Isolation, structure, and biological activities of fellutamides C and D from an undescribed Metulocladosporiella (Chaetothyriales) using the genome-wide Candida albicans fitness test

Article abstract of DOI:10.1021/np2001573

In a whole-cell mechanism of action (MOA)-based screening strategy for discovery of antifungal agents, Candida albicans was used, followed by testing of active extracts in the C. albicans fitness test (CaFT), which provides insight into the mechanism of a

Full text of DOI:10.1021/np2001573

ARTICLE
pubs.acs.org/jnp
Isolation, Structure, and Biological Activities of Fellutamides C and D
from an Undescribed Metulocladosporiella (Chaetothyriales) Using the
Genome-Wide Candida albicans Fitness Test
Deming Xu,† John Ondeyka,‡ Guy H. Harris,‡ Deborah Zink,‡ Jennifer Nielsen Kahn,^ Hao Wang,†
Gerald Bills,§ Gonzalo Platas,§ Wenxian Wang,|| Alexander A. Szewczak,|| Paul Liberator,^ Terry Roemer,†
and Sheo B. Singh*,‡
‡Department of Natural Products and Medicinal Chemistry and ^Infectious Diseases, Merck Research Laboratories, Rahway,
New Jersey 07065, United States
†Merck-Frosst Center for Therapeutic Research, Merck-Frosst Canada, Kirkland, Quebec, Canada H9H 3L1
§Centro de Investigaciꢀon Bꢀasica (CIBE), Merck Sharp & Dhome de Espan~a, S. A., Madrid, Spain
Merck Research Laboratories, Boston, Massachusetts, United States
S Supporting Information
b
ABSTRACT: In a whole-cell mechanism of action (MOA)-
based screening strategy for discovery of antifungal agents,
Candida albicans was used, followed by testing of active extracts
in the C. albicans fitness test (CaFT), which provides insight
into the mechanism of action. A fermentation extract of an
undescribed species of Metulocladosporiella that inhibited pro-
teasome activity in a C. albicans fitness test was identified. The chemical genomic profile of the extract contained hypersensitivity of
heterozygous deletion strains (strains that had one of the genes of the diploid genes knocked down) of genes represented by
multiple subunits of the 25S proteasome. Two structurally related peptide aldehydes, named fellutamides C and D, were isolated
from the extract. Fellutamides were active against C. albicans and Aspergillus fumigatus with MICs ranging from 4 to 16 μg/mL and
against fungal proteasome (IC50 0.2 μg/mL). Both compounds showed proteasome activity against human tumor cell lines, potently
inhibiting the growth of PC-3 prostate carcinoma cells, but not A549 lung carcinoma cells. In PC-3 cells compound treatment
produced a G2M cell cycle block and induced apoptosis. Preliminary SAR studies indicated that the aldehyde group is critical for the
antifungal activity and that the two hydroxy groups are quantitatively important for potency.
atural products continue to provide a rich chemical diversity
of biologically active molecules. The conventional strategy
lipopeptide echinocandins (inhibiting β-1,3-glucan synthesis).
All treatment options have limitations including resistance, toxicity
leading to a very narrow therapeutic window, and/or lack of oral
efficacy, thus creating a serious need for new antifungal agents.
Our chemical genomic approach, the C. albicans fitness test
(CaFT, adopted from an analogous assay developed in the yeast
Saccharomyces cerevisiae),4 relies on the nature of obligatory diploidy
of the fungus. For a vast majority of genes of the pathogens,
heterozygous deletion of the genes results in no detectable growth
defects of the pathogens under standard conditions. However, in
the presence of sublethal concentrations of an antifungal com-
pound, only some heterozygotes display growth/fitness varia-
tions (hypersensitivity or resistance) with significant deviation
from the rest of the heterozygotes. In many cases, they include
those corresponding to the specific target, and in the other cases
they represent aspects of the mechanism of action (MOA) of the
compound.5À10 The strains with significant deviations in their
growth induced by the chemical perturbation collectively com-
prise the chemical genetic profile of a given antifungal compound.
