Pyrrolomycins as antimicrobial agents. Microwave-assisted organic synthesis and insights into their antimicrobial mechanism of action

Article abstract of DOI:10.1016/j.bmc.2019.01.010

New compounds able to counteract staphylococcal biofilm formation are needed. In this study we investigate the mechanism of action of pyrrolomycins, whose potential as antimicrobial agents has been demonstrated. We performed a new efficient and easy metho

Full text of DOI:10.1016/j.bmc.2019.01.010

Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Pyrrolomycins as antimicrobial agents. Microwave-assisted organic
synthesis and insights into their antimicrobial mechanism of action
⁎
⁎
Maria Valeria Raimondia,1, Roberta Listrob,1, Maria Grazia Cusimanoa, , Mery La Francaa,
,
Teresa Faddettaa, Giuseppe Galloa, Domenico Schillacia, Simona Collinab, Ainars Leonchiksc,
Giampaolo Baronea
a Department of Biological, Chemical and Pharmaceutical Sciences and Technologies, (STEBICEF), University of Palermo, via Archirafi 32, 90123 Palermo, Italy
b Drug Sciences Department, Medicinal Chemistry and Pharmaceutical Technology Section, University of Pavia, via Taramelli 12, 27100 Pavia, Italy
c APP Latvian Biomedical Research and Study Centre (BMC), Rātsupītes iela 1, LV-1067 Rīga, Latvia
A R T I C L E I N F O
A B S T R A C T
Keywords:
New compounds able to counteract staphylococcal biofilm formation are needed. In this study we investigate the
mechanism of action of pyrrolomycins, whose potential as antimicrobial agents has been demonstrated. We
performed a new efficient and easy method to use microwave organic synthesis suitable for obtaining pyrro-
lomycins in good yields and in suitable amount for their in vitro in-depth investigation. We evaluate the in-
hibitory activity towards Sortase A (SrtA), a transpeptidase responsible for covalent anchoring in Gram-positive
peptidoglycan of many surface proteins involved in adhesion and in biofilm formation. All compounds show a
good inhibitory activity toward SrtA, having IC50 values ranging from 130 to 300 µM comparable to berberine
hydrochloride. Of note compound 1d shows a good affinity in docking experiment to SrtA and exhibits the
highest capability to interfere with biofilm formation of S. aureus showing an IC50 of 3.4 nM. This compound is
also effective in altering S. aureus murein hydrolase activity that is known to be responsible for degradation,
turnover, and maturation of bacterial peptidoglycan and involved in the initial stages of S. aureus biofilm for-
mation.
Antimicrobial resistance
Pyrrolomycins
Sortase A
Staphylococcus aureus
In-silico docking studies
MAOS
Pharmacokinetics studies
Murein hydrolase activity
1. Introduction
Surveillance System (GLASS) in order to deal with growing emergency
of super bacteria that do not respond to standard antibiotics. Using the
Antimicrobial resistance (AMR) is one of the causes for hundreds of
thousands of deaths worldwide every year. The new report of the World
Health Organization (WHO) in January 2018 deals with the dramatic
phenomenon of the antibiotic resistance, and a group of pathogens
including Carbapenem-resistant Enterobacteriaceae (CRE), Acinetobacter
baumannii, Pseudomonas aeruginosa, Salmonella, Escherichia coli,
Klebsiella pneumoniae, Staphylococcus aureus etc. are listed on the basis
of a priority (critical, high, medium) regarding AMR.1 Already in Oc-
tober 2015, the WHO launched the new Global Anti-microbiotic
data provided by GLASS, the WHO detected more than 500,000 infec-
tion cases in 22 countries.2
Adhesion to the host tissues is a step of central importance in bac-
terial pathogenesis,3 and novel antimicrobial compounds targeting such
mechanism could also be useful to inhibit the formation of biofilms
intrinsically resistant to conventional antibiotics. Sortase A (SrtA) is an
enzyme of 206 amino acids present in Gram-positive bacteria, which
includes an amino-terminal zone (with nonpolar residues) and a cata-
lytic domain for the transpeptidation reaction. Its action consists in
Abbreviations: MAOS, microwave-assisted organic synthesis; SrtA, Sortase A; AMR, antimicrobial resistance; WHO, World Health Organization; GLASS, Global Anti-
microbiotic Surveillance System; MSCRAMMs, Microbial Surface Components Recognizing Adhesive Matrix Molecules; FnbpA, fibronectin binding protein A; FnbpB,
fibronectin binding protein B; ClfA and ClfB, clumping factors; Can, collagen-binding protein; NBS, N-bromosuccinimide; NCS, N-chlorosuccinimide; MW, micro-
wave; ADME, absorption distribution metabolism and excretion; DMSO, dimethyl sulfoxide
⁎ Corresponding authors.
E-mail addresses: mariavaleria.raimondi@unipa.it (M.V. Raimondi), roberta.listro01@universitadipavia.it (R. Listro),
mariagrazia.cusimano@unipa.it (M.G. Cusimano), mery.lafranca@unipa.it (M. La Franca), teresa.faddetta@unipa.it (T. Faddetta),
giuseppe.gallo@unipa.it (G. Gallo), domenico.schillaci@unipa.it (D. Schillaci), simona.collina@unipv.it (S. Collina), ainleo@biomed.lu.lv (A. Leonchiks),
giampaolo.barone@unipa.it (G. Barone).
1 These authors contributed equally to this work.
https://doi.org/10.1016/j.bmc.2019.01.010
Received 7 November 2018; Received in revised form 10 January 2019; Accepted 13 January 2019
0968-0896/©2019PublishedbyElsevierLtd.
Pleasecitethisarticleas:Raimondi,M.V.,Bioorganic&MedicinalChemistry,https://doi.org/10.1016/j.bmc.2019.01.010

M.V. Raimondi et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
covalently linking a number of proteins – the Microbial Surface Com-
ponents Recognizing Adhesive Matrix Molecules (MSCRAMMs) – to
peptidoglycan; these are cell wall-associated proteins that have a direct
role in the first stage of pathogenesis and biofilm formation.
MSCRAMMs are also relevant virulence factors of S. aureus because they
are involved in pathogenic processes such as evasion of immune de-
fense – due to protein A (SpA) – adhesion to host tissues, such as two
fibronectin binding proteins (FnbpA and FnbpB), two clumping factors
(ClfA and ClfB) and a collagen-binding protein (Cna). For some of these
proteins a key role in biofilm formation has been reported.5–9 There-
fore, SrtA is considered as a good target for the design of new anti-
bacterial drugs, or in general against the bacterial adhesion and the
biofilm formation.4 In 2000, Muzmanian et al. studied knockout mu-
tations of SrtA in S. aureus, and they focused their attention on the
different modifications of the expression of the protein with LPxTG
domain.5–9 The loss of activity of SrtA with consequent attenuation of
virulence are evident in S. aureus and also in other important Gram-
positive pathogens as Listeria monocytogenes, Streptococcus pneumoniae,
Streptococcus mutans.10–13
2. Results
2.1. Chemistry
Pyrrolomycins 1d,e,h,i, were already obtained by authors using N-
bromosuccinimide (NBS) or N-chlorosuccinimide (NCS) as source of
halogen atoms. All reactions were conducted under magnetic stirring
for 24 h and at room temperature.22,23 However, this approach is labor
intensive and therefore, a different strategy has been explored to
identify a convenient procedure in terms of cost and timing. The main
criticism is related to the formation of the intermediates 4,5,6. Indeed,
the reaction is not selective, it gives rise to complex reaction mixtures
and, as a consequence, subsequent chromatographic steps are needed to
isolate the compounds of our interest.
