On the antibiotic and antifungal activity of pestalone, pestalachloride a, and structurally related compounds

Article abstract of DOI:10.1021/np400301d

Pestalone (1) is a prominent marine natural product first isolated by M. Cueto et al. in 2001 from a co-fermentation of a marine fungus with a marine bacterium. For more than 10 years, 1 had been considered as a promising new antibiotic compound, the repo

Full text of DOI:10.1021/np400301d

Note
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On the Antibiotic and Antifungal Activity of Pestalone,
Pestalachloride A, and Structurally Related Compounds
Daniel Augner,† Oleg Krut,*,‡ Nikolay Slavov,† Dario C. Gerbino,† Hans-Georg Sahl,§ Jurgen Benting,⊥
̈
Carl F. Nising,⊥ Stefan Hillebrand,⊥ Martin Kronke,‡ and Hans-Gunther Schmalz*,†
̈
̈
†Department of Chemistry, University of Cologne, Greinstrasse 4, 50939 Koln, Germany
̈
‡Institute for Medical Microbiology, Immunology and Hygiene, University of Cologne, Goldenfelsstrasse 19-21, 50935 Koln,
̈
Germany
§Institute for Medical Microbiology, Immunology and Parasitology, University of Bonn, Meckenheimer Allee 168, 53155 Bonn,
Germany
⊥Bayer CropScience, Research−Chemistry Disease Control, Alfred-Nobel-Strasse 50, 40789 Monheim, Germany
ABSTRACT: Pestalone (1) is a prominent marine natural
product first isolated by M. Cueto et al. in 2001 from a co-
fermentation of a marine fungus with a marine bacterium. For
more than 10 years, 1 had been considered as a promising new
antibiotic compound, the reported MIC against methicillin-
resistant Staphylococcus aureus (MRSA) being 37 ng/mL. After
overcoming the limited availability of 1 by total synthesis (N.
Slavov et al., 2010) we performed new biological tests, which
did not confirm the expected degree of antibiotic activity. The observed activity of pestalone against different MRSA strains was
3−10 μg/mL, as determined independently in two laboratories. A number of synthetic derivatives of 1 including pestalachloride
A and other isoindolinones (formed from 1 by reaction with amines) did not exhibit higher activities as compared to 1 against
MRSA and a series of plant pathogens.
he story of pestalone (1) started in 2001 in the context of
Ta research program by Fenical and co-workers, who tried
to trigger the expression of new antibiotics against drug-
resistant pathogens from marine microorganisms in response to
bacterial challenge.1 After co-culturing a fungus of the genus
Pestalotia and a unicellular marine bacterium (strain CNJ-328)
they isolated 1 as a new antibiotic agent. Structural elucidation
revealed 1 to be a highly functionalized, chlorinated
benzophenone derivative (Figure 1).2
models of infectious disease”.1 Due to its pronounced activity, 1
was highlighted in several publications as a promising new lead
structure in the struggle against multiresistant microorganisms.3
Nevertheless, because of its limited availability, no further
biological investigations of 1 have been reported so far. In 2008,
Che and co-workers reported the discovery of the pestala-
chlorides A−C (Figure 2) from the plant endophytic fungus
Pestalotiopsis adusta.4 These compounds are structurally closely
related to 1, from which they may be biosynthetically derived.
Recently, we demonstrated that 1 can be easily converted into
pestalachloride A (rac-2a) by treatment with ammonia.5 The
fact that this compound occurs in nature also as a racemic
Figure 1. The marine natural product pestalone (1).
Pestalone (1) was reported to exhibit strong antibiotic
activity against methicillin-resistant Staphylococcus aureus
(MRSA, minimum inhibitory concentration (MIC) = 37 ng/
mL) and vancomycin-resistant Enterococcus faecium (VRE, MIC
78 = ng/mL). The authors even underlined that “the potency
of this agent toward drug-resistant pathogens suggests that
pestalone should be evaluated in more advanced, whole animal
Figure 2. The natural products pestalachloride A (rac-2a),
pestalachloride B (3), and pestalachloride C (4).
Received: April 15, 2013
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
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Journal of Natural Products
Note
mixture indicates its nonenzymatic “biosynthesis” from pest-
alone (1).