N
of natural product discovery has been seriously challenged by the
frequent rediscovery of known molecules.1 One of the keys to
enhance success in natural products research is to identify mechan-
istic imprints present in the crude extract prior to chemical
fractionation so that the chemical resources will be allocated to
those extracts with potential novel biological activities and hence
likely to yield novel chemical structures. We have implemented a
chemical genomic approach using the principal human fungal
pathogen Candida albicans that genetically profiles mechanistic
imprints of biologically active molecules from various sources. As
part of our effort to discover new antimicrobial compounds, we
also expanded our isolation of microorganisms from diverse
geographical regions and habitats and improved high-throughput
fermentation methods by miniaturization and automation.2
C. albicans and other Candida spp. are the most significant
causes of fungal infections in the hospital setting worldwide in
terms of total number of cases and mortality.3 The standard
antifungal therapy is restricted to drugs in three mechanistic
categories, namely, azoles (inhibiting ergosterol biosynthesis),
macrocyclic polyenes (perturbing the cell membrane), and cyclic
Received: February 16, 2011
Published: July 13, 2011
r
Copyright
2011 American Chemical Society and
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As the assay contained ∼2900 strains in the published version5
and ∼5400 (i.e., ∼90% genomic coverage for essential genes) in
the upgraded version, it is possible to genetically profile an
antifungal compound of interest at the genomic level. Applying
this approach to crude extracts of natural products, we were able
to discern novel biological activities present in the crude extracts,
which in turn led to the discovery of novel chemical structures.7,11,12
For example, we identified an extract with a chemical genomic
profile indicative of the RNA cleavage/polyadenylation complex
as the target. Although the profile of the extract overlapped with
that of cordycepin (an analogue of adenosine), the latter con-
tained specific resistance of a heterozygous deletion strain for the
fungal nucleoside transporter, an indication of uptake of cordy-
cepin by this transporter, and thus differentiated itself. The
purified compound from this extract is a novel isoxazolidinone
that inhibits the RNA poly(A) polymerase.7,11
Applying the same approach we identified a fungal fermenta-
tion extract that contained proteasome-inhibiting activity. Here
we present bioassay-guided isolation and structure elucidation of
two new antifungal agents, fellutamides C (1) and D (2), from
the extract. Both compounds are potent inhibitors of fungal
proteasome activity with broad spectrum antifungal activity. In
addition, they showed significant potency against the human
tumor cell line PC-3.
Figure 1. Chemical genomic profiling and characterization of extract
ECC2567. (A) CaFT profiles of the original extract (ECC2567),
regrowth extract, mixture of fellutamides C and D, and gliotoxin. Two
independent CaFT experiments were selected for each extract/com-
pound, with IC indicated on the top. In the hierarchical clustering
analysis, heterozygous deletion strains were selected on the basis of the
absolute values of their normalized z-scores g4.0 in at least two
experiments. The result is displayed by heat map with scale indicated
at the bottom. The genes for subunits of the 20S core particle are
indicated in red (with R, β indicating the rings), the 19S regulatory
particle in green, and those involved in nuclear transport in blue. The
arrows indicate the genes whose heterozygous deletion strains were
tested in Figure 3A and B. (B) In vitro assay for the fungal proteasome
activity. The activities in the presence of compounds at the concentra-
tions indicated were normalized with that of mock treatment. Shown are
averages of three measurements with bars indicating standard deviations.
Note that the antifungal MIC of gliotoxin was ∼4 μg/mL, and lacta-
cystin was not active against C. albicans (at 200 μg/mL).
’ RESULTS AND DISCUSSION
CaFT Profiling of the Fungal Extract ECC2567. In an attempt
to discover novel antifungal agents from microbial sources, we
screened and tested extracts of fermentation broths exhibiting
whole-cell Candida activity (see below) in the C. albicans fitness
test. The extract under investigation, ECC2567, generated a profile
indicating the target as the fungal 26S proteasome. It induced
specific hypersensitivity of heterozygotes for genes of several
subunits (see below for details) (Figure 1A). The in vitro protea-
some assay confirmed the potent inhibitory effect of ECC2567, with
MIC e 0.25% WBE (whole-broth equivalents, Figure 1B). Glio-
toxin is known to inhibit the chymotrypsin-like activity of the 20S
proteasome in vitro13À15 and inhibited the fungal proteasome
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activity at concentrations much higher than the whole-cell activity
(Figure 1B). It generated a nonoverlapping profile in the fitness test
(Figure 1A). These results indicated that the antifungal activity of
this extract targets the proteasome and is distinct from that of
Gliotoxin and lactacystin (another proteasome inhibitor but pro-
duced by Streptomyces sp.), thus prompting us to isolate the active
component(s) present in ECC2567.