We then revised the reaction conditions, experimenting the
Microwave-Assisted Organic Synthesis (MAOS). So, we have directly
verified that MAOS has numerous benefits, including faster chemistry
(it reduces reaction times from days or hours to minutes), greater ver-
satility and formation of cleaner products, compared to conventional
synthetic methods.29 Moreover, starting from the observation that the
presence of acyl group influences the reactivity of the free positions of
the pyrrole, we postulated that the halogenation degree can be appro-
priately modulated. Accordingly, we performed the reaction by mi-
crowave heating, using only one reagent equivalent at a time, isolating
the product of interest and then moving on to the subsequent halo-
genation. This new protocol allowed to increase the yield of O-methy-
lated pyrrolomycins 4b,c, 5c,d and 6b,c up to 93%, and to synthesize
the new compounds 4a,d, 5b,e and 6d,e (Table 1). In details, com-
pounds 4a-d and 5a-e were obtained by irradiation with a microwave
power of 60 W at 90 °C for 5 min. To introduce the halogen in 3-position
on the pyrrolic nucleus (compounds 6a-e) it was necessary to carry out
the reaction with four cycles of 15 min each of microwave power of
60 W at 90 °C. The final reaction step, i.e. the O-demethylation, was
conducted as previously described, with slight modification, using a
large excess of AlCl3 at room temperature, allowing to obtain 1d,e,h,i,
C and F2a with very good yields (Scheme 1).22,23
For several years, pyrrolomycins have attracted the attention of the
scientific community for their antibacterial properties and for their
capability to inhibit the formation of biofilms responsible for the re-
sistance to conventional antibiotics.14–20 Pyrrolomycins are polihalo-
genated compounds (Fig. 1) isolated from the fermentation broth of
Actinosporangium and Streptomyces species. They are active against
Gram-positive and a few Gram-negative bacterial strains as Pseudo-
monas aeruginosa.12–14,21–27
Natural pyrrolomycins, produced by terrestrial and marine micro-
organisms, are generally active against all preformed staphylococcal
biofilms, with percent of inhibition above 60% at the concentration of
1.5 µg/mL27 Nevertheless, their mechanism of action is still unknown.
We have been active in this field for several years. To discover new
active antibacterial agents, we prepared several synthetic pyrrolomy-
cins (Fig. 2), characterized by the presence of chlorine or bromine
atoms on the pyrrolomycin scaffold. We observed that their anti-
bacterial property is closely linked to their substitution pattern and that
their biological activity is strictly correlated to the different halogena-
tion degree.12,13,21–23,26–28 Particularly, some of our synthetic pyrrolo-
mycin derivatives showed huge antibacterial activity against S. aureus,
with MIC in the range of 0.003–0.016 µM.12,13,21–23,26
Analytical and spectroscopic measurements were used to determine
the molecular structures of all compounds and the identity of all
compounds was confirmed by comparison with spectroscopic literature
data.12,14,19,22-24
Starting from our previous findings, herein we focus on two natural
pyrrolomycins (C and F2a) and on our most promising synthetic pyr-
rolomycins (compounds 1d,e,h,i, see Scheme 1). In detail, we have
optimized and scaled-up the synthetic procedure to dispose of an
amount of 1d,e,h,i suitable for in-depth biological investigation. The
development of a quick and easy to use methodology for synthesizing
pyrrolomycins has a great relevance from a medicinal chemistry
standpoint, since the extraction yield from culture broth is slow,
moreover, their production is expensive. Indeed, after several con-
trolled pH extractions and chromatographic purifications steps, only
2.5 mg/L of pyrrolomycins from culture broth can be obtained.14,18–20
As subsequent step of the research, in order to investigate whether
pyrrolmycins C, F2 and 1d,e,h,i would bind to the active site of SrtA,
we performed in silico docking studies using the crystal structure of
SrtA and hypothesized the interactions of compounds with the enzyme
at the molecular level. Afterwards, we evaluated in vitro their ability to
interact with SrtA, to inhibit biofilm formation of reference staphylo-
coccal strains and P. aeruginosa. For the most interesting compound, the
effect on murein hydrolase activity of S. aureus extracellular proteins
was also evaluated. As a last step of our work we studied the ‘drug-
likeness’ of pyrrolomycins C, F2 and 1d,e,h,i.
Going into details on new pyrrolomycins 4a,d, 5b,e and 6d,e, in 1H
NMR spectra, all compounds showed a singlet attributable to a meth-
oxylic group in the range of 3.72–3.77 δ, signals in the range of
6.51–7.87 δ for the aromatic protons and a broad singlet for the pyrrolic
NH in the range of 12.41–13.56 δ, it was verified also by IR spectra that
showed a broad band in the range of 3188–3250 cm−1
elemental analysis confirmed their structures.
.
13C NMR and
It is interesting to note that when compounds 3a-c react with one
molar equivalent of NCS or NBS, the aromatic signal of 3-proton in
pyrrolic nucleus shows an unusual chemical shift. In fact, compounds
4a-d exhibit two doublets in the range of 6.51–6.69 δ for 3-H and
7.33–7.43 δ for 5-H. The 3-proton is more shielded than that awaited
from 2-carbonyl pyrroles, probably due to the anisotropic effect of
phenyl ring, twisted with respect to the pyrrolic ring. According to what
previously observed by Hodge and Rickards,25 the introduction of
chlorine (or bromine) substituents into pyrrole rings causes only minor
shifts in the resonance frequencies of the remaining hydrogens.
2.2. Molecular modelling
In order to predict whether pyrrolomycins are able to interact with
the active site of SrtA, we performed docking studies using the crystal
This paper describes the results of our studies.
2

M.V. Raimondi et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
Cl
Cl
NO2
Cl
Cl
Cl
NO2
Cl
Cl
Cl
Cl
Cl
N
H
N
H
N
H
OH
OH
O
A
B
C
Cl
Cl
NO2
Cl
NO2
Cl
Cl
Cl
Cl
N
H
Cl
Cl
Cl
Cl
N
H
N
H
HO
OH
Cl
OH
OH
O
Cl
D
E
G
Cl
Cl
Cl
Cl
Cl
Cl
NO2
Cl
Cl
Cl
Cl
Cl
Cl
N
H
N
H
N
H
OCH3
OCH3
Br
O
OCH3
O
OH
J
I
H
Br
Cl
Cl
Br
Br
Br
Cl
NO2
Br
Br
Cl
Cl
N
H
N
H
N
H
OH
OH
O
O
O
O
F2a
F1
Dioxapyrrolomycin
Br
Br
Cl
Br
Br
Br
Cl
Cl
N
N
H
OH
OH
H
O
F3
O
F2b
Fig. 1. Structure of natural pyrrolomycins.