Pestalachlorides A−C were reported to exhibit antimicrobial
activity against certain plant pathogenic fungi (Table 1). The
Table 1. Activity (Minimum Inhibitory Concentration) of
Pestalachloride A against Plant Pathogenic Fungi: Fusarium
culmorum, Gibberella zeae, and Verticillium aibo-atrum, As
Reported by Che4
MIC (μg/mL)
compound
F. culmorum
G. zeae
V. aibo-atrum
rac-2a
3.2
20.1
50.1
5.0
50.1
5.0
3
4
>100
>100
>100
Figure 3. Further synthetic analogues of pestalone (1) employed in
this study.
highest antifungal activity was observed for pestalachloride A
(rac-2a) against Fusarium culmorum (MIC = 3.2 μg/mL). In
contrast, pestalachloride B (3) was most effective against
Gibberella zeae (MIC = 5.0 μg/mL) and Verticillium aibo-atrum
(MIC = 5.0 μg/mL), while no biological activity was observed
for pestalachloride C (4) in these in vitro test systems.4
The limited availability of 1 from natural sources challenged
efforts toward its chemical synthesis. A synthesis of deformyl-
pestalone and some related analogues was achieved in 2003,6
and a first total synthesis was disclosed a little later.7 However,
no further biological experiments were reported since then.
Recently, we developed an efficient total synthesis and were
able to produce substantial amounts of pestalone (1), the
identity of which was secured by X-ray crystal structure
analysis.5 In the course of this work, we also demonstrated that
1 reacts with ammonia to give rac-2a or with primary amines to
afford isoindolinones rac-2b and rac-2c, respectively. In
addition we found that the conversion of 1 into pestalalactone
rac-5 proceeds smoothly either under photochemical con-
ditions or in the presence of a nucleophilic catalyst such as
cyanide (Scheme 1).8
reference strain, ATCC 29213. The results are shown in Table
2.
Table 2. Activity of Pestalone (1) and Analogues against
Different S. aureus Strains
a
MIC [μg/mL] against different S. aureus strains
compound
USA300
10
Mu50
10
MW2
ATCC29213
1
16
10
rac-2a
10
>50
>50
rac-2b
>50
>50
rac-2c
rac-5
25
6
10−15
30−35
5−10
7−10
0.25 - 0.5
15−20
30−45
5−10
7−10
4
7
8
9
vancomycin
1
1
a
105 cfu/mL; controls with vancomycin and 1.
The availability of material enabled us to further explore the
antibacterial potency of pestalone (1), the related isoindoli-
nones (rac-2a−c), pestalalactone (rac-5), and analogues
(Figure 3). For this purpose, we utilized a standardized broth
microdilution method to determine the activity against three
different clinically relevant Staphylococcus aureus strains, i.e.,
USA300 (epidemic community acquired MRSA), Mu50
(hospital acquired MRSA, vancomycin-intermediate S. aureus,
VISA), S. aureus MW2 (community acquired MRSA), and one
Our experiments revealed a significant biological effect of
pestalone (1), pestalachloride A (rac-2a), and some analogues
against methicillin-resistant Staphylococcus aureus, however,
only at comparably high concentrations (MIC = 10 μg/mL
for 1). Pestalone (1) was found to be about 270 times less
active then reported.1 Pestalachloride A (rac-2a) also exhibited
a certain activity (comparable to 1), in contrast to the N-
alkylated derivatives (rac-2b/c), which were not active at any
a
Scheme 1. Conversion of Pestalone (1) into Pestalachloride A and Derivatives (rac-2a−c) or Pestalalactone (rac-5)
a
Conditions: (a) RNH2, 1,4-dioxane/H2O, AcOH; (b) cat. NaCN, DMSO (or hν, DMSO).
B
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Journal of Natural Products
Note
reasonable concentration. Pestalalactone (rac-5) displayed
lower antibacterial potency (MIC = 25 μg/mL). Deformyl-
pestalone (8) showed a similar activity to 1, against both
USA300 and Mu50 (MIC = 5−10 μg/mL).
that pestalalactone (rac-5), pestalachloride A (rac-2a), and
related isoindolinones, which are readily formed from 1 under
mild conditions, also do not exhibit a particular level of activity.
Compounds 1, rac-2a, and the deformyl derivatives 8 and 9
were found to be active against methicillin-resistant Staph-
ylococcus aureus strains at concentrations of 3−10 μg/mL
(MIC). The natural products 1 and rac-2a as well as a series of
synthetic derivatives were also tested against a spectrum of
plant pathogenic fungi. While 1 displayed the broadest activity,
none of the compounds proved to be sufficiently active to
qualify as a potential crop-protecting agent.
