The Effect of Cytochalasans on the Actin Cytoskeleton of Eukaryotic Cells and Preliminary Structure-Activity Relationships

Abstract

In our ongoing search for new bioactive fungal metabolites, two new cytochalasans were isolated from stromata of the hypoxylaceous ascomycete Hypoxylon fragiforme. Their structures were elucidated via high-resolution mass spectrometry (HR-MS) and nuclear magnetic resonance (NMR) spectroscopy. Together with 23 additional cytochalasans isolated from ascomata and mycelial cultures of different Ascomycota, they were tested on their ability to disrupt the actin cytoskeleton of mammal cells in a preliminary structure-activity relationship study. Out of all structural features, the presence of hydroxyl group at the C7 and C18 residues, as well as their stereochemistry, were determined as important factors affecting the potential to disrupt the actin cytoskeleton. Moreover, reversibility of the actin disrupting effects was tested, revealing no direct correlations between potency and reversibility in the tested compound group. Since the diverse bioactivity of cytochalasans is interesting for various applications in eukaryotes, the exact effect on eukaryotic cells will need to be determined, e.g., by follow-up studies involving medicinal chemistry and by inclusion of additional natural cytochalasans. The results are also discussed in relation to previous studies in the literature, including a recent report on the anti-Biofilm activities of essentially the same panel of compounds against the pathogenic bacterium, Staphylococcus aureus.

Full text

biomolecules
Article
The Effect of Cytochalasans on the Actin
Cytoskeleton of Eukaryotic Cells and Preliminary
Structure–Activity Relationships
Robin Kretz 1,2, Lucile Wendt 1, Sarunyou Wongkanoun 3, J. Jennifer Luangsa-ard 3,
Frank Surup 1, Soleiman E. Helaly 1,4 , Sara R. Noumeur 1,5, Marc Stadler 1, *
and Theresia E.B. Stradal 6, *
1Department of Microbial Drugs, Helmholtz Centre for Infection Research (HZI), Inhoffenstrasse 7,
38124 Braunschweig, Germany; kretzrob@hs-albsig.de (R.K.); lucile.wendt@helmholtz-hzi.de (L.W.);
frank.surup@helmholtz-hzi.de (F.S.); soleiman.helaly@helmholtz-hzi.de (S.E.H.);
noumeur_sara@yahoo.fr (S.R.N.)
2University of Applied Sciences Albstadt-Sigmaringen, Faculty of Life Sciences, Anton-Günther-Strasse 51,
72488 Sigmaringen, Germany
3
National Centre for Genetic Engineering and Biotechnology (BIOTEC), NSTDA, 113 Thailand Science Park,
Phahonyothin Road, Klong Nueng Klong Luang, Pathum Thani 12120, Thailand;
sarunyou.wong@biotec.or.th (S.W.); jajen@biotec.or.th (J.J.L.-a.)
4Department of Chemistry, Faculty of Science, Aswan University, 81528 Aswan, Egypt
5Department of Microbiology-Biochemistry, Faculty of Natural and Life Sciences, University of Batna 2,
Batna 05000, Algeria
6Department of Cell Biology, Helmholtz Centre for Infection Research (HZI), Inhoffenstraße 7,
38124 Braunschweig, Germany
*Correspondence: marc.stadler@helmholtz-hzi.de (M.S.); theresia.stradal@helmholtz-hzi.de (T.E.B.S.);
Tel.: +49-531-6181-4240 (M.S.); Fax: +49-531-6181-9499 (M.S.)
Received: 21 January 2019; Accepted: 14 February 2019; Published: 19 February 2019


Abstract:
In our ongoing search for new bioactive fungal metabolites, two new cytochalasans were
isolated from stromata of the hypoxylaceous ascomycete Hypoxylon fragiforme. Their structures were
elucidated via high-resolution mass spectrometry (HR-MS) and nuclear magnetic resonance (NMR)
spectroscopy. Together with 23 additional cytochalasans isolated from ascomata and mycelial cultures
of different Ascomycota, they were tested on their ability to disrupt the actin cytoskeleton of mammal
cells in a preliminary structure–activity relationship study. Out of all structural features, the presence
of hydroxyl group at the C7 and C18 residues, as well as their stereochemistry, were determined as
important factors affecting the potential to disrupt the actin cytoskeleton. Moreover, reversibility
of the actin disrupting effects was tested, revealing no direct correlations between potency and
reversibility in the tested compound group. Since the diverse bioactivity of cytochalasans is interesting
for various applications in eukaryotes, the exact effect on eukaryotic cells will need to be determined,
e.g., by follow-up studies involving medicinal chemistry and by inclusion of additional natural
cytochalasans. The results are also discussed in relation to previous studies in the literature, including
a recent report on the anti-Biofilm activities of essentially the same panel of compounds against the
pathogenic bacterium, Staphylococcus aureus.
Keywords:
actin cytoskeleton; Ascomycota; chromatography; secondary metabolites; structure
elucidation; Xylariales
Biomolecules 2019,9, 73; doi:10.3390/biom9020073 www.mdpi.com/journal/biomolecules