Producing Fungus and Fermentation. The producing or-
ganism of extract ECC2567, F-192,783 (Figure S1, Supporting
Information), was isolated from a soil sample collected from
Equatorial Guinea. Sequence analysis of the rDNA (28s D1-D2
and ITS regions, Figures S2, S3, Supporting Information) indicated
that it was closely related to and possibly congeneric with a complex
of species in the genus Metulocladosporiella (Ascomycotina,
Chaetothyriales), including M. musae, M. musicola, and the closely
related Cladosporium adianticola.16 In vitro, the fungus produces
melanized micronematous to macronematous conidiophores
(up to 150 μm tall) that terminate apically in two to three levels
of polyblastic metulae that give rise to acropetal chains of conidia.
The conidia are fusoid to cylindrical, smooth, hyaline to only
faintly melanized, predominantly 1-sepate, mostly 15À50 μm
long by 5À10 μm wide, with the extremes narrowing to an apical
scar (Figure S1, Supporting Information). The consistently
septate conidia easily distinguish the fungus from either M. musae
or M. musicola, and in some aspects, the fungus more closely
resembles C. adianticola, a fungus known only from foliage of an
Adiantum sp. (Pteridaceae) from Cuba.17 However, the conidio-
phores of C. adianticola branch in a more open pattern, and its
septate conidia are more ellipsoidal, ovoid, or subglobose.
Alignments and bootstrapped neighbor-joining analysis of the
both the ITS and D1-D2 regions of the rDNA of this new fungus
(Figures 2, 3 and Supporting Information) support the conclu-
sion that it is distinct.
The antifungal activity produced by F-192,873 was initially
detected in a pilot study that compared microfermentation in the
96-deepwell plates2 with fermentations in the standard 12 mL
Figure 2. ESIMS fragmentation of fellutamides C (1) (R = isopropyl)
and D (2) (R = isobutyl).
Figure 3. Characterization of fellutamides C (1) and D (2). (A and B) Selected heterozygous deletion strains (see Figure 1A) and the control (HIS3)
were tested against fellutamides C and D at the concentrations indicated in YNBD. The growth of each strain in the presence of fellutamide was
normalized by that of mock treatment. Shown are averages of three measurements with coefficient variation less than 10% in most cases (not shown).
(C) In vitro fungal proteasome assay. The proteasome activity in the presence of fellutamide was normalized by that of mock treatment. Shown are
averages of three measurements with bars indicating standard deviations. (D) Anti-aspergillus activity of fellutamides C and D (in mixture). A. fumigatus
strain CEA10 was seeded in solid ACM medium with aliquots of compound/extract (ECC2567) with indicated amounts spotted on the surface. Shown
is 24 h growth at 37 ꢀC.
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vials.18 The study employed 320 newly isolated fungal strains
grown in three and 12 media in aerated vial fermentations and in
static 96-deepwell microfermentations, respectively. Consequently,
52 of the 320 strains (16%) produced antifungal activities in
deepwell microfermentations, compared to 24 (7.5%) in vial
fermentations. The antifungal activity of F-192,873 was detected
in only one medium among 15 different vial and deepwell mi-
crofermentations. The production of antifungal activity by F-192,873
was extremely sensitive to the growth conditions, and activity was
produced only in one medium (YES) in a static microfermenta-
tion. The fermentation was scaled-up by growing the fungus in
200 mL of static liquid YES in 2.8 L Fernbach flasks for 14 days.
Aerated fermentations in the same medium and under otherwise
the same conditions were inactive.
Isolation of Fellutamides C and D. The fermentation broth
was extracted with methyl ethyl ketone (MEK). When tested in
the CaFT, the extract reproduced the original profile (Figure 1A),
suggesting the preservation of the same (or similar) biological
activities in the MEK extract, which was then processed by
liquidÀliquid extraction with EtOAc and water. The EtOAc extract,
which retained the antifungal activity, was chromatographed on
Sephadex LH20. The resulting antifungal fractions were further
separated on an Amberchrome column, followed by reverse-
phase HPLC to yield fellutamides C (10.4 mg, 4.2 mg/L) and D
(7.5 mg, 3.1 mg/L). Both compounds showed poor solubility in
most of the solvents tested, particularly in aqueous systems,
leading to low recovery from HPLC. Prior to HPLC, a mixture of
both compounds was tested in the CaFT and generated a profile
that is largely consistent with those of the original and regrowth
extracts (Figure 1A), suggesting that they were the components
in the extracts associated with the specific biological activities
sought.
