepsins nor the
NS2B/NS3 protease, while it was the most potent inhibitor in the
fibrinogen adherence assay on S. aureus (Fig 7). This is a
7g
12b
16a
222
381
222
381
>500
>500
All results include the median value from three biological replicates with two
technical replicates each.
While the inhibitors 7e and 7f showed medium cell growth
inhibition at higher concentrations (MIC=7–16 µM), all other
inhibitors did not exhibit any effect at <100 µM. The MIC for all
inhibitors tested on E. coli was higher than the upper test limit of
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remarkable improvement compared to the weak selectivity of
parent compound 1.
A
B
Cytotoxicity on HeLa cells
O
O
R1
R1
In vitro cytotoxicity assessment of disulfanylbenzamides was
accomplished on HeLa cells using the MTT-assay. The results
demonstrated the non-toxic properties of disulfanylbenzamides at
relevant treatment concentrations (Table 5). While the parent
compound 1 displayed a CC50 of 87 µM, the cytotoxicity of the
disulfanylbenzamides was between 156 µM and >1000 µM.
(Tables 5).[79]
N
N
O
H
[O]
H
HS
R1
N
S
-H2O
S
S
R3
S
R2
S
S
R3
H
R2
O
O
R1
N
R1
N
S
HS
Table 5. Cytotoxicity (CC50) toward HeLa cells and srtA inhibition constant
values (Ki) of representative compounds.
Scheme 4. Hypothesis for
a
proposed metabolic conversion of
disulfanylbenzamides to benzisothiazolinones in adaption to Nikolayevskiy et
al.[81] A) Transfer of the thioalkyl fragment (R2) to cellular thiols (R3) leaves the
2-mercaptobenzamide which can be metabolized to benzisothiazolinones. B)
By means of the N-methylation, this metabolic conversion is blocked.
Cpd.
structure
CC50 [µM]
Ki [µM]
87
±2.8
34.2
±5.87
1
409
±3.9
367
±24.4
In this case, the N-methylation of 7h prevented the conversion to
the benzisothiazolinone 1 (Scheme 4b). A recent metabolism
study suggested the thermodynamics of 2-sulfanylbenzamides’
3a
7e
7f
316
±31
16.2
±2.87
metabolism having a strong effect on its biological activity.[81]
A
metabolic involvement is supported by the fact that only prolonged
cellular incubation times (48 h) with all disulfanylbenzamides lead
to significant cytotoxic effects.
156
±8.5
14.3
±2.87
253
±67
17.0
±3.04
7g
7h
7m
Conclusions
20.5
±3.53
>1000
>1000
Based on a warhead chemotype transformation strategy, we
discovered a novel class of small-molecule SrtA inhibitors. We
established the structure-activity relationship of a broad series of
substituted disulfanylbenzamides and defined the structural
requirements for efficient SrtA inhibition. The choice of a warhead
for irreversible inhibitors was guided by the particular biochemical
properties of the catalytic Cys126. We concluded from our
findings that covalent targeting is much more effective by
disulfanylbenzamides than by conventional Michael-acceptor
warheads. The pH-independent transfer of a thioethyl fragment
(+60.0 Da) was found to be the predominant mode of action for
SrtA inhibition. While showing low mammalian cytotoxicity
(CC50=253 µM), weak bacterial growth inhibition (MIC=116 µM),
and low off-target protease inhibition, compound 7g was the most
effective inhibitor in diminishing S. aureus fibrinogen adherence
(–66% at 10 µM). Therefore, we concluded that, as a lead
structure, compound 7g should be investigated in further studies.
The selectivity differences in adherence inhibition between the
S. aureus strains SA113 and USA300 should also be addressed
in further studies, just as the potential of any bacterial resistance
development towards disulfanylbenzamides.
6.40
±0.84
762
±137
8.24
±1.12
7o
331
±36
10.3
±2.3
12a
All results include the mean value and standard deviations from triplicate
measurements.
Compounds 7f and 7h differed only by the N-methylation of the
amide bond. The affinity (Ki) and reactivity (kinact) of both inhibitors
did not deviate significantly, but the methylated substance 7h
showed reduced cytotoxicity by a factor of >8. The metabolic
conversion
benzisothiazolinones could be
cytotoxicity behavior (Scheme 4).[80,81]
of
substituted
disulfanylbenzamides
cause for this different
to
a
9
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Differential scanning fluorimetry: Thermal shift assays were conducted
in triplicate using a C1000/CFX384 qPCR system (Bio-Rad, Hercules,
California) using the FRET channel and contained SrtA (2.2 µg), the
respective test compound (50 μM) and Sypro Orange (5×) in 25 µL of
assay buffer (50 mM Tris, 150 mM NaCl, 5 mM CaCl2, pH 7.50). The
samples were heated at 0.5 °C/s, from 25 to 75 °C. The fluorescence
intensity was plotted as a function of the temperature. The melting point
was given by the inflection point of the fluorescence curve as calculated
by the High-Precision-Melt software (Bio-Rad).