Biomolecules 2019,9, 73 2 of 14
1. Introduction
Cytochalasans are a class of fungal metabolites, derived from mixed polyketide synthase/
nonribosomal peptide synthetase (PKS/NRPS) biosynthesis that are widely distributed among the
Ascomycota [
1
], and they occur particularly frequently in the genera of the order Xylariales [
2
].
Over the past decades, many of these compounds have been discovered in the course of natural
product screening campaigns due to their prominent activities in biological systems and, in particular,
their strong effects on eukaryotic cells [
3
]. Their biological activities have been attributed to their
interactions with the actin cytoskeleton [
4
,
5
], even though this has so far only been established for
a small portion of the representatives of this class of molecules [
6
]. The classic therapeutic indication for
actin inhibitors is cancer, and some studies have been conducted in the past to evaluate the feasibility
of obtaining a drug candidate based on this compound class [
7
], however, so far these activities have
not been successful.
We have recently obtained a number of cytochalasans, including some new natural products,
from different sources in the course of our ongoing search for novel bioactive fungal metabolites,
and surprisingly found that several of them are able to significantly inhibit biofilm formation in the
pathogenic bacterium, Staphylococcus aureus [
8
], while others did not show any activity. Since bacteria
lack actin, this observation cannot be attributed to the known mechanism of action (MOA) of the
compound class. Biofilm formation inhibitors may be of great utility for use in combination therapy
with antibiotics since they may enhance the efficacy of the latter compounds [
9
,
10
]. On the other
hand, an ideal biofilm inhibitor with therapeutic potential should neither possess activity against
the pathogenic target microbes, in order to avoid upcoming resistance, nor should it be toxic to the
cells of the host. We therefore decided to evaluate the panel of cytochalasans previously studied by
Yuyama et al. [8], and some additional cytochalasans that have become available in our laboratory in
the meantime, for their effects on mammalian cell lines using fluorescence microscopy, in order to find
out more about the structure–activity relationships; our results are reported in the present paper.
2. Materials and Methods
2.1. Fungal Material
Stromata of Hypoxylon fragiforme were collected from Fagus sylvatica by L. Wendt in the
vicinity of Braunschweig, Germany in 2017. A voucher specimen of the material is kept in the
fungarium of M. Stadler at the Helmholtz Centre for Infection Research, Braunschweig, Germany
(Acc. No. STMA18022). Stromata of Daldinia spp. were collected in Thailand, Chiang Mai Province,
Ban Hua Thung
community forest, on decaying wood by P. Srikitikulchai and S. Wongkanoun. Voucher
specimens of the material are kept in the fungarium (BBH) and culture collection (BCC) of BIOTEC
(Panthum Thani, Thailand). The stromata of both specimens were extracted as described previously [
8
].
The culture of Preussia simillis G22 was isolated from healthy roots of the medicinal plant Shrubby
globularia (Globularia alypum) collected from Batna (Algeria) in March 2014 by S. R. Noumeur and
was identified using the methods described previously [
11
]. The culture has been deposited with
DSMZ, Braunschweig, Germany (designation No. DSM 32328) as well as in the culture collection of
the Helmholtz Center for Infection Research (HZI).
2.2. Purification of the Compounds
Compounds
1
to
5
,
7
,
11
and
17
were purified from stromatal crude extracts by a preparative
reversed-phase high-performance liquid chromatography (RP-HPLC) system (Gilson, Middleton,
WI, USA) equipped with a GX-271 liquid handler, a diode array detector (DAD) 172 and a 305 and
306 pump. For the experiments, deionized water (solvent A), acetonitrile (ACN; solvent B, Avantor
Performance Materials, Center Valley, PA, USA) and a VP Nucleodur C18ec (150
×
40 mm, 7
µ
m;
Macherey-Nagel, Düren, Germany) column were used. The experiments were performed with a flow
rate of 20 mL/min. To remove fatty acids and debris, the crude extracts were dissolved in acetonitrile