2.3. Biological investigation
structure of PDB-ID: 1T2W.30 The results obtained suggested that all
considered compounds have a similar affinity toward the chain A of
SrtA. The picture of the binding complex involving compound 1d is
shown in Fig. 3. In the same figure, the amino acidic residues inter-
acting with the docked molecule are also shown in the insert. In all
cases, a hydrogen bond between SER116 and the carbonyl oxygen of
pyrrolomycin is also present. Data relative to the other pyrrolomycin
compounds are reported in the SI. The studied pyrrolomycins have
score values between −6.0 and −6.4 kcal/mol (see Table 3). Given all
the above, the designed compounds were considered suitable for the
purpose of this study. The analysis of Fig. 3 and of Figs. S1–S6 in the
Supplementary Information show that all considered pyrrolomycins are
able to similarly bind SrtA, essentially by the combination of both hy-
drophobic and H-bond interactions.
After synthesis and chemical characterization, compounds 1d,e,h,i
together with pyrrolomycins C and F2a were subjected to biological
assays, evaluating their ability to inhibit the enzyme SrtA and the
biofilm formation of S. aureus ATCC 25923 and of P. aeruginosa ATCC
15442.31
For testing the SrtA inhibitory activity, the assay checking enzy-
matic hydrolysis of Srt A FRET substrate analogue dabcyl-QALPET-
GEE-edans (Table 2) was employed. All compounds resulted active,
with IC50 values in the range of 130–300 µM. Synthetic pyrrolomycins
resulted more effective than natural ones with an IC50 comparable to
that of the standard berberine hydrochloride (IC50 = 120 µM).
Encouraged from these results, we evaluated the capability of pyr-
rolomycins to inhibit staphylococcal biofilm formation, since the
3

M.V. Raimondi et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
R4
Table 1
R2
R3
Yield for compounds 4a-d, 5a-e, 6a-e, C, F2a, 1d,e,h,i.
Compound R1 R2 R3 R4 R5 Previous protocol Yield %[Ref.] MW Yield %
R4
R2
R3
X
R1
R5
N
H
4a
4b
4c
4d
5a
5b
5c
5d
5e
6a
6b
6c
6d
6e
C
H
H
H
H
Br
Br
Cl
Cl
H
H
H
H
H
H
H
H
H
Br Br
–
80
83
79
75
89
83
83
85
76
93
90
90
91
82
–
R1
O
Cl Cl 7122
Cl Cl 6122
N
H
HO
OH
O
R5
Br
H
–
Br Br
Br Br
Cl Cl
Cl Br
Br Cl
Br Br 9723
2a-d
1a-i
Cl Cl
–
Cl Cl 6822
Cl Cl 8122
Compound R1 R2 R3 R4 R5
X
-
-
-
-
-
-
-
-
Br
H
–
Br Br
Br Br
H
H
H
H
H
H
Br
Br
1a
1b
1c
1d
1e
1f
1g
1h
1i
2a
2b
2c
2d
Br Br Br Br Br 9823
Br Br Br Cl Cl 6822
Cl Cl Br Cl Cl 7422
Br Br Br
Br Br Br Br Br
Br Br Br Cl Cl
Br Cl Br Cl Cl
Cl Br Br Cl Cl
Cl Cl Br Cl Cl
Cl Br Cl Cl Cl
Cl Br Cl Cl Cl
–
–
Br Cl Br Br
H
Cl Cl
H
Cl Cl 9523
F2a
1d
1e
1 h
1i
Br Cl Br Br
H
9423
–
Br Br Br Br Br 9112
Br Br Br Cl Cl 8922
Cl Cl Br Cl Cl 9222
Cl Br Cl Cl Cl 8912
–
-
–
Br Br
Br Br Br Br Br CH2
Br Br Br Br CO
Br Br Br Br Br CO
H
Br Br CH2
–
–
H
Fig. 2. Synthetic pyrrolomycins previously prepared by us.
All tested pyrrolomycins were active in preventing S. aureus ATCC
25923 from producing biofilm at IC50 values ranging from 3.4 nM to
20.3 µM, in particular the compound 1d showed the best IC50 value
(3.4 nM) against S. aureus. The pyrrolomycins proved to be selective
towards staphylococcal biofilm formation, as P. aeruginosa biofilm
formation was weakly affected at IC50 concentrations ranging from 18.6
to 235.7 µM. The highest potency against P. aeruginosa was observed for
the compound 1i whose IC50 value was 18.6 µM.
R4
R4
R2
(a)
R5
R5
N
H
N
H
OCH3
OCH3
O
O
3a-c
4a-d
Among all pyrrolomycins, the most active in inhibiting the staphy-
lococcal biofilm were the synthetic pyrrolomycin 1d (IC50 = 3.4 nM)
and the natural pyrrolomycin F2a (IC50 = 8.7 nM), this underlines the
importance of the bromine atoms on phenolic ring.
3a: R4 = R5 = Br
3b: R4 = R5 = Cl
3c: R4 = Br; R5 = H
(a)
Finally, the most performant pyrrolomycins, in terms of inhibition
of biofilm formation (F2a and 1d), were assayed on murein hydrolase
activity of S. aureus extracellular proteins. Murein hydrolases - hydro-
lytic enzymes acting in degradation, turnover and maturation of bac-
terial peptidoglycan - are involved in the initial stages of S. aureus
biofilm formation.32,33 In S. aureus, the most prominent murein hy-
drolase is AtlA. that is synthetized as a 137.5 kDa pro-protein cleaved to
generate different proteolytic species.32–34 Indeed, murein hydrolase
activity was abolished in S. aureus AltA mutants.32–34 Thus, compound
1d and F2a were added to the inoculated growth medium of S. aureus
static cultivations at the concentration of 1 µM (≃0.5 µg/mL), respec-
tively. The addition of the compounds caused up to 80% of inhibition of
biofilm formation in the respect of untreated cultures after 24 h of in-
cubation. Using the extracellular proteins of the spent medium collected
after 24 h of incubation from the treated and untreated S. aureus static
cultivations, the zymography analysis was carried out and it highlights
the activity of a high Mw murein hydrolases (i.e. between 148 and
98 kDa) as a sharp band (Fig. 4a). Furthermore, other appreciable
hydrolytic activities can be observed at lower Mw with a total of 6
hydrolysis bands (Fig. 4a). According to the observed inhibition of
biofilm formation, the activity of the high Mw murein hydrolase (band
1) diminished at least 2-fold in treated cultivations in the respect of
untreated cultivation (Fig. 4b). However, the overall effect of com-
pound 1d and F2a on murein hydrolase activities of S. aureus extra-
cellular proteins was not identical but it revealed a different hydrolytic
pattern. As an example, an increment of murein hydrolytic activity in
correspondence of lower Mw proteins (bands 4, 5 and 6) was observed
for the spent medium of F2a-treated cultivations (Fig. 4b). According to
Sahukhal et al. (2015), this last observation could be ascribed to an
increased proteolytic activity producing different low Mw Alt species.32
Interestingly, the increment of murein hydrolytic activity due to low
R4
R4
R3
R2
R2
(b)
R5
R5
R1
R1
N
H
N
OCH3
OCH3
H
O
O
6a-e
5a-e
(c)
(c)
For pyrrolomycin C
R4
R3
R2
R5
R1
N
H
OH
O
C, F2a, 1d, 1e, 1h, 1i
Scheme 1. Experimental protocol to obtain compounds: 4a-d, 5a-e, 6a-e, C,
F2a, 1d,e,h,i. Reagents and reaction conditions: (a) acetonitrile as solvent,
equimolar amounts of NBS or NCS, MW heating: 90 °C, 60 W, 5 min; (b) acet-
onitrile as solvent, equimolar amounts of NBS or NCS, MW heating: 90 °C, 60 W,
4 cycles of 15 min each; (c) dichloromethane anhydrous as solvent, AlCl3
(30 mmol excess), r.t., magnetic stirrer, 12 h; quenching with a 5% H2SO4 so-
lution.