β-carbon of leucinal, as determined by the COSY spectrum. The
mass spectrum showed a fragmentation pattern similar to 1
except for changes attributable to the substitution of valinal with
leucinal in 2 (Figure 2). Epimerization of C-2 of valinal and
leucinal was noticed for compounds 1 and 2 during purification.
The absolute configuration of the amino acids and the HTDA
was deduced as follows. A mixture of the two compounds (1 and 2)
was reduced to that of a mixture of corresponding primary alcohols
(3 and 4) by reaction with NaBH4. The mixture of the alcohols
was hydrolyzed by 6 N HCl to yield components, leucinol,
valinol, aspartic acid, 3-hydroxyglutamic acid, and 3-hydroxy
myristic acid. 3-Hydroxymyristic acid was recovered by extrac-
tion with CHCl3. The resulting mixture of amino alcohols and
amino acids was reacted with Marfey’s reagent and compared by
HPLC with the Marfey’s derivatives of both enantiomers of
corresponding amino acids/alcohols. This led to the identifica-
tion of L-valinol, L-leucinol, L-aspartic acid (L-asparagine), and
3R-L-glutamic acid (3R-L-glutamine). The recovered 3R-hydro-
xymyristic acid was purified by RP HPLC and characterized by
NMR and MS. The absolute configuration was confirmed by
comparison of specific rotation (observed [R]D À10, lit. [R]D À16).
Thus the structures 1 and 2 were assigned to fellutamides C and D,
respectively.
These compounds are very closely related to fellutamides A
and B isolated from Penicillium fellutanum recovered from the
gastrointestinal tract of the fish Apogon endekataenia.19 Both
fellutamides A and B contain (3R)-hydroxydodecanoic acid and
leucinal. Fellutamide A contains a glutamine, and fellutamide B
contains a hydroxyglutamine. In addition the other structural
differences between fellutamides B and D (2) lie in the length of
β-hydroxyl aliphatic tails (C14 in B and C12 in D). Fellutamide B
is known to inhibit human proteasome and induce nerve growth
factor synthesis20 and is a potent inhibitor of the Mycobacterium
tuberculosis proteasome.21 However, no antifungal activity has
been reported for fellutamide A or B.
Synthesis of Myristyl-(S)-Asn-(S)-Gln-(S)-Leucinal. Felluta-
mides are peptide aldehydes with β-hydroxyl aliphatic tails of
varying length: (3R)-hydroxydodecanoic acid in fellutamides A
and B and (3R)-hydroxymyristic acid in C and D. Both fell-
utamides C and D contain a hydroxy-(S)-glutamine. We report
here the total synthesis of a simpler version of the fellutamide D
(Scheme 1), which provided structureÀactivity relationship
information. The standard PyBop coupling of L-CBZ-Asn and
L-Gln-O-t-Bu gave an excellent yield of the dipeptide 5 (Scheme 1).
Deprotection of the CBZ group by hydrogenolysis afforded
the dipeptide 6, which was coupled with myristic acid to furnish
myristylated dipeptide 7 in 93% yield. Deprotection of the tert-
butyl ester with TFA followed by PyBop coupling with L-leucinol
produced the tripeptide 8 in 76% yield. The primary alcohol was
oxidized with SO3ÀPy complex to afford the aldehyde 8 in 60%
yield. The overall reaction yield (36%) of the aldehyde was
excellent, but product was lost during HPLC purification due to
poor aqueous solubility.
Biological Activities of Fellutamides C and D. When the
mixture of fellutamides C and D was tested in the CaFT, the
resulting profile reproduced the hypersensitivity of heterozygous
deletion strains for genes of four R and four β subunits of the 20S
proteasome core particle and four other proteins involved in
nuclear transport that was observed in the original and regrowth
extracts tested at comparable inhibitory concentrations (Figure 1A).