Biomolecules 2019,9, 73 3 of 14
and filtered through a Strata X-33
µ
m polymere reversed phase tube (Phenomenex, Aschaffenburg,
Germany) prior to preparative liquid chromatography (LC) experiments. Fractions from preparative
LC experiments were collected in round bottle flasks according to the ultraviolet (UV) absorption
at 210 nm. Acetonitrile was removed from the fractions via evaporation in vacuo and the aqueous
residues were frozen. An Alpha 1–4 LSC freeze dryer (Christ, Osterode, Germany) was used to remove
the remaining water from the fractions.
Compounds
1
,
2
, and
17
were purified from stromata of H. fragiforme using the following gradient:
The crude extracts were dissolved in ACN and the compounds purified by using an Agilent 1100 series
preparative HPLC system (Agilent Technologies, Waldbronn, Germany). A Kromasil RP C18 (7mm,
250
×
25 mm; AkzoNobel, Mainz, Germany) and the mobile phase ACN and water was used (Milli-Q,
Millipore, Schwalbach, Germany); flow rate 20 mL min
−1
. Isocratic conditions at 53% ACN were
applied, followed by a linear gradient for 15 min to 67% ACN. Afterwards, another linear gradient to
100% ACN was applied. Fractions were combined according to UV adsorption at 220, 254 and 325 nm,
solvents were evaporated, and liquid chromatography-mass spectrometry (LC-MS) analyses were
performed. Fragiformin C (
1
) was eluted at t
R
10.2 min, fragiformin D (
2
) at t
R
8.9 min and compound
(
17
) at t
R
9.5 min. The yields were ca. 2.7 mg of (
1
), 0.5 mg of (
2
) and 1 mg of (
17
) from 100 mg of
crude extract.
Compound 3was purified from stromata of Daldinia sacchari as described in [8].
Compounds
4
,
5
,
7
and
11
were purified from P. simillis DSM 32328 using the following conditions:
The crude extracts were dissolved in methanol and purified by using an Agilent 1100 series preparative
HPLC system (Agilent Technologies, Waldbronn, Germany); Kromasil RP C18 (7 mm, 250
×
25 mm;
AkzoNobel, Mainz, Germany) column was used; mobile phase ACN and water (Milli-Q, Millipore,
Schwalbach, Germany); flow rate 20 mL min
−1
. Isocratic conditions at 48% ACN and 52% water for
30 min were applied; fractions were combined according to UV adsorption at 220, 254 and 325 nm, solvents
were evaporated, and LC-MS analyses were performed. Cytochalasin B (
4
) was eluted at t
R
10.2 min,
deoxaphomin (
5
) at t
R
10.8 min, cytochalasin F (
7
) at t
R
11.7 min and cytochalasin Z2 (
11
) at t
R
13.5 min.
The yields were ca. 17.8 mg of (
4
), 5 mg of (
5
), 0.8 mg of (7) and 1 mg of (11) from 256 mg of crude extract.
Finally, compound (6) was purchased from Sigma-Aldrich (C8273, St. Louis, MO, USA).
The identification of the compounds was confirmed by high-resolution electrospray ionization
mass spectrometry (HR-ESIMS) using the instrumental conditions described by Narmani et al. [
12
].
NMR spectra for structure elucidation were recorded with a Bruker Avance III 700 spectrometer
with a 5 mm TCI cryoprobe (
1
H 700 MHz,
13
C 175 MHz) and a Bruker Avance III 500 (
1
H 500 MHz,
13
C 125 MHz) spectrometer (Bruker, Bremen, Germany). Chemical shifts
δ
were referenced to the
solvents chloroform-d (
1
H,
δ
= 7.27 ppm;
13
C,
δ
= 77 ppm; Sigma-Aldrich, St. Louis, MO, USA) and
acetonitrile-d3 (
1
H,
δ
= 1.94 ppm;
13
C,
δ
= 1.39 ppm; Sigma-Aldrich, St. Louis, MO, USA). Optical
rotations were determined using a 241 MC polarimeter (Perkin Elmer, Waltham, MA, USA).
2.3. Spectral Data
2.3.1. Fragiformin C
Colorless oil. [
α
]
25
D
= +18.0 (c 1.0, AcN).
1
H NMR (500 MHz, CDCl
3
): see Table 1;
13
C NMR
(125 MHz, CDCl3): see Table 1. HR-ESIMS m/z 434.2688 ([M + H]+, calcd for C28H36NO3434.2695).

Biomolecules 2019,9, 73 4 of 14
Table 1. Nuclear magnetic resonance (NMR) spectroscopic data for fragiformins C (1) and D (2).
1a2b
δC, mult. δH, mult. δC, mult. δH, mult.
1174.2, C 173.2, C
25.56, br s 8.35, br s
359.1, CH 3.41, m 52.5, CH 3.59, m
447.0, CH 3.64, br s 43.9, CH 3.01, br d (6.3)
5126.2, C 35.3, CH 1.45, m
6131.6, C 57.5, C
769.5, CH 4.08, d (9.5) 61.7, CH 2.78, d (5.8)
OH: 1.26, br s
853.5, CH 2.09, m 49, CH 1.94, m
962.6, C 65.5, C
10 42.8, CH22.69, dd (13.4, 7.5)2.63, dd (13.4, 7.5) 43.3, CH22.71, dd (13.0, 4.1)
2.19, dd (13.0, 9.2)
11 17.1, CH31.44, s 12.1, CH30.56, d (7.2)
12 14.1, CH31.70, s 19.1, CH31.12, s
13 127.2, CH 6.04, ddd (15.7, 10.1, 1.0) 127.3, CH 5.85, ddd (15.5, 9.6, 1.0)
14 138.6, CH 5.20, ddd (15.7, 10.9, 4.8) 135.4, CH 4.94, ddd (15.5, 10.8, 4.5)
15 42.6, CH22.01, m 42.5, CH21.93, m
1.84, ddd (12.0, 11.0, 10.9) 1.69, m
16 32.7, CH 1.33, m 28.3, CH 1.61, m
17 49.2, CH21.70, m 53.9, CH21.69, m
1.50, dt (13.8, 3.8) 1.52, m
18 34.8, CH 2.44, m 73, C OH: 4.83, s
19 155.4, CH 7.14, dd (16.4, 7.2) 155.4, CH 6.58, d (16.5)
20 130.6, CH 7.01, br d (16.4) 129.2, CH 6.73, d (16.5)
21 196.7, C 195.5, C
22 25.0, CH31.03, d (7.0) 26.2, CH30.98, d (6.8)
23 20.8, CH31.10, d (6.9) 30, CH31.21, s
10137.4, C 136.8, C
20/60129.2, CH 7.21, br d (7.8) 129.7, CH 7.18, br d (7.7)
30/50128.7, CH 7.33, br t (7.8) 128.2, CH 7.29, br t (7.7)
40
126. 9, CH
7.25, br t (7.8) 126.5, CH 7.21, br t (7.7)
a700 Mhz for 1H, 175 MHz for 13C in CHCl3-d,b500 Mhz for 1H, 125 MHz for 13 C in DMSO-d6.
2.3.2. Fragiformin D
Colorless oil ([
α
]
25
D
not determined for lack of material).
1
H NMR (500 MHz, DMSO-d
6
): see
Table 1;
13
C NMR (125 MHz, DMSO-d
6
): see Table 1. HR-ESIMS m/z 450.2644 ([M + H]
+
, calcd for
C28H38 NO4450.2639).
2.4. Cytochalasans
All cytochalasans used are listed with their names in Table 2. For treatment of the cells,
the cytochalasans were dissolved in DMSO (Carl Roth GmbH, Karlsruhe, Germany).