The activity of pestalone (1) was confirmed in an
independent series of broth microdilution experiments employ-
ing other bacterial strains (Table 3). Staphylococcus aureus strain
Table 3. Activity of Pestalone (1) against Different
Pathogenic Bacteria
MIC [μg/mL]
At this point we have no explanation for the discrepancy
between our results and the reported data concerning the
antibiotic activity of pestalone (1). Since no reference strain
was used in the susceptibility testing of natural pestalone, it is
possible that the employed isolate was particularly sensitive by
chance. However, our data, which are based on repeated testing
in different laboratories using both reference stains and clinical
isolates, should reflect the susceptibility of S. aureus to
pestalone rather precisely. Finally, having identified 1 as a
reactive agent (e.g., forming isoindolinones with primary
amines), we cannot exclude the possibility that a highly active
derivate of 1 had formed and caused the adventitious
antimicrobial effect.
compound
B. subtilis 168
S. aureus SG511
MRSA LT-1334
1
1.6
3.1
6.25
SG511 was affected by 1 at a MIC of 3.1 μg/mL, while the
growth of a randomly chosen clinical isolate, MRSA LT-1334,
was inhibited at a MIC of 6.25 μg/mL. As a reference, a strain
of Bacillus subtilis 168 was also tested (MIC = 1.6 μg/mL). It
was demonstrated again that 1 acts as an antibacterial agent,
however, not at the very low concentrations originally
reported.1
We also examined the antifungal potency of these
compounds against various plant pathogens.9 The results
summarized in Table 4 indicate that the compounds behaved
very differently. Pestalone (1) displayed significant antimicro-
bial activity against several of the microorganisms tested (7 out
of 15 plant pathogens), while pestalachloride A (rac-2a)
interfered only with Pyrenophora teres (ED50 = 33.1 μg/mL)
and Fusarium culmorum (ED50 = 31.9 μg/mL) with moderate
activities. (ED50 = effective dose that causes 50% growth
inhibition.) Pestalalactone (rac-5) selectively affected Phytoph-
thora cryptogea (MIC = 18.9 μg/mL), Trametes versicolor (ED50
= 13.1 μg/mL), and Pyrenophora teres (ED50 = 4.3 μg/mL).
The dibromo derivative (7) was selective against the most
sensitive organism, i.e., Fusarium culmorum (ED50 = 0.014 μg/
mL). While compounds 6 and 10 are active, only compound 11
showed no antimicrobial activities (at ≤50 μg/mL).
EXPERIMENTAL SECTION
■
General Experimental Procedures. MIC determinations (Table
2) were carried out in microtiter plates according to CLSI standards
with 2-fold serial dilutions of the compounds (Clinical and Laboratory
Standards Institute; NCCLS, 2006: Methods for Dilution Antimicro-
bial Susceptibility Tests for Bacteria That Grow Aerobically; Approved
Standard M7-A6). Strains were grown in Mueller−Hinton broth
(Oxoid). Bacteria were added at an inoculum of 105 cfu/mL in a final
volume of 0.2 mL. After incubation for 24 h at 37 °C the MIC was
read as the lowest compound concentration causing inhibition of
visible growth. S. aureus reference strains were obtained from
American Type Culture Collection (ATCC 29213) or from the
Network on Antimicrobial Resistance in Staphylococcus aureus
(USA300, Mu50, MW2). The bacterial stocks were stored at −70
°C and recovered on Mueller-Hinton agar a few days before
experiments.
In conclusion, we have demonstrated that the antimicrobial
activity of synthetic pestalone (1) is significantly less than that
reported for the natural product.1 In addition, we demonstrated
MIC determinations (Table 3) were carried out in 96-well
microtiter plates according to CLSI standards with 2-fold serial
Table 4. Activity (ED50 Values) of Pestalone (1), Pestalachloride A (rac-2a), and Other Derivatives (see Figure 3) against
Different Plant Pathogens
ED50 [μg/mL]
organism
1
rac-2a
rac-5
6
7
10
n.d.
11
Phytophthora infestans
Phytophthora cryptogea
Pythium aphanidermatum
Alternaria mali
>50
>50
>50
>50
>50
>50
>50
33.1
>50
31.9
>50
>50
>50
>50
>50
>50
n.d.
18.9
>50
>50
>50
>50
4.3
n.d.
>50
>50
n.d.