interference with growth as a biofilm is strictly related to antivirulence
properties of antimicrobial agents. All pyrrolomycins inhibited biofilm
formation with inhibition percentages higher than 50% (IC50 values are
determined and reported in Table 2).
4

M.V. Raimondi et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
Fig. 3. Best pose of pyrrolomycin 1d, one of the derivatives with the highest docking score for segment A of Sortase A. In the insert, the amino acidic residues
interacting with the molecule are shown.
properties of each pyrrolomycin compound (Table 3): (b) percentage of
oral dose absorbed (%OA); (c) partition coefficient (ClogP); (d) loga-
rithm for aqueous solubility (ClogS); (e) serum protein binding (ClogK);
(f) blood–brain barrier permeation as pLog (ClogBB); (g) HERG
K + channel blockage. These parameters are the most representative
features to determine the ADME profile. In Table S1 of Supplementary
Information, the calculated ADME properties of the considered pyrro-
lomycin molecules have been summarized. Almost all parameters taken
into consideration, with the exception of the weakly polar component
of SASA and of the possibility to accept H-bonds, are included within
the range of properties of 95% of known drugs. The results obtained
show that the considered pyrrolomycins are promising drug candidates.
The values of Human Oral Absorption reported in Table S1 are 100%
for all pyrrolomycins considered. This result indicates that these com-
pounds could be orally administered drugs. In general, all parameters
reported in Table S1 show that they all possess optimal pharmacoki-
netic properties. Since the reference values of serum protein binding
constant (logK) are within the range −1.5 – 1.5, the results show that
all the title compounds possess an optimal value of binding to plasmatic
proteins.
Table 2
In vitro activity of pyrrolomycins as SrtA inhibitors and IC50 values (µM) of
tested pyrrolomycins.
SrtA
Biofilm inhibition
inhibition
Compound
IC50 (µM)
S.aureus ATCC
P.aeruginosa ATCC 15442,
25923, IC50 (µM)
IC50 (µM)
C
300
250
140
130
130
160
0.92
235.7
78.9
F2a
1d
1e
1 h
1i
0.0087
0.0034
20.30
0.10
74.8
106.5
170.3
18.6
0.0247
Table 3
Calculated properties of the selected pyrrolomycins.
(c)
(g)
Docking
%OA(b) logP
logS(d)
logK(e) logBB(f) log IC50
score(a)
C
−6.1
100
100
100
100
100
100
4.475
4.256
4.891
4.737
4.626
4.594
−5.068 0.381
−5.419 0.420
−6.292 0.579
−6.083 0.531
−5.679 0.488
−5.650 0.481
0.165
0.119
0.347
0.321
0.306
0.285
−4.524
−4.419
−4.413
−4.364
−4.304
−4.294
F2a −6.0
3. Conclusions
1d
1e
−6.4
−6.4
1 h −6.4
In the present paper, we identified two pyrrolomycins as promising
antimicrobial agents. Of particular interest is compound 1d, for which a
quick, easy to use and scalable synthetic methodology was developed.
Conversely, the extraction procedure of F2a from culture broth is slow
and expensive.
1i
−6.4
(a)
(b)
(c)
(d)
(e)
(f)
Best pose score for docking into SrtA (in kcal/mol).
Percentage of oral dose absorbed.
Partition logaritm for octanol/water.
Logarithm for aqueous solubility.
Serum protein binding.
The results of molecular modelling studies and enzymatic assays
have confirmed the involvement of SrtA in the biological activity of
pyrrolomycins. In particular, the results of the computational in-
vestigations, and the analysis of the binding modes, show that the title
compounds can be promising drug candidates to be used to treat in-
fections. Results on the inhibition of staphylococcal biofilm formation,
coupled with an altered pattern of murein hydrolase activity, have
shown the antivirulence property of pyrrolomycins.
blood–brain barrier permeation as pLog.
HERG K+ channel blockage.
(g)
Mw protein species is associated with increased cell death and a defect
in biofilm maturation.32 Therefore, this analysis showed a common
negative effect on the activity of the high Mw hydrolase (band 1) of
both compounds and, more in general, a compound-specific effect on
the overall murein hydrolase activities that paralleled the inhibition of
biofilm formation.
Lastly, results of in silico drug likeness study evidenced that both
compounds have good ADME properties, thus prompting us to further
studies.
2.4. In silico ADME properties
4. Experimental section
Often, promising potential drugs fail because of unsatisfactory
ADME properties, so we performed studies in silico to predict ADME
and toxicity, starting from the docking complexes (see paragraph 2.2).
Molecular descriptors were computed to predict six physicochemical
4.1. Chemistry
4.1.1. Materials and instruments
Melting points were determined on a Büchi 530 capillary melting
5

M.V. Raimondi et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
Fig. 4. Zymography assay of S. aureus extracellular proteins. Panel a): murein hydrolytic activity was assayed using extracellular proteins of spent medium aliquots
that were collected from static cultivations untreated (A) or treated with 0.1% DMSO (B), 1 µM F2a (C) and 1 µM compound 1d (D). Panel b): murein hydrolase
activity was quantified as lysozyme µg equivalents based on electrophoretic band intensities.
point apparatus and are uncorrected. IR spectra were recorded at room
temperature in Nujol mulls with a Perkin Elmer Infrared 137 E spec-
trometer. 1H NMR (300 MHz) and APT (75 MHz) spectra were recorded
with a Bruker AC-E spectrometer at room temperature in DMSO‑d6,
unless otherwise specified, using tetramethylsilane as internal standard;
chemical shifts (δ) are expressed as ppm values. Microwave reactions
(acetone‑d6): δ 62.1, 113.8, 118.2, 124.3, 127.4, 128.6, 130.7, 131.5,
136.0, 152.8, 180.5, 205.3. IR (cm−1): 3242 (NH), 1619 (CO). Anal.
calc. for C12H9BrClNO2: C, 45.82%; H, 2.88%; N, 4.45%. Found: C,
45.87%; H, 2.93%; N, 4.44%.