In addition, the hypersensitivity of heterozygous deletion of sub-
units of the 19S proteasome regulatory particle extended from
Structural Elucidation of Fellutamides C and D. HRE-
SIFTMS analysis of fellutamide C (1) showed a molecular formula
of C28H51N5O8. Its UV spectrum exhibited end absorption, and
the IR spectrum showed absorption bands for carbonyl and
hydroxy groups. The 13C NMR spectrum (Table 1) showed five
carbonyl carbons in the amide carbonyl region along with three
resonances appearing in the region of R-carbons typical of amino
acids, suggesting that this compound was a peptide. Analysis of
the DEPT spectrum revealed the presence of three methyls, six
1
methines, 13 methylenes, and an aldehyde (Table 1). The H
NMR spectrum in a mixture of DMSO-d6 + CD3CO2D sug-
gested a large methylene envelope indicating the presence of a
fatty chain along with the resonances for three doublets for amide
NH protons and four amide NH singlets assignable to two primary
amides. The COSY spectrum of 1 showed the spin systems
assignable to a valinal, a β-hydroxyglutamine (HyGln), aspar-
agine (Asn), and a β-hydroxytetradecanoic acid (HTDA, hydroxy
myristic acid) (Table 1). The HRESIFTMS spectrum showed
fragment ions corresponding to the loss of a molecule of water
and sequential losses of valinal (m/z 485) and HyGln (m/z 341),
indicating that the valinal was present at the carboxy end and the
Asn formed the N-terminus, which was acylated with HTDA
(Figure 2). The sequence was confirmed by the HMBC correla-
tions of the R-CH and NH to the corresponding carbonyl
carbons (Table 1).
HRESIFTMS analysis of fellutamide D (2) showed a molec-
ular formula of C29H53N5O8 consisting of an additional methy-
lene moiety compared to fellutamide C (1). The NMR spectra
of 2 were identical to the corresponding spectra of 1 except for
the presence of an extra methylene, which was assigned to the
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1
Table 1. H (500 MHz) and 13C (125 MHz) NMR Assignments of Fellutamides C (1) and D (2) in DMSO-d6 + CD3CO2D (9:1)
1
2a
position
δC
δH, mult (J in Hz)
HMBC (HfC)
δC
δH, mult (J in Hz)
leucinal/valinal
1
201.6
63.2
27.9
18.1
19.0
9.38, d (1.5)
3.94, ddd (7.0, 5.5, 1.5)
2.15, m
C-2
201.9
56.8
36.4
23.8
21.4
23.1
9.34 s
2
C-1, 3, 4, 5, HyGln (C-1)
4.01, m
3
1.47, m
4
0.88, d (7.0)
0.84, d (7.0)
C-2, 3, 5
C-2, 4, 4
1.65 m
5
0.85, d (7.0)
0.85, d (7.0)
8.21, d (7.0)
6
NH
1
8.07, d (7.0)
HyGln (C-1)
C-1, 3
β-hydroxyglutamine (HyGln)
170.3
57.0
170.4
57.0
2
4.27 dd (8.5, 3.0)
4.35, brm
4.23, dd (8.5, 3.0)
4.37, brm
3
67.7
67.5
4
39.8
2.2, m
C-3
40.0
2.20, m
5
172.3
172.4
NH
OH
NH2
7.75, d (8.5)
5.12, brs
6.84, s
Asn (C-1)
7.78, d (8.5)
5.14 brs
6.82, s
C-4
C-5
7.15, s
7.15, s
asparagine (Asn)
1
2
3
171.0
49.9
36.9
171.4
49.9
36.9
4.55, apparent q (7.0)
2.45, m
C-1, 2, HTDA (C-1),
C-2, 4
4.5, apparent q (7.0)
2.50, m
2.55, dd (15, 7.0)
2.58, dd (14, 7.0)
4
171.9
172.0
NH
NH2
8.16, d (7.0)
6.94, s
HTDA (C-1)
8.20, d (6.5)
6.95, s
C-3
C-4
7.45, s
7.47, s
3-hydroxy-teradecanoic acid (HTDA)
1
2
3
4
171.4
43.5
67.4
37.0
171.4
43.4
67.5
36.9
2.2, m
C-1, 4
C-1, 5
2.2, m
3.75, brs
1.3, m
3.77, brs
1.33, m
1.2, m
1.20, m
5
25.2
28.8
29.1
31.4
22.1
14.0
1.2, m
25.2
28.8
29.1
31.4
22.1
14.0
1.29À1.35
1.29À1.35
1.29À1.35
1.29À1.35
1.29À1.35
0.85, t (7.0)
6
1.2, m
7À11
12
13
14
1.2, m
1.2, m
1.2, m
0.9, t (7.5)
C-12, 13
a Compound 2 showed HMBC data similar to 1 (data not listed).
two (orf1299/RPN6 and orf19.3168/RPN8) to four (plus orf19.4032/
RPN5 and orf19.4102/RPN10). We concluded that these two com-
pounds were largely responsible for the chemical genetic profile of
the extract and hence the biological activities present in the original
fermentation extract.