Biomolecules 2019,9, 73 5 of 14
Cytochalasin B (
4
), F (
7
), Z2 (
11
) and deoxaphomin (
5
) were isolated from cultures of P. simillis
as described above—cytochalasans “6” (
13
), “9” (
14
), “10” (
15
), “11” (
16
), 18-epi-cytochalasan 12 (
2
),
L-696,474 (
9
), 21-O-deacyl-L-696,474 (
10
), 18-fragiformin A (
1
) and 18-epi-fragiformin B (
12
) were
isolated previously from mycelial cultures of H. fragiforme, as described [
8
]. Compound
17
was
isolated from Daldinia spp. as described in 2.2. Cytochalasin H (
8
), 19,20-epoxycytochalasin C,
19,20-epoxycytochalasin D, 19,20-epoxycytochalasin N, 18-deoxy-19,20-epoxycytochalasin Q (
18–21
)
and the phenochalasins C (
22
) and D (
23
) were isolated from Daldinia spp. by RP-HPLC as described
in [
8
]. Chaetoglobosins were isolated previously from Ilyuha vitellina [
13
]. The organism “Hypoxylon
kretzschmarioides”, from which the phenochalasins C and D were derived [
8
], has meanwhile been
subjected to a taxonomic study that resulted in its transfer to the genus Daldinia and the herbarium
specimen represents the epitype of Daldinia kretzschmarioides, comb. nov. [14].
2.5. Cell Culture
U2OS, a human osteosarcoma cell line [ATCC HTB-96] was cultured in Dulbecco’s modified
minimum essential medium (DMEM, Life Technologies, Carlsbad, CA, USA) containing 10% fetal
bovine serum, 1% L-glutamine, 1% minimum essential medium nonessential amino acids (MEM
NEAA) and 1% sodium-pyruvate (Life Technologies, Carlsbad, CA, USA) at 36 ◦C and 5% CO2.
2.6. Cytochalasan Treatment
For cytochalasan treatment, the cells were grown on glass coverslips. Prior to cell growth,
the coverslips were coated with 25
µ
g/mL fibronectin in phosphate buffered saline (PBS) for one hour.
Cytochalasans were applied at a concentration of 1 and 5
µ
g/mL for 1 h. Washout experiments were
conducted by exchanging the cytochalasan-containing medium with DMEM after incubation time and
incubating for another hour. Further details can be found in the Supplementary Information.
2.7. Immunofluorescence
Treated cells were fixed with 4% paraformaldehyde (AppliChem, Darmstadt, Germany) in PBS
for 20 min at room temperature. Fixed cells were washed with PBS, permeabilized with 0.1% Trition
X-100 (Bio-Rad Laboratories, Hercules, CA, USA) in PBS for 1 min at room temperature and again
transferred to PBS. The actin cytoskeleton was stained using fluorescently labelled phalloidin ATTO
594 (1:200 ATTO-Tec, Siegen, Germany) in PBS for 1 h. The cover slips were mounted in Prolong
Diamond antifade mountant with DAPI (inVitrogen, Carlsbad, CA, USA) to stain the nucleus. Pictures
were taken with a Axio Vert 135 TV inverse microscope with phase contrast and CoolSnap 4k camera
(Zeiss, Oberkochen, Germany). Pictures were processed using Image J (NIH, Bethesda, MD, USA).
3. Results and Discussion
3.1. Structure Elucidation of the New Compounds
A fruiting body extract of H. fragiforme was fractionated by reversed-phase HPLC and provided
the new metabolites
1
and
2
as colorless oils. For
1,
the molecular formula C
28
H
35
NO
3
was deduced
based on its [M + H]
+
and [M+Na]
+
peaks at m/z 434.2688 and 456.2502 in the HRESIMS spectrum,
implying 12 degrees of unsaturation.
1
H and HSQC NMR spectra revealed the presence of four
methyls, three methylenes, and seven olefinic (two with dual intensity) as well as six aliphatic methines.
In addition, the
13
C spectrum specified a conjugated ketone, an amide carbon, and four further carbons
devoid of bound protons. Subsequently, spin systems were constructed by
1
H,
1
H COSY and TOCSY
correlations, which were connected by HMBC correlations, to form a cytochalasan skeleton (Figure S1).
The closest structural relative for the planar structure of
1
is fragiformin A, the 18-hydroxyl derivative
of
1
[
15
]. The stereochemistry of
1
was assigned by ROESY data: ROESY correlations between 13–H
and 20–H as well as 14–H and 19–H supported the characteristic conformation described for the
eleven-membered ring system [
15
]. ROESY correlations between 23–H
3
and 20–H, located below the