4.1.2.1.3. (4,5-dibromo-1H-pyrrol-2-yl)(3,5-dichloro-2-
methoxyphenyl)methanone (5b). White solid, Yield 83%, mp
162–163 °C. 1H NMR: δ 3.71 (s, 3H, OCH3), 6.80 (s, 1H, H-3), 7.51
(d, 1H, H-6ꞌ, Jm = 2.5 Hz), 7.82 (d, 1H, H-4ꞌ, Jm = 2.5 Hz), 13.58 (br s,
1H, NH). 13C NMR (acetone‑d6): δ 62.1, 96.4, 121.6, 127.4, 128.6,
129.0, 130.4, 131.5, 135.4, 152.7, 179.7, 206.4. IR (cm−1): 3188 (NH),
1616 (CO). Anal. calc. for C12H7Br2Cl2NO2: C, 33.68%; H, 1.65%; N,
3.27%. Found: C, 33.78%; H, 1.71%; N, 3.36%.
were performed with an Anton Paar GmbH
- Monowave 300
(Microwave synthesis reactor). Microanalyses (C, H, N) were carried
out with Elemental Vario EL III apparatus and were in agreement with
theoretical values
0.4%. All reactions were monitored by TLC on
precoated aluminium sheets 20 × 20 (0.2 mm Kieselgel 60 G F254,
Merck) and C-18 reverse phase (RP-18 F254, Merck) using UV light at
254 nm for visualization. All reagents and solvents were from Aldrich,
Fluka, Merck, Across or J.T. Baker.
4.1.2.1.4. (5-bromo-4-chloro-1H-pyrrol-2-yl)(5-bromo-2-
methoxyphenyl)methanone (5e). White solid, Yield 76%, mp
134–135 °C. 1H NMR: δ 3.73 (s, 3H, OCH3), 6.58 (s, 1H, H-3), 7.10
(d, 1H, H-3ꞌ, Jo = 8.7 Hz), 7.46 (d, 1H, H-6ꞌ, Jm = 2.5 Hz), 7.63 (dd,
1H, H-4ꞌ, Jo = 8.75 Hz, Jm = 2.45 Hz), 13.28 (br s, 1H, NH). 13C NMR
(acetone‑d6): δ 62.2, 96.4, 113.78, 121.7, 127.5, 128.7, 129.0, 130.5,
131.6, 135.4, 179.8, 206.4. IR (cm−1): 3206 (NH), 1611 (CO). Anal.
calc. for C12H8Br2ClNO2: C, 36.63%; H, 2.05%; N, 3.56%. Found: C,
36.78%; H, 2.13%; N, 3.63%.
4.1.2. Methods
Known pyrrolomycins 1d,e,h,i, C, F2a, 4b,c, 5a,c,d and 6a-c were
prepared and characterized as previously described.12,14,19,22–24
4.1.2.1. General procedure for preparation of substituted (2-
methoxyphenyl-1H-pyrrol-2-yl)methanones (4a,d, 5b,e, 6d,e) under
4.1.2.1.5. (4-bromo-3,5-dichloro-1H-pyrrol-2-yl)(3,5-dichloro-2-
methoxyphenyl)methanone (6d). White solid, Yield 91%, mp
193–194 °C. 1H NMR: δ 3.78 (s, 3H, OCH3), 7.58 (d, 1H, H-6ꞌ,
microwave heating. To
a solution of 1 mmol of 3a-c in 5 mL of
acetonitrile, 1 mmol of crystallized NBS or NCS was added. The
reaction mixture was irradiated with a microwave power of 60 W at
90 °C for 5 min (compounds 4a-d and 5a-e) or 60 W at 90 °C for 4 cycles
of 15 min each (compounds 6a-e). The reaction mixture was evaporated
under reduced pressure and the residue was partitioned between water
(10 mL) and diethyl ether (10 mL) and, subsequently, extracted twice
with diethyl ether (10 mL). The combined extracts were washed with
water, dried over anhydrous sodium sulfate and evaporated. The crude
product was crystallized from ethanol.
J
m = 2.6 Hz), 7.87 (d, 1H, H-4ꞌ, Jm = 2.6 Hz), 13.86 (br s, 1H, NH).
13C NMR (acetone‑d6): δ 61.88, 105.1, 113.6, 121.1, 127.2, 128.8,
129.1, 131.6, 135.5, 152.5, 179.6, 205.4. IR (cm−1): 3201 (NH), 1626
(CO). Anal. calc. for C12H6BrCl4NO2: C, 34.49%; H, 1.45%; N, 3.35%.
Found: C, 34.58%; H, 1.51%; N, 3.43%.
4.1.2.1.6. (5-bromo-2-methoxyphenyl)(3,5-dibromo-4-chloro-1H-
pyrrol-2-yl)methanone (6e). White solid, Yield 82%, mp 184–185 °C. 1
H
NMR: δ 3.73 (s, 3H, OCH3), 7.10 (d, 1H, H-3ꞌ, Jo = 9.15 Hz), 7.46 (d,
1H, H-6ꞌ, Jm = 2.5 Hz), 7.67 (dd, 1H, H-4ꞌ, Jo = 9.2 Hz, Jm = 2.5 Hz),
13.67 (br s, 1H, NH). 13C NMR (acetone‑d6): δ 55.6, 103.8, 106.6,
107.5, 112.1, 113.7, 130.1, 130.9, 134.4, 156.6, 180.7, 205.2. IR
(cm−1): 3207 (NH), 1611 (CO). Anal. calc. for C12H7Br3ClNO2: C,
30.51%; H, 1.49%; N, 2.97%. Found: C, 30.62%; H, 1.58%; N, 3.08%.
4.1.2.1.1. (4-bromo-1H-pyrrol-2-yl)(3,5-dibromo-2-methoxyphenyl)
methanone (4a). White solid, Yield 80%, mp 116 °C. 1H NMR: δ 3.71 (s,
3H, OCH3), 6.67 (s, 1H, H-3), 7.48 (s, 1H, H-5), 7.49 (d, 1H, H-6ꞌ,
J
m = 2.5 Hz), 7.81 (d, 1H, H-4ꞌ, Jm = 2.5 Hz), 12.58 (br s, 1H, NH). 13
C
NMR (acetone‑d6): δ 62.1, 113.5, 118.1, 124.2, 127.4, 128.6, 130.7,
131.4, 135.9, 152.7, 180.4, 205.2. IR (cm−1): 3250 (NH), 1630 (CO).
Anal. calc. for C12H8Br3NO2: C, 32.91%; H, 1.84%; N, 3.20%. Found: C,
33.05%; H, 1.93%; N, 3.24%.