The 26S proteasome of eukaryotes, consisting of the 20S core
particle and the 19S regulatory particle, is an ATP-dependent
proteolytic complex responsible for ubiquitin-dependent protein
degradation.22 The core particle, in turn, is comprised of two
inner β rings (prominently catalytic) and two outer R rings
(largely structural), with each ring containing seven subunits.
The regulatory particle consists of 19 subunits, of which 10 form
the base that binds to the R ring and the remaining nine
constitute the lid to which polyubiquitin is bound. Fellutamides
C (1) and D (2) induced specific hypersensitivity of hetero-
zygous deletion strains for subunits of both rings of the core
particles and the four subunits of the lid of the regulatory particles
(Figure 1A). Collectively they suggest that the cellular target of
fellutamides C and D is the fungal proteasome.
We selected four representative heterozygous deletion strains
(orf19.5378/SCL1, an R subunit; orf19.4025/PRE1, a β subunit;
orf19.3168/RPN8, a subunit of the regulatory particle; and orf19.4088/
NUP188, a subunit of the nuclear pore complex) from the CaFT
profile, as highlighted by arrows in Figure 1A, and tested them for
susceptibility to both fellutamides C and D. As expected, all these strains
were hypersensitive to both compounds with minor quantitative
differences (Figure 3A and B). Preliminary results indicated that when
compounds were tested in combination, only additive activity was
observed. Although fellutamide C (1) was more potent than D (2) in
the whole-cell antifungal activity, with MIC values of 2.5 and 10 μg/mL
(in minimal medium YNBD), respectively (Figure 3A and B), both
fellutamides were equally potent in inhibiting the proteasome activity,
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Scheme 1. Synthesis of Tripeptide Aldehyde (10)
Table 2. Antifungal Activity of Fellutamides C (1) and D (2)
MIC (μg/mL)
organism
C. albicans
strain
2323
fellutamide C
fellutamide D
4
4
16
8
C. tropicalis
C. glabrata
C. lusitaniae
C. krusei
MY1012
MY1381
4
8
MY1396
16
4
32
8
ATCC6258
ATCC22019
MF5668
C. parapsilosis
A. fumigatus
T. mentagrophytes
S. aureus
2
8
16
>32
>32
32
>32
>32
MF7004
MB2865
Figure 4. Antifungal activities of fellutamide D (fell D), compound 9
(CMP9), and compound 10 (CMP10) at concentrations indicated
against the control (WT) and the PRE4 heterozygous deletion
(PRE4) strains. The growth of each strain in the presence of drug
treatments was normalized by that of the mock treatment.
with an IC50 of ∼0.2 μg/mL (Figure 3C) and an MIC
of ∼1.25 μg/mL. Their proteasome activities were at least 10
times more potent than gliotoxin and lactacystin (Figure 1B).
Collectively, these results suggest that the whole-cell and the
proteasome inhibitory activities in the original extract are attri-
butable to the additive effects of both fellutamides.
The antifungal spectrum of fellutamides C and D was evaluated
against a panel of fungal pathogens in the medium used in the fitness
test (modified RMPI5). Indeed, both compounds were reason-
ably active against all the Candida species tested, with MIC
ranging from 2 to 32 μg/mL. Fellutamide C (1) was consistently
more potent than D (2) against each fungus (Table 2). Both
compounds were also active against Aspergillus fumigatus in liquid
(Table 2) and solid (Figure 3D) media. However, no significant
activity was observed against the dermatophyte Trichophyton
mentagrophytes or the bacterium Staphylococcus aureus (Table 2).