Biomolecules 2019,9, 73 6 of 14
molecular main plain, in addition to those between 18–H and 16–H, located above the molecular main
plain, confirm the downwards orientation of 23–H
3
and upwards orientation of 18–H and thus an 18S
configuration. We propose the trivial name fragiformin C for compound
1
, whose systematic IUPAC name
is (7S,13E,16S,18S,19E)-16,18-dimethyl-7-hydroxy-10-phenyl-[
11
]-cytochalasa-5,13,19-triene 1,21-dione [
16
].
Metabolite
2
, isolated from the stromatal (fruiting body) extract of H. fragiforme, was analyzed
for its molecular formula C
28
H
35
NO
4
by HRESIMS, indicating a formal addition of an oxygen
atom compared to
1
. The NMR data of
2
showed a similarity to those of
1
for specifying the
cytochalasin scaffold. An analysis of
1
H,
13
C and HSQC data identified the key differences as the
replacement of the C–5/C–6 double bond by a methine and an oxygenated carbon devoid of bound
protons, and the replacement of methine C–18 by oxygenation. Consequently,
2
was elucidated as
16,18-dimethyl-6,7epoxy-18-hydroxy-10-phenyl-[
11
]-cytochalasa–13,19-diene-1,21-dione, which has been
described previously [
17
]. However, a careful examination of
13
C chemical shifts revealed significant
differences, specifically for C–17 and C–23. Therefore, the configuration of
2
was examined by ROESY data.
Analogously to
1
, ROESY correlations between 23–H
3
and 20–H and between 18–H and 16–H confirmed
the downwards orientation of 23–H
3
and the upwards orientation of 18–H, respectively. Consequently,
compound
2
with its 18Rconfiguration is the 18-epimer of the known compound
17
isolated from a Daldinia
species [
18
], which was later identified as D. eschscholtzii [
19
]. Its systematic name is (7S,13E,16S,18R,19E)-
16,18-dimethyl-6,7epoxy-18-hydroxy-10-phenyl-[
11
]-cytochalasa–13,19-diene-1,21-dione [
13
], and here we
propose the trivial name fragiformin D for the compound.
Interestingly, the two novel cytochalasins that we report in this study were actually discovered in
the course of another project aimed at the identification of complex azaphilone pigments that were
detected in the stromata of carbonized, fossil H. fragiforme originating from excavations in France [
20
].
However, they were not detected in the ancient specimens but only in the recently collected reference
material that was used to isolate the metabolites in sufficient quantities for structure elucidation.
The producer organism is actually the type species of the recently resurrected family Hypoxylaceae [
21
]
and belongs to the most frequently encountered macromycetes in the Northern hemisphere.
Cytochalasans B (
4
), F (
7
), Z2 (
11
) and deoxaphomin (
5
) were concurrently isolated from the
endophytic fungus Preussia similis DSM 32328, and their structures were identified by comparing
their
1
H and
13
C chemical shifts as well as the HRMS data to those reported in the literature
(references [22,23]
for compound
4
; reference [
23
] for compound
5
; references [
24
,
25
] for compound
7
;
reference [
26
] for compound
11
). An authentic sample of commercially available cytochalasin B was
used for comparison, and the NMR data of the isolated cytochalasin B (
4
) were identical with the
commercial sample.
Further known cytochalasans (Table 2, Figure 1) were isolated from other Sordariomycetes species
and identified comparing the
1
H and
13
C chemical shifts and the HRMS data to those reported in the
literature as previously described (see Table 2).

Biomolecules 2019,9, 73 7 of 14
Table 2.
Effects of cytochalasans on mammalian cells and against biofilms of Staphylococcus aureus.
Actin
disruption
:
+++
complete disruption at 1
µ
g/mL,
++
complete disruption at 5
µ
g/mL,
+
incomplete
disruption at 5
µ
g/mL,
-
no disruption; Reversibility:
+
reversible effect,
+/-
partially reversible effect,
-
irreversible;
nd
: not determined because it was not active in the first place.
Anti-Biofilm activity
:
activities taken from the study by Yuyama et al. [
8
];
nt
: compound not tested, due to insufficient
amounts available or apparent instability.
Trivial Name Actin Disruption Reversible Anti-Biofilm [8] Biological source
1Fragiformin C + +/- nd
Hypoxylon fragiforme (this study)
2Fragiformin D +++ - nd H. fragiforme (this study)
3Saccalasin A - nt + Daldinia sacchari [12]
4Cytochalasin B ++ + -Preussia similis (this study)
5Deoxaphomin +++ - + P. similis (this study)
6Cytochalasin D +++ +/- - Zygosporium mansorii (Sigma)
7Cytochalasin F + + nd P. similis (this study)
8Cytochalasin H +++ + - H. fragiforme [8]
9L-696,474 +++ + ++ H. fragiforme [8]
10 21-O-Deacyl-L-696,474 +++ + + H. fragiforme [8]
11 Cytochalasin Z2 + + nd P. similis (this study)
12 “Cytochalasin 6” [16]+++ - +++ D. eschscholtzii [8]
13 “Cytochalasin 9” [16]++ - - D. eschscholtzii [8]
14 “Cytochalasin 10” [17]+ +/- +++ D. eschscholtzii [8]
15 “Cytochalasin 11” [17]} + +/- +++ D. eschscholtzii [8]
16 “Cytochalasin 12” [18]- nt + D. eschscholtzii [8]
17 New Cytochalasin + +/- nd D. eschscholtzii [8]
18 19,20-Epoxycytochalasin C +++ + ++ Rosellinia rickii [8]
19 19,20-Epoxycytochalasin D +++ +/- - R. rickii [8]
20 19,20-Epoxycytochalasin N + + - R. rickii [8]
21
18-Deoxy-19,20-Epoxy-cytochalasin Q
++ + - R. rickii [8]
22 Phenochalasin C ++ - + H./D. kretzschmarioides [8]
23 Phenochalasin D - nt ++ H./D. kretzschmarioides [8]
24 Chaetoglobosin A + + +++ Ijuhya vitellina [13]
25 Chaetoglobosin D ++ - nd Il. vitellina [13]