4.1.2.2. General procedure for preparation of pyrrolomycins C, F2a,
1d,e,h,i.12,22,23. In a typical experiment, to a solution of 1 mmol of
6a-e (or 5c for pyrrolomycin C) in 20 mL of anhydrous
dichloromethane in an ice-salt bath, 4 g of AlCl3 (30 mmol) was
added. The reaction mixture was left to stir overnight at room
temperature. The solution was cautiously decomposed with ice-
4.1.2.1.2. (5-bromo-2-methoxyphenyl)(4-chloro-1H-pyrrol-2-yl)
methanone (4d). White solid, Yield 75%, mp 115–116 °C. 1H NMR: δ
3.73 (s, 3H, OCH3), 6.51 (s, 1H, H-3), 7.12 (d, 1H, H-3ꞌ, Jo = 8.95 Hz),
7.33 (s, 1H, H-5), 7.45 (d, 1H, H-6ꞌ, Jm = 2.35 Hz), 7.65 (dd, 1H, H-4ꞌ,
Jo = 8.92 Hz, Jm = 2.3 Hz), 12.41 (br s, 1H, NH). 13C NMR
6

M.V. Raimondi et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
sulfuric acid 5% (30 mL), then 50 mL of diethyl ether was added. The
mixture was stirred vigorously for 10 min, the organic layer was
separated and the aqueous phase extracted with diethyl ether (2x30
mL). The combined extracts were washed with water until neutrality,
dried over anhydrous sodium sulfate, and evaporated. The crude
product was crystallized from dichloromethane to give pure
pyrrolomycins. The yields coincide with those reported in the
literature.12,22,23
parameter variable slope sigmoidal function in SigmaPlot 12.5 using a
nonlinear least squares algorithm.
(max min)
y = min +
HillSlope
1 + (x/IC50)
4.3.2. Inhibition of biofilm formation (Crystal violet method)
Bacterial strains S. aureus ATCC 25923 and P. aeruginosa ATCC
15442 were incubated in test tubes with Tryptic Soy Broth (TSB) (5 mL)
containing 2% w/v glucose at 37 °C for 24 h. After that, the bacterial
suspensions were diluted to achieve a turbidity equivalent to a 0.5
McFarland standard. The diluted suspension (2.5 μL) was added to each
well of a single cell culture polystyrene sterile, flat-bottom 96-well plate
filled with TSB (100 μL) with 2% w/v glucose. A screening concentra-
tion of 100 μg/mL of all compounds were directly added to the wells to
assess inhibition percentages values, or in the case of determination of
IC50 (concentration at which a percentage of inhibition equal to 50% is
observed), concentrations in the range among 100 and 0.001 μM. Plates
were incubated at 37 °C for 24 h. After biofilm growth, the content of
each well was removed, wells were washed twice with sterile PBS 1X
and stained with 150 μL of 0.1% w/v crystal violet solution for 30 min
at room temperature. Excess solution was removed and the plate was
washed twice using tap water. A volume of 125 μL of acetic acid of 33%
v/v was added for 15 min to each stained well to solubilize the dye.
Optical density (OD) was read with wavelength of 540 nm using a
microplate reader (Glomax Multidetection System Promega).
4.2. Molecular modelling
The molecular structures have been drawn with the Avogadro
program, version 1.2.0,35 while their geometry optimization was per-
formed with the LigPrep routine of the Maestro Schrödinger Suite
version 2017-4.36
The X-ray crystallographic structure of SrtA (Fig. 3) was obtained
from the Protein Data Bank,37 PDB-ID: 1T2W.30 The chosen structure of
SrtA is complexed with the LPETG peptide, since it has been reported
that all SrtA inhibitors bind the protein similarly to such peptide.
All co-crystallized water molecules and counterions were removed
from the PDB protein structure and non-resolved hydrogen atoms were
added in some amino acid residues.
The Chimera program,38 UFCS Chimera version 1.11.2, was used to
create the pdb files of the ligands and of SrtA. Molecular docking cal-
culations were performed with Autodock Vina 39 on the protein binding
site of the LPETG inhibitor. Pharmacokinetic studies, to estimate the
Absorption, Distribution, Metabolism, and Excretion (ADME) and the
toxicity of the ligands, were performed with QikProp routine of
Maestro.36
All tests involved two replicates and were repeated in at least three
independent experiments. The percentage of inhibition was calculated
through the formula:
4.3. Biological evaluation
% of inhibition = [(OD growth control OD sample)/OD growth control]
× 100
4.3.1. Screening as Sortase A (SrtA) inhibitors
All of the compounds were prepared as 10 mM stock solutions in
dimethyl sulfoxide (DMSO) and used for the IC50 determination. The
compounds were tested at concentrations ranging from 200 to 20 µM in
black 384-well plates (Greiner Bio-One). Berberine chloride, a known
Sortase A inhibitor, was used as the positive control.40 The inhibitory
activity of all the compounds was assessed by quantifying the increase
in fluorescence intensity upon cleavage of the FRET-peptide
4.3.3. Murein hydrolase assays
S. aureus [108 cfu/mL] was inoculated in 5 mL of TSB containing 2%
(w/v) glucose and incubated overnight at 37 °C with shaking (200 rpm).
To evaluate the effect on S. aureus murein hydrolytic activity, an aliquot
of 20 µL of culture was inoculated into 2 mL of TSB with 2% (w/v) glu-
cose containing 1 µM of compound 1d or F2a in flat 24-well polystyrene
cell culture plate (Costar). Compound 1d and F2a were prepared as 0.5
µg/µL stock solutions in DMSO. Untreated and 0.1% (v/v) DMSO-con-
taining S. aureus cultivations were used as control conditions. The test and
control cultivations were performed in triplicated replicas. The cell cul-
ture plate was incubated statically at 37 °C for 24 h. Following incubation,
spent medium samples were collected without taking well-surface ad-
herent cells. The samples were centrifuged (10 min at 2000 rcf) to sepa-
rate possible planktonic cells and, then, 1 mL of supernatants was lyo-
philized and suspended in 200 µL of 1 M Tris-HCl buffer (pH 7.4). The
activity of extracellular murein hydrolases were assayed by zymographic
analyses as previously described using lysozyme as positive control.33 In
particular, 10 µL for each sample was separated using SDS-PAGE on a
12% acrylamide gel containing 1 mg/mL Micrococcus lysodeikticus ATCC
No 4698 cells (Sigma Aldrich, St. Louis, MO) at 100 V for approximately
2 h. After electrophoresis, the gel was washed twice in 100 mL of H2O for
30 min at room temperature under constant agitation. After washing, the
gel was incubated with renaturing buffer (20 mM Tris, 50 mM NaCl,
20 mM MgCl2, 0.5% Triton X-100, pH 7.4) for 30 min at room tempera-
ture and then for 24 h at 37 °C with replaced renaturing buffer under
constant agitation until clear hydrolytic bands appeared (between 16 h
and 48 h). To highlight the presence of digestion bands, the gels were
stained with 0.1% Methylene Blue (Sigma-Aldrich). Thus, bands without
staining were indicative of muerein hydrolysis. In particular, S. aureus
dabcyl-QALPETGEE-edans, which was used as the sortase substrate. A
40,41
previously published method
was used with slight modifications.