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
33.9
>50
22.4
>50
26.7
>50
44.4
>50
>50
>50
>50
>50
>50
42
>50
>50
>50
>50
>50
Botrytis cinerea
>50
>50
Septoria tritici
Pyrenophora teres
Leptosphaeria nodorum
Fusarium culmorum
Gibberella zeae
2.2
5.4
22.4
>50
>50
>50
>50
>50
>50
13.1
>50
22.5
1.2
>50
0.12
>50
28.6
>50
0.014
2.2
>50
12.6
>50
>50
>50
>50
>50
>50
>50
Pyricularia oryzae
Ustilago avenae
5
>50
>50
>50
>50
>50
>50
>50
>50
Aspergillus niger
>50
>50
>50
Trametes versicolor
Pseudomonas fluorescens
C
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Journal of Natural Products
Note
dilutions of the compounds. Strains were grown and tested in half-
concentrated Mueller−Hinton broth (Oxoid). Bacteria were added at
an inoculum of 105 cfu/mL in a final volume of 0.2 mL. After
incubation for 24 h at 37 °C the MIC was read as the lowest
compound concentration causing inhibition of visible growth.
In Vitro Test for the Calculation of the ED50 Value with
Microorganisms (Table 4). Wells of 96-well microtiter plates were
filled with 10 μL of a solution of the test compound in methanol
together with the emulsifier alkylaryl polyglycol ether. Thereafter, the
solvent was evaporated in a hood. At the next step, into each well 200
μL of liquid potato dextrose medium was added that had been
amended with an appropriate concentration of spores or mycelium
suspension of the test fungus. With the aid of a photometer the
extinction in all wells was measured at the wavelength of 620 nm. The
microtiter plates were then transferred for 3−5 days onto a shaker at
20 °C and 85% relative humidity. At the end of the incubation time
the growth of the test organisms was measured again photometrically
at the wavelength of 620 nm. The difference between the two
extinction values (taken before and after incubation) was proportional
to the growth of the test organism. Based on the Δ extinction data
from the different test concentrations and that of the untreated test
organism (control) a dose−response curve was calculated. The
concentration that is necessary to give 50% growth inhibition is
defined and reported as ED50 value in ppm (= μg/mL).
AUTHOR INFORMATION
Corresponding Author
*(O. Krut) Phone: +49-221-478-32103. E-mail: oleg.krut@uni-
koeln.de. (H.-G. Schmalz) Phone: +49-221-470-3063. E-mail:
schmalz@uni-koeln.de.
■
Notes
The authors declare no competing financial interest.
REFERENCES
■
(1) Cueto, M.; Jensen, P. R.; Kauffman, C.; Fenical, W.; Lobkovsky,
E.; Clardy, J. J. Nat. Prod. 2001, 64, 1444−1446.
(2) A closely related structure, formally derived from 1 by cleavage
(O-demethylation) of the methoxy group, was reported: Wachi, Y.;
Yamashita, T.; Komatsu, K.; Yoshida, S. JP Patent, JKXXAF JP
07061950 A2 19950307, 1995.
(3) (a) Donia, M.; Hamann, M. T. Lancet Infect. Dis. 2003, 3, 338−
348. (b) Scherlach, K.; Hertweck, C. Org. Biomol. Chem. 2009, 7,
1753−1760. (c) Pettit, R. K. Appl. Microbiol. Biotechnol. 2009, 83, 19.
(d) Villa, F. A.; Gerwick, L. Immunopharm. Immunotox. 2010, 32,
228−237. (e) Hughes, C. C.; Fenical, W. Chem.Eur. J. 2010, 16,
12512−12525. (f) Rahman, H.; Austin, B.; Mitchell, W. J.; Morris, P.
C.; Jamieson, D. J.; Adams, D. R.; Spragg, A. M.; Schweizer, M. Mar.
Drugs 2010, 8, 498−518.
(4) Li, E.; Jiang, L.; Guo, L.; Zhang, H.; Che, Y. Bioorg. Med. Chem.
2008, 16, 7894−7899.
(5) Slavov, N.; Cvengros, J.; Neudorfl, J.-M.; Schmalz, H.-G. Angew.
̈
Chem., Int. Ed. 2010, 49, 7588−7591.
(6) Kaiser, F.; Schmalz, H.-G. Tetrahedron 2003, 59, 7345−7355.
(7) Iijima, D.; Tanaka, D.; Hamada, M.; Ogamino, T.; Ishikama, Y.;
Nishiyama, S. Tetrahedron Lett. 2004, 45, 5469−5471.
(8) (a) Augner, D.; Gerbino, D. C.; Slavov, N.; Neudorfl, J.-M.;
̈
Schmalz, H.-G. Org. Lett. 2011, 13, 5374−5377. (b) Gerbino, D. C.;
Augner, D.; Slavov, N.; Schmalz, H.-G. Org. Lett. 2012, 14, 2338−
2341.
(9) For a review on the relevance of natural products in crop
protection, see: Nising, C. F.; Hillebrand, S.; Rodefeld, L. Chem.
Commun. 2011, 47, 4062−4073.
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