Experimental Section
Synthesis: Protocols for the synthesis of all final products and
intermediates with their respective analytical data can be found in the SI.
Protein expression and purification: Expression of the S. aureus SrtA
was performed as described previously.[41] E. coli strain BL21-Gold (DE3)
cells (Agilent Technologies, Santa Clara, California) were transformed with
a pET23b expression construct and grown in LB medium containing
100 µM ampicillin at 37 °C to an OD600 of ~0.7. Expression was induced
with 1 mM isopropyl-D-thiogalactoside (IPTG) for 16 h at 20 °C. After
harvesting, cells were resuspended in lysis buffer (20 mM Tris-HCl, pH 6.9,
300 mM NaCl, 0.1% Triton X-100, RNase, DNase, lysozyme) and lysed by
sonication (Sonoplus, Bandelin, Berlin, Germany). The lysate was cleared
by centrifugation (45 min at 15 krpm) and the protein was purified from the
supernatant by IMAC (HisTrap HP 5 mL column, GE Healthcare, Chicago,
Illinois). Eluted proteins were subsequently subjected to a gel-filtration step
(HiLoad 16/60 Superdex 200 column, GE Healthcare) and eluted in the
storage buffer (20 mM Tris-HCl, pH 7.50, 150 mM NaCl, 5 mM CaCl2).
Purified proteins were concentrated, shock frozen in liquid nitrogen and
stored at –80 °C until further usage. Throughout all steps, protein
concentrations were measured via absorbance at 280 nm and sample
purity was assessed via SDS-PAGE.
Cell viability assay (HeLa Cells): Cell culturing was performed in a
humidified incubator at 37 °C with 5% CO2 atmosphere. HeLa cells were
grown in cell culture flasks according to standard protocols (Dulbecco’s
Modified Eagle Medium [DMEM], 10% (v/v) fetal calf serum, 1% pyruvate,
and 1% penicillin-streptomycin) and seeded to 96-well microplates at a
concentration of 15,000 cells in a volume of 100 μL of DMEM. Inhibitors
were dissolved at a concentration of 7.8–250 µg/mL in DMEM containing
DMSO (0.08%–2.5%) and added in triplicates to the HeLa cells. Negative
inhibition control was performed by mock treatment with DMEM with
DMSO in the same concentration as the compound solutions were used.
After an incubation time of 48 h (37 °C, 5% CO2) a solution of 3-(4,5-
dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) in
DMEM (40 μL, 3.0 mg/mL) was added directly to each well and the plate
was incubated for additional 20 min. The medium was aspirated and
replaced by 200 μL of DMSO and 25 μL of glycine buffer (0.1 M glycine,
0.1 M NaCl, pH 10.5). After shaking for 20 min, the absorbance was
measured at 595 nm using an Infinite M200 Pro plate reader (Tecan). The
background at 670 nm and the absorbance of the compounds at the same
wavelength was subtracted from the data obtained from the first readout.
Cell viability was normalized to the absorbance measured from DMSO-
DMEM threated cells.
Inhibition of sortase A: Inhibition of SrtA-catalyzed in vitro
transpeptidation was performed as described previously.[20,82] Briefly, the
recombinantly expressed SrtA (final concentration: 1 µM) was incubated
in assay buffer (50 mM Tris, 150 mM NaCl, 5 mM CaCl2, pH 7.50) with
25 µM of the FRET-pair substrate Abz-LPETG-Dap(Dnp)-OH (Genscript,
Piscataway, New Jersey) and 0.5 mM tetraglycine (Sigma Aldrich, St.
Louis, Missouri). Inhibitors were added from DMSO stocks. Negative
inhibition control was performed by mock treatment with DMSO. Reactions
were initiated by addition of SrtA and monitored for 30 min at 25 °C in an
Infinite M200 Pro plate reader with λex 320 nm/λem 430 nm (Tecan,
Männedorf, Switzerland). Three technical replicates were carried out for
each inhibitor in black flat-bottom 96-well plates (Greiner bio-one,
Kremsmünster, Austria). The enzyme kinetics were analyzed as described
previously.[83] To determine first-order inactivation rate constants (kobs) for
Bacterial growth inhibition: The MIC of different inhibitors was
determined against S. aureus USA300[86], SA113[87] and a laboratory strain
of E. coli using the microbroth dilution assay according to standard
protocols in 96-well, polystyrene tissue culture plates (Greiner Bio-One,
Cellstar, F-form). The MIC was determined as the concentration of the
inhibitor where the lowest OD595 values were recorded with a Tecan Infinite
200Pro (Tecan).