Briefly, the reactions were performed in a volume of 100 µL containing
50 mM Tris-HCl, 5 mM CaCl2, 150 mM NaCl, pH 7.5, 10 µM S. aureus
SrtA, 20 µM fluorescent peptide substrate dabcyl-QALPETGEE-edans,
and the prescribed concentrations of the test compounds or positive
controls. The peptide substrate without the recombinant SrtA was in-
cubated in the same manner and used as a negative control. The reac-
tions were conducted for 24 h at 37 °C, and the fluorescence emitted
with an excitation wavelength of 350 nm and an emission wavelength
of 495 nm after substrate cleavage was recorded. End-point determi-
nation of product formation was used as a criterion for the primary
screening. This determination was made by measuring the total product
fluorescence 24 h after the initiation of the reaction. The relative in-
hibition activity was determined as %I = 100% − (Fsample
/
Fcontrol*100%), where Fsample is the fluorescence intensity of the well
containing the corresponding test compound and Fcontrol is the fluor-
escence of the positive control reaction without inhibition. For the IC50
determination, 10 μM S. aureus SrtA was preincubated in the reaction
buffer with increasing concentrations of the inhibitory compounds
(x–y μM) for 1 h at 37 °C prior to the addition of the dabcyl-QALPET-
GEE-edans substrate to a final concentration of 50 µM. The total
fluorescence was recorded at 1-min intervals for 1 h, and the progress
curves were constructed. The initial velocities of the biphasic reactions
were obtained through nonlinear regression. The IC50 values were de-
termined by fitting the obtained data to the following default four-
hydrolytic activity was calculated using triplicate measurements by Im-
42
ageJ
analysis performed on digitalized zymography images and was
reported as lysozyme µg equivalents.
7

M.V. Raimondi et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
Acknowledgments
16. Koyama M, Kodama Y, Tsuruoka T, Ezaki N, Niwa T, Inouye S. Structure and
synthesis of pyrrolomycin A, a chlorinated nitro-pyrrole antibiotic. J Antibiot.
1981;34(12):1569–1576. https://doi.org/10.7164/antibiotics.34.1569.
17. Kaneda M, Nakamura S, Ezaki N, Iitaka Y. Structure of pyrrolomycin B, a chlorinated
nitro-pyrrole antibiotic. J Antibiot. 1981;34(10):1366–1368. https://doi.org/10.
7164/antibiotics.34.1366.
This work was supported by Ministero dell’Istruzione dell’Università
e della Ricerca (MIUR) of the Italian Government [grant number
PJ_RIC_FFABR_2017_160599]. A special thanks to Professor Salvatore
Petruso for His valuable advice.
18. Ezaki N, Koyama M, Shomura T, Tsuruoka T, Inouye S. Pyrrolomycins C, D and E new
members of pyrrolomycins. J Antibiot. 1983;36(10):1263–1267. https://doi.org/10.
7164/antibiotics.36.1263.
19. Ezaki N, Koyama M, Kodama Y, et al. Pyrrolomycins F1, F2a, F2b and F3, new
metabolites produced by the addition of bromide to the fermentation. J Antibiot.
1983;34(11):1431–1438. https://doi.org/10.7164/antibiotics.36.1431.
20. Charan RD, Schlingmann G, Bernan VS, Feng X, Carter GT. Dioxapyrrolomycin
biosynthesis in streptomyces fumanus. J Nat Prod. 2006;69(1):29–33. https://doi.
org/10.1021/np0503404.
Author contributions
M.V.R., D.S. and G.B. conceived the work and were responsible for
the correctness of the whole study. M.V.R., M.G.C. and G.G. were re-
sponsible for the experimental design and for data analysis of the whole
study. A.L., R.L., M.G.C., T.F. and M.L.F. performed the experiments.
M.V.R., D.S., G.B., and S.C. wrote and revised the manuscript.
Conflict of interest
21. Schillaci D, Petruso S, Sciortino V. 3,4,5,3’,5’-Pentabromo-2-(2’-hydroxybenzoyl)
pyrrole: a potential lead compound as anti-gram-positive and anti-biofilm agent. Int J
Antimicrob Agents. 2005;25(4):338–340. https://doi.org/10.1016/j.ijantimicag.
2004.11.014.
22. Raimondi MV, Cascioferro S, Schillaci D, Petruso S. Synthesis and antimicrobial
activity of new bromine-rich pyrrole derivatives related to monodeoxypyoluteorin.
Eur J Med Chem. 2006;41(12):1439–1445. https://doi.org/10.1016/j.ejmech.2006.
07.009.
The authors declare no competing financial interest.
Appendix A. Supplementary data
23. Raimondi MV, Schillaci D, Petruso S. Synthesis and anti-staphylococcal activity of
new halogenated pyrroles related to pyrrolomycins F. J Heterocyclic Chem.
2007;44(6):1407–1411. https://doi.org/10.1002/jhet.5570440626.
24. Renneberg B, Kellner M, Laatsch H. Synthese halogenierter benzyl-und benzoylpyr-
role. Liebigs Ann Chem. 1993:847–852. https://doi.org/10.1002/jlac.
1993199301134.
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bmc.2019.01.010.
References
25. Hodge P, Rickards RWJ. The halogenation of methyl pyrrole-2-carboxylate and of
some related pyrroles. Chem Soc. 1965:459–470. https://doi.org/10.1039/
JR9650000459 and references therein cited.
1. Jan 29, C. D. | N. R. | C. N. |; 2018. WHO releases its first report on global antibiotic
resistance http://www.cidrap.umn.edu/news-perspective/2018/01/who-releases-
its-first-report-global-antibiotic-resistance. (accessed 2.05.18.).
2. Organization, W. H. O. Global Antimicrobial Resistance Surveillance System (GLASS)
Report: Early Implementation 2016–2017; World Health Organization, 2018.
Number of pages: 164. ISBN: 978-92-4-151344-9. http://www.who.int/glass/
resources/publications/early-implementation-report/en/ (accessed 2.05.18.).
3. Dodson KW, Pinkner JS, Rose T, Magnusson G, Hultgren SJ, Waksman G. Structural
basis of the interaction of the pyelonephritic E. Coli adhesin to its human kidney
receptor. Cell. 2001;105(6):733–743. https://doi.org/10.1016/S0092-8674(01)
00388-9.
26. Schillaci D, Petruso S, Cascioferro S, Raimondi MV, Haagensen Ja.J, Molin S. In vitro
anti-gram-positive and antistaphylococcal biofilm activity of newly halogenated
pyrroles related to pyrrolomycins. Int J Antimicrob Agents. 2008;31(4):380–382.
https://doi.org/10.1016/j.ijantimicag.2007.10.013.
27. Schillaci D, Petruso S, Raimondi MV, et al. Pyrrolomycins as potential anti-staphy-
lococcal biofilms agents. Biofouling. 2010;26(4):433–438. https://doi.org/10.1080/
08927011003718673.
28. Cascioferro S, Raimondi MV, Cusimano MG, et al. Pharmaceutical potential of syn-
thetic and natural pyrrolomycins. Molecules. 2015;20(12):21658–21671. https://doi.
org/10.3390/molecules201219797.