Biomolecules 2019,9, 73 8 of 14
Biomolecules 2019, 9, x FOR PEER REVIEW 8 of 14
Figure 1. Chemical structures of cytochalasans employed in this study.
Figure 1. Chemical structures of cytochalasans employed in this study.
It is interesting to compare the chemical shift of methyls C–22 and C–23 of
1
and
2
to those of
compounds that are epimeric at C–18, but otherwise possess an identical carbon backbone, such as
2

Biomolecules 2019,9, 73 9 of 14
and
17
. Whereas the chemical shifts
δC
of C–22 are nearly identical in all cases (
9
: 25.3;
10
: 25.2;
3
: 26.1;
13: 26.2), the C–23 chemical shifts differ significantly (9: 22.1; 10: 22.4; 3: 17.5; 13: 17.6).
The same fact was observed for compounds that are oxidized at C–18, such as
12
and its 18-epimer,
fragiformin B (see NMR data in reference [
15
] for comparison); C–23 is significantly shifted downfield
in fragiformin B (
δC
30.2) compared to
12
(
δC
25.7). Consequently, the chemical shift of methyl CH
3
-23
might be assessed as an indicator for the variable stereochemistry at C–18. The chemical structures of
all compounds tested are shown in Figure 1.
3.2. Effects of Cytochalasans on Cell Cultures
The effects of the compounds on the actin cytoskeleton of mammalian cells were analyzed by
fluorescence microscopy upon staining with fluorescently labelled phalloidin to stain for filamentous
actin (F-actin) and with 4
0
,6-diamidino-2-phenylindole (DAPI) to stain for nuclear DNA. To enable
comparability between the compounds, the effects on cellular F-actin structures were analyzed at two
different concentrations (and at two different exposure times, not shown) and classified using stepwise
gradations from “
+++
” to “
-
“ (see Table 2) as follows: compounds leading to complete disruption of
the actin cytoskeleton at a concentration of 1
µ
g/mL were marked with “
+++
”; compounds leading
to complete disruption of the cytoskeleton at concentrations of 5
µ
g/mL were classified as “
++
”;
compounds showing an incomplete disruption of the cytoskeleton at 5
µ
g/mL were categorized as
“
+
”; and finally, compounds that did not affect the actin cytoskeleton at all in this study were classified
as “-“.
The reversibility of cytochalasan-mediated effects was assessed in a 1 h-washout experiment and
classified as follows: full recovery of actin structures was categorized as reversible “
+
”; partial recovery
as partially reversible “
+/-
“; and, when disruption of the actin cytoskeleton remained severe even after
washout, the effects were classified as irreversible “
-
“. Results of these experiments are summarized in
Table 2and representative examples of fluorescently labelled cells are displayed in Figure 2.

Biomolecules 2019,9, 73 10 of 14
Biomolecules 2019, 9, x FOR PEER REVIEW 10 of 14
Figure 2. Immunofluorescence staining with phalloidin of U2OS cells treated with cytochalasans. (A)
Treated with 1 µM cytochalasin H (8). (B) Treated with 5 µM cytochalasin H. (C) Treated with 1 µM
cytochalasin B (4). (D) Treated with 5 µM cytochalasin B. (E) treated with 1 µM chaetoglobosin D
(25). (F) Treated with 5 µM chaetoglobosin D. (G) Treated with 5 µM DMSO (negative control). (H)
Washout after treatment with 5 µM cytochalasin H.
Of all the cytochalasans, cytochalasin B (4) and its effect on the actin cytoskeleton has been
characterized best, as revealed by various reports in the literature [6,27,28]. Therefore, this
metabolite was used as the reference compound in this study. Notably, most cytochalasans tested
here showed characteristic phenotypic changes of the actin cytoskeleton upon treatment. However,
the extent of actin cytoskeleton disruption was highly variable. Less severe effects (e.g., compounds
7 and 17) showed small patches of aggregated actin and rearrangement but no loss of stress fibers. In
contrast, cells treated with more potent compounds showed complete actin disruption and the
formation of dense, asterisk-like F-actin aggregates (e.g., compounds 2 and 18).
These phenotypic changes of the actin cytoskeleton have been reported previously in several
studies [6,29,30]. Treatment with the two chaetoglobosins studied, which—like the majority of the
compounds used in our study, were evaluated here for the first time—resulted in a different
phenotype, which also showed actin aggregation. However, this was not associated with a
significant loss of the pre-existing actin filaments.
Figure 2.
Immunofluorescence staining with phalloidin of U2OS cells treated with cytochalasans.
(
A
) Treated with 1
µ
M cytochalasin H (
8
). (
B
) Treated with 5
µ
M cytochalasin H. (
C
) Treated with 1
µ
M
cytochalasin B (
4
). (
D
) Treated with 5
µ
M cytochalasin B. (
E
) treated with 1
µ
M chaetoglobosin D (
25
).
(
F
) Treated with 5
µ
M chaetoglobosin D. (
G
) Treated with 5
µ
M DMSO (negative control). (
H
) Washout
after treatment with 5 µM cytochalasin H.
Of all the cytochalasans, cytochalasin B (
4
) and its effect on the actin cytoskeleton has been
characterized best, as revealed by various reports in the literature [
6
,
27
,
28
]. Therefore, this metabolite
was used as the reference compound in this study. Notably, most cytochalasans tested here showed
characteristic phenotypic changes of the actin cytoskeleton upon treatment. However, the extent of
actin cytoskeleton disruption was highly variable. Less severe effects (e.g., compounds
7
and
17
)
showed small patches of aggregated actin and rearrangement but no loss of stress fibers. In contrast,
cells treated with more potent compounds showed complete actin disruption and the formation of
dense, asterisk-like F-actin aggregates (e.g., compounds 2and 18).
These phenotypic changes of the actin cytoskeleton have been reported previously in several
studies [
6
,
29
,
30
]. Treatment with the two chaetoglobosins studied, which—like the majority of the
compounds used in our study, were evaluated here for the first time—resulted in a different phenotype,