the irreversible inhibition, progress curves were analyzed by nonlinear
ꢀ
[
푃
]
(
1 −
regression analysis (t=0–10 min) using the equation: 퐹 =
∙ꢆ
ꢃꢄꢅ
)
푒ꢁꢂ
+ 표푓푓푠푒푡 . Fitting of the kobs values against the inhibitor
S. aureus adherence assay: S. aureus adherence was tested in 96-well,
polystyrene tissue culture plates (Greiner Bio-One, Cellstar, F-form) as
previously described[88] with the following modifications. Before starting the
experiment, the plates were coated with fibrinogen.[89] Fibrinogen from
human plasma (Sigma Aldrich) was dissolved in NaCl solution (0.9%) to
10 mg/mL. A fibrinogen solution of 100 µg/mL was prepared in PBS and
100 µL were dispensed into each well of the plate. After sealing, the plate
was incubated at 4 °C overnight to allow fibrinogen coating of the well. The
next day, the fibrinogen solution was aspirated. Bacterial strains
(OD600∼0.05 in tryptic soy broth) were incubated under static conditions in
the presence of different dilutions of inhibitors in 1.6% DMSO at 30 °C for
18 h. The next day, the planktonic bacteria were discarded, the plates were
rinsed twice with PBS (1×) and the biofilm was heat-fixed at 65 °C for 1 h.
Plates were stained with 10 mg/mL crystal violet for 2 min, washed twice
with double-distilled water before measuring the absorbance at OD492 with
an ELISA plate reader (Multiskan Ascent, Thermo Fisher Scientific,
Waltham, Massachusetts).
[ ]
concentrations to the hyperbolic equation 푘ꢇꢈꢉ = (푘ꢊꢋꢌꢍꢆ 퐼 )/(퐾ꢎꢌꢏꢏ + [퐼])
gave the individual values of KIapp and kinact. Progress curves and kobs vs
[I] plots of all active compounds can be found in the SI figures 5–7. KIapp
values were corrected to the zero-substrate concentration by the Cheng-
[
Prusoff equation 퐾 = 퐾ꢎꢌꢏꢏ/(1 + ] ). For [S]<<KM we assumed KIapp = Ki.
ꢐ
ꢊ
ꢑ
ꢒ
Protease inhibition selectivity: Fluorometric assays of the ZIKV
NS2B/NS3 protease were performed as described previously.[84] The
assay was carried out in triplicates at 25 °C in assay buffer (50 mM Tris,
pH 9.0, 20% glycerol and 1 mM CHAPS). 100 µM Boc-GRR-AMC
(Bachem, Bubendorf, Switzerland) was used as a substrate on a Tecan
Infinite M200 Pro plate reader (λex 380 nm/λem 460 nm). Fluorometric
assays for cathepsin B and cathepsin L (Calbiochem, Merck Millipore,
Burlington, Massachusetts) were performed as described previously.[85]
Cbz-Phe-Arg-AMC was used as substrate (80 μM for cathepsin B, 5 μM
for cathepsin L) in assay buffer (20 mM Tris, pH 6.0, 5 mM EDTA, 200 mM
NaCl, 0.005% Brij).
Incorporation of synthetic SrtA substrates on S. aureus: The FAM-
GSLPETGGS-NH2 substrate was synthesized using a 3D-printed solid-
phase peptide synthesizer[90] (detailed procedure in the SI). The
incorporation of a synthetic substrate on the S. aureus cell wall was
conducted as described previously with minor modifications.[25] USA300
and SA113 were grown in tryptic soy broth medium in the presence of
0.3 mM FAM-GSLPETGGS-NH2 and different concentrations of
compound 7f. After 15 h, cells (OD600~8) were harvested from all cultures
in a final volume of 500 µL and washed with cold PBS (1×). Non-covalently
bound substrate was removed by treatment with 5% SDS for 5 min at
Protein mass spectrometry: An S. aureus SrtA stock solution (3.8 μL,
760ꢀµM) was diluted in 500 μL enzyme dilution buffer (50 mM Tris, 150 mM
NaCl, 5 mM CaCl2, pH 7.50). The compounds 7h and 16b were dissolved
in DMSO to generate stock solutions. Inhibitors were added to SrtA at a
final concentration of 400 μM and were allowed to react for 1 h at room
temperature. Samples were stored until MS analysis at –20 °C. The
detailed procedure for the proteolytic digestion and the LC/MS can be
found in the SI.
10
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60 °C. Cells were washed twice with cold PBS and then suspended in
200 µL PBS. The fluorescence of incorporated substrate was measured
with an Infinite M200 Pro plate reader (λex 485 nm/λem 535 nm).
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Entry for the Table of Contents
Benzisothiazolinone to disulfanylbenzamide warhead chemotype transformation and systematic activity relationship uncovered novel
S. aureus sortase A inhibitors with potent inhibition of fibrinogen attachment. The transfer of a thioalkyl fragment to the Cys126 was
identified as the mode of action. Warhead reactivity fine-tuning yielded inhibitors with selectivity over other cysteine proteases.
13
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