29. Rossino G, Raimondi MV, Rui M, et al. 400/cerium ammonium nitrate combined
with microwave-assisted synthesis for rapid access to beta-amino ketones. an easy-to-
use protocol for discovering new hit compounds. Molecules. 2018;23((4), 775):1–12.
https://doi.org/10.3390/molecules23040775.
4. Cascioferro S, Raffa D, Maggio B, Raimondi MV, Schillaci D, Daidone G. Sortase A
inhibitors: recent advances and future perspectives. J Med Chem.
2015;58(23):9108–9123. https://doi.org/10.1021/acs.jmedchem.5b00779.
5. Mazmanian SK, Liu G, Jensen ER, Lenoy E, Schneewind O. Staphylococcus Aureus
Sortase mutants defective in the display of surface proteins and in the pathogenesis of
animal infections. Proc Natl Acad Sci USA. 2000;97(10):5510–5515. https://doi.org/
10.1073/pnas.080520697.
30. Zong Y, Bice TW, Ton-That H, Schneewind O, Narayana SV. Crystal structures of
Staphylococcus aureus sortase A and its substrate complex. J Biol Chem.
2004;279:31383–31389. https://doi.org/10.1074/jbc.M401374200.
31. Cascioferro S, Totsika M, Schillaci D, Sortase A. An ideal target for antivirulence drug
development. Microb Pathog. 2014;77C. https://doi.org/10.1016/j.micpath.2014.10.
007 105e112.
6. Oh K-B, Oh M-N, Kim J-G, Shin D-S, Shin J. Inhibition of sortase-mediated
Staphylococcus aureus adhesion to fibronectin via fibronectin-binding protein by
sortase inhibitors. Appl Microbiol Biotechnol. 2006;70(1):102–106. https://doi.org/
10.1007/s00253-005-0040-8.
32. Sahukhal GS, Batte JL, Elasri MO. msaABCR operon positively regulates biofilm
development by repressing proteases and autolysis in Staphylococcus aureus. Nakane
A, ed. FEMS Microbiol Lett. 2015;362(4):1–10.
7. Sibbald MJJB, Yang X-M, Tsompanidou E, et al. Partially overlapping substrate
specificities of staphylococcal group A sortases. Proteomics.
33. Bose JL, Lehman MK, Fey PD, Bayles KW. Contribution of the Staphylococcus aureus
Atl AM and GL murein hydrolase activities in cell division, autolysis, and biofilm
formation. e42244 PLoS ONE. 2012;7(7) https://doi.org/10.1371/journal.pone.
0042244.
2012;12(19–20):3049–3062. https://doi.org/10.1002/pmic.201200144.
8. Bierne H, Mazmanian SK, Trost M, et al. Inactivation of the SrtA gene in listeria
monocytogenes inhibits anchoring of surface proteins and affects virulence. Mol
Microbiol. 2002;43(4):869–881. https://doi.org/10.1046/j.1365-2958.2002.
02798.x.
34. Oshida T, Sugai M, Komatsuzawa H, Hong YM, Suginaka H, et al. A Staphylococcus
aureus autolysin that has an N-acetylmuramoyl-L-alanine amidase domain and an
endo-beta-N-acetylglucosaminidase domain: cloning, sequence analysis, and char-
acterization. Proc Natl Acad Sci USA. 1995;92:285–289.
9. A.S. Kharat A. Tomasz Inactivation of the SrtA Gene affects localization of surface
proteins and decreases adhesion of Streptococcus pneumoniae to human pharyngeal
cells in vitro Infect Immun 2003 71 5 2758 2765 https://doi.org/ 10.1128/IAI.71.5.
2758-2765.2003.
35. Hanwell MD, Curtis DE, Lonie DC, Vandermeersch T, Zurek E, Hutchison GR.
Avogadro an advanced semantic chemical editor, visualization, and analysis plat-
form. J Cheminform. 2012;4(1):1–17. https://doi.org/10.1186/1758-2946-4-17.
36. Maestro, Schrödinger, LLC, New York, NY, 2017, version 11.4.011.
37. Berman HM, Westbrook J, Feng Z, et al. The protein data bank. Nucleic Acids Res.
2000;28:235–242.
10. Hu P, Huang P, Chen WM. Curcumin inhibits the Sortase A activity of the
Streptococcus mutans UA159. Appl Biochem Biotechnol. 2013;171(2):396–402. https://
doi.org/10.1007/s12010-013-0378-9.
11. Vanier G, Sekizaki T, Domínguez-Punaro MC, et al. Disruption of SrtA gene in
streptococcus suis results in decreased interactions with endothelial cells and ex-
tracellular matrix proteins. Vet Microbiol. 2008;127(3–4):417–424. https://doi.org/
10.1016/j.vetmic.2007.08.032.
38. Pettersen EF, Goddard TD, Huang CC, et al. UCSF Chimera—A visualization system
for exploratory research and analysis. J Comput Chem. 2004;25:1605–1612. https://
doi.org/10.1002/jcc.20084.
12. Petruso S, Bonanno S, Caronna S, Ciofalo M, Maggio B, Schillaci D. Oxidative ha-
logenation of substituted pyrroles with Cu(II). Part IV. bromination of 2-(2′-
Hydroxybenzoyl)pyrrole. A new synthesis of bioactive analogs of mono-
deoxypyoluteorin. J Heterocyclic Chem. 1994;31(4):941–945. https://doi.org/10.
1002/jhet.5570310442.
39. Trott O, Olson AJ. J Comput Chem. 2010;31:455–461. https://doi.org/10.1002/jcc.
21334.
40. Maggio B, Raffa D, Raimondi MV, et al. Discovery of a new class of sortase a
transpeptidase inhibitors to tackle gram-positive pathogens: 2-(2-phenylhy-
drazinylidene)alkanoic acids and related derivatives. Molecules.
2016;21(2):241–256. https://doi.org/10.3390/molecules21020241.
41. Cascioferro S, Maggio B, Raffa D, Raimondi MV, Cusimano MG, Schillaci D,
Manachini B, Leonchiks A, Daidone G. A new class of phenylhydrazinylidene deri-
vatives as inhibitors of Staphylococcus aureus biofilm formation. Med Chem Res.
2016;255:870–878. https://doi.org/10.1007/s00044-016-1535-9.
42. Rueden CT, Schindelin J, Hiner MC. Image J2: imageJ for the next generation of
scientific image data. BMC Bioinf. 2017;18:529https://doi:10.1186/s12859-017-
1934-z.
13. Schillaci D, Maggio B, Petruso S. In vitro activity of synthetic bromo analogs of
pyrrolomycins against clinical staphylococcus aureus strains. Pharmazie.
1996;51(1):64.
14. Koyama M, Ezaki N, Tsuruoka T, Inouye S. Structural studies on pyrrolomycins C D
and E. J Antibiot. 1983;36(11):1483–1489. https://doi.org/10.7164/antibiotics.36.
1483.
15. Carter GT, Nietsche JA, Goodman JJ, et al. LL-F42248α, A novel chlorinated pyrrole
antibiotic. J Antibiot. 1987;40(2):233–236. https://doi.org/10.7164/antibiotics.40.
233.
8