Biomolecules 2019,9, 73 11 of 14
which also showed actin aggregation. However, this was not associated with a significant loss of the
pre-existing actin filaments.
To reveal structural features of cytochalasans that correlate with the potency of the compounds to
disrupt the actin cytoskeleton, we performed a meta-analysis and inspected the chemical structures of
the different potency groups in depth. Our analysis revealed common features among the grouped
compounds. Hydroxyl groups at C7 and C18 were most prominent in the “
+++
” group, with each
compound showing at least one of these. In the “
-
“ group, both functional groups were completely
absent, emphasizing the importance of these structural feature for actin disruption. The importance
of the C7 hydroxyl group also was reported for anticancer activity [
27
], which may be related to the
effect on the actin cytoskeleton. Most striking are differences in the activities of structurally closely
related compounds, such as phenochalasins C (
22
) and D (
23
), and the compound
17
and fragiformin
D (
2
). Phenochalasin D (
23
) differs from phenochalasin C (
22
) by the addition of a hydroxyl group at
C7, as well as a methyl group instead of an exocyclic double bond at C6. However, the cells showed
strong actin disruption (“
++
”) upon treatment with phenochalasin D, but no actin disruption upon
treatment with phenochalasin C, highlighting the importance of the C7 hydroxyl group for activity
once more (see Table S1).
Comparison of compound
17
(“
+
”) and fragiformin D (
2
) (“
+++
”) revealed the importance
of stereochemistry of functional groups for actin disruption activity (see Table S1). Also,
the stereochemistry of the C18 group seems to affect the binding affinity towards actin, since
fragiformin D showed an irreversible, and compound
17
a reversible, actin disruption. Also, a high
abundance of compounds showing an acetylated C21 residue is located in the “
+++
” group, indicating
a possible correlation of this feature with high actin disruption capacity. Lastly, higher conformational
freedom of the macrocycle of cytochalasans was reported to positively affect the anticancer activity
of cytochalasans, whereas the size of the macrocycle did not [
31
]. Here, no evidence for a correlation
between conformational freedom of the macrocycle and actin disruption capacity was found. However,
all highly potent compounds (“
+++
”) with the exception of deoxaphomin (
5
), showed a 11-membered
macrocycle, which is the smallest possible macrocycle reported for cytochalasans. In support of this
notion, higher activity of cytochalasans with 11-membered macrocycles compared to 14-membered
macrocycles has been reported previously [
32
]. Comparing the trait of reversibility of the compounds,
none of the different potency groups correlated with being reversible or irreversible. This indicates
that the reversibility of cytochalasan effects is not structurally linked to their potency concerning
actin disruption. However, all compounds showing irreversible effects on actin filaments carry only
few functional groups in addition to hydroxylated C–7 residue. Also, none of these compounds is
acetylated at C–21. The absence of additional functional groups in the irreversible compounds groups
indicates that, in general, the small size of the compounds contributes to irreversibility.
As mentioned in the introduction, we recently have studied a largely overlapping panel of
cytochalasans that were examined here for their effect on the biofilm formation of S. aureus [
8
]
and found likewise that some of the tested molecules had rather strong inhibitory effects, while
others were completely inactive in this bioassay. According to preliminary results, the degree
of biofilm inhibition can be influenced by, e.g., the presence of an isomeric double bond in the
macrocycle, the degree of acetylation of the hydroxyl groups, or the presence of a phenol at C4
0
phenyl group, as well as epoxy groups in the macrocyclic ring. Notably, these results were mostly
deduced from pairwise comparison of closely related congeners, and not in the classical manner for
establishing structure–activity relationships, i.e., by semisynthetic modification of the same basic
structure. Therefore, the results must be regarded as tentative and can only guide medicinal chemists
in a future concise optimization program to find the best inhibitor. Interestingly, despite the two traits
(biofilm inhibition vs. actin modulation) probably having different MOA, there are three compounds,
i.e., L-696,474 (
9
), epoxycytochalasin C (
18
) and compound
12
, which show strong biofilm inhibition
as well as strong actin disruption. However, further correlations between the two traits could not
be determined. Recently, it was shown that cytochalasans can cause either cytotoxic or cytostatic

Biomolecules 2019,9, 73 12 of 14
effects in human cancer cells [
31
]. Those compounds that could not be removed from the actin
filaments, indicating that they irreversibly inhibited actin-related processes in cells and ultimately will
prolong apoptotic signaling, may constitute good candidates for development of anticancer drugs
by a rational, medicinal chemistry-driven optimization program. On the other hand, compounds
14
and
15
from D. eschscholtzii, and in particular the chaetoglobosins, might serve as initial structures
for development of biofilm inhibitors if their toxicity could be further reduced by semisynthesis, or
even total synthesis, because they showed relatively low potency against actin but were among the
strongest inhibitors of biofilm in S. aureus in our previous study. However, this topic will have to be
addressed in future studies.
4. Conclusions
This study described the isolation of two new cytochalasans from H. fragiforme, adding to the
growing the number of cytochalasans. Recently, with the discovery of cytotoxic, cytostatic, antiviral
and anti-biofilm activities of cytochalasans, interest in using these compounds as therapeutic agents
is reemerging. However, the use in human therapy requires excellent knowledge and in-depth
characterization of the molecular effects caused by these fungal metabolites. The aim of this study was
to systematically analyze the effects of cytochalasans on the actin cytoskeleton
in vivo
and link these
to structural features of the compounds. This meta-analysis aims at enabling the prediction of the
molecular effects of novel cytochalasan derivatives. Our data strongly suggest that the presence of C–7
and C–18 hydroxylation is correlated with high potency to disrupt the actin cytoskeleton of eukaryotes,
very likely related to their cytotoxic and cytostatic activities. Also, we show here that the capability of
the compounds to disrupt the actin cytoskeleton does not directly correlate with the reversibility of
this effect, which in turn seems to be connected to the compound size. This important information
helps to understand the effects of these metabolites and paves the way for future approaches to
synthesize compounds with the desired features but lacking unwanted activities. We therefore propose
that certain compounds should be isolated from the available fungal strains in larger quantities and
subjected to a microderivatization program, or obtained by total synthesis in larger quantities and
finally subjected to a broad biological characterization including the evaluation of their biological
activities against mammalian cells, viruses and pathogenic microbes. This endeavor would afford
substantial capacities for biotechnological production of the molecules and medicinal chemistry.
Supplementary Materials:
Supplementary material can be found at http://www.mdpi.com/2218- 273X/9/2/
73/s1. Figures S1 and S2: COSY and TOCSY (blue arrows), HMBC (green arrows) and ROESY (violet arrows)
correlations indicating the structures of fragiformin C (1) and fragiformin D (2). Figure S3: HPLC-HRESIMS data
of 1. Figure S4:
1
H NMR spectrum (700 MHz, CDCl3) of 1. Figure S5:
13
C NMR spectrum (175 MHz, CDCl3) of
1. Figure S6: COSY NMR spectrum (700 MHz, CDCl3) 1. Figure S7: ROESY NMR spectrum (700 MHz, CDCl3)
1. Figure S8: HSQC NMR spectrum (700 MHz, CDCl3) 1. Figure S9: HMBC NMR spectrum (700 MHz, CDCl3)
1. Figure S10: HPLC-HRESIMS data of 2. Figure S11:
1
H NMR spectrum (500 MHz, DMSO-d6) of 2. Figure
S12:
13
C NMR spectrum (125 MHz, DMSO-d6) of 2. Figure S13: COSY NMR spectrum (500 MHz, DMSO-d6)
of 2. Figure S14: ROESY NMR spectrum (500 MHz, DMSO-d6) of 2. Figure S15: HSQC NMR spectrum (500
MHz, DMSO-d6) of 2. Figure S16: HMBC NMR spectrum (500 MHz, DMSO-d6) of 2. Figure S17:
1
H NMR
spectrum (500 MHz, CDCl3) of 17. Figure S18:
13
C NMR spectrum (125 MHz, CDCl3) of 17. Figure S19:
1
H NMR
spectrum (500 MHz, Acetone-d6) of cytochalasin B (4). Figure S20:
13
C NMR spectrum (500 MHz, Acetone-d6)
of cytochalasin B (4). Figure S21: HSQC NMR spectrum (500 MHz, Acetone-d6) of cytochalasin B (4). Figure
S22:
1
H NMR spectrum (500 MHz, Acetone-d6) of deoxaphomin (5). Figure S23:
13
C NMR spectrum (500 MHz,
Acetone-d6) of deoxaphomin (5). Figure S24:
1
H NMR spectrum (500 MHz, Acetone-d6) of cytochalasin F (7).
Figure S25:
13
C NMR spectrum (500 MHz, Acetone-d6) of cytochalasin F (7). Figure S26:
1
H NMR spectrum (500
MHz, Acetone-d6) of cytochalasin Z2 (11). Figure S27:
13
C NMR spectrum (500 MHz, Acetone-d6) of cytochalasin
Z2 (11). Table S1: Cytochalasan treatment of U2OS cells.
Author Contributions:
R.K. contributed to isolation of compounds, compound testing and manuscript writing;
F.S. contributed to structure elucidation and manuscript writing; S.W. contributed to the isolation of compounds;
L.W. contributed to experiment guiding; S.E.H. and S.R.N. contributed to the isolation of compounds; T.E.B.S.
contributed to experiment guiding and edited the manuscript; M.S. and J.J.L.-a. contributed to experiment guiding
and edited the manuscript.

Biomolecules 2019,9, 73 13 of 14
Funding:
This project received funding from the European Union’s Horizon 2020 research and innovation
programme (RISE) under the Marie Skłodowska-Curie grant agreement No. 645701, project acronym “GoMyTri”,
lead beneficiaries J.J.L.-a. and M.S. T.E.B.S. was supported by the Helmholtz Society (HGF impulse fund
W2/W3-066). L.W. gratefully acknowledges a stipend from the HSBDR Graduate School, Leibniz University
Hannover. S.R.N. gratefully acknowledges a stipend from the Algerian government and S.E.H. is grateful to the
Alexander-von-Humboldt Foundation for a postdoctoral stipend.
Acknowledgments:
We thank A. Gollasch, S. Reinecke, K. Schober and C. Bergmann for conducting HPLC-MS
measurements, and V. Stiller and A. Skiba for expert technical assistance in the mycological laboratory. We further
thank A. Otto for expert technical assistance in cell culture. We also thank K. Wittstein, K.T. Yuyama, W.-R.
Abraham and all other coauthors of the preceding study that had embarked on the evaluation of biofilm inhibition
of cytochalasans in Staphylococcus aureus.
Conflicts of Interest: The authors declare no conflicts of interest.
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2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
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