We then tested the antifungal activities of the bis-des-hydroxy
version of fellutamide D (i.e., compound 10) and its leucinol
version (compound 9) using the wild-type and the heterozygous
deletion strain of PRE4/orf19.4230 (see Figure 1A), encoding
another β subunit of the proteasome (Figure 4). Compound 10
showed lower activity than fellutamide D (2) in inhibiting the
wild-type fungal cells; however both compounds showed almost
equal potency against heterozygous deletion strain (PRE4/
orf19.4230 (see Figure 1A), encoding another β subunit of the
proteasome (Figure 4)). Compound 9 was inactive against both
strains at a concentration as high as 10 μg/mL (Figure 4). These
results suggest that the β-hydroxy group of the aliphatic tail and
the hydroxyl group of the central amino acid of the tripeptide are
not essential for antifungal whole-cell activity and that the aldehyde
group of the C-terminal amino acid is critical for biological
activities.
The inhibition of the human proteasome by fellutamide B20
was reported during the course of our study. As part of our
examination of the properties of fellutamides C and D, we tested
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Figure 5. Effects of fellutamides C (1) and D (2) on human cancer cells. (A) Treatment with purified fellutamide C or D for 48 h results in potent
inhibition of the growth of PC-3 cells (IC50 = 440 ( 60 and 160 ( 20 nM, respectively), while having relatively little effect on A549 cells. (B) PC-3 cells
treated with fellutamide C/D are blocked at G2M, which subsequently results in apoptosis. (C) In PC-3 cells treated with fellutamide C/D/after 24 h
caspase activity is apparent, and after 48 h treatment annexin V and propidium iodide staining can be seen.
the compounds for activity against PC-3 and A549 human cancer
cell lines (Figure 5). PC-3 cells are known to be sensitive to
proteasome inhibitors such as bortezumib, while A549 cells are
relatively unaffected.23,24 As shown in Figure 5A, both fellutamides
inhibited PC-3 cells, with observed IC50 values of approximately
440 and 160 nM, respectively. To further characterize this effect,
we then examined the cell cycle profile of PC-3 cells treated with
a mixture of fellutamide C/D. In this experiment, the felluta-
mides produce a clear G2M block that develops within 24 h
(Figure 5B). Finally, we stained fellutamide C/D-treated PC3
cells for caspase, annexin V, and propidium iodide, common markers
for apoptosis and cell death (Figure 5C). Taken together, these
results are consistent with proteasome inhibition within the PC-3
cells, namely, that fellutamide C/D treatment creates a G2M
block and induces apoptosis.
Fellutamides are peptide aldehyde inhibitors of the protea-
some and contain β-hydroxy aliphatic tails of various lengths and
C-terminal aldehyde.20 The aldehyde functional group forms a
hemiacetal covalent adduct at the active site of the proteasome,
leading to its inhibition.25 However, while peptide aldehydes
such as MG132 and bortezomib are potent inhibitors of the
chymotrypsin-like activity of the human proteasome that results
in rapid cell cycle arrest and induction of apoptosis (thus killing
the cells), they do not inhibit growth of the yeast S. cerevisiae
partially due to poor uptake.26,27 Fellutamide B is known to inhibit
the chymotrypsin-, caspase-, and trypsin-like activities of the human
proteasome.20 We confirmed that fellutamides C and D also inhibited
the chymotrypsin-like activity of the human proteasome (Figure 5).
As expected, both were cytotoxic against human PC-3 prostate
carcinoma cells, but were not active against A549 lung carcinoma
cells. In PC-3 cells, fellutamides treatment caused a G2M cell cycle
arrest and induced apoptosis. This pattern of activity matches that of
known proteasome inhibitors, including the tripeptide boronic acid
bortezomib.23,24,28 The human proteasome has emerged as a new
therapeutic target for cancer and neurodegenerative diseases.20,25
This raises the question whether fellutamides are suitable antifungal
agents with therapeutic potential due to polypharmacy, even though
their target, the fungal proteasome, is essential for virulence in an
animal model of candidiasis.29 In addition, application of felluta-
mides to other therapeutic areas must address the mechanistic
disadvantages associated with other peptide aldehyde inhibitors. As
in the case of MG132, the formation of the aforementioned covalent
hemiacetal adduct between the inhibitor and the catalytic center of
the proteasome is a reversible reaction under physiological condi-
tions. The aldehyde inhibitors are rapidly inactivated by oxidation
inside the cells and effluxed by the multidrug resistance carrier
system.25 If this is also true for fellutamides, their application in
antifungal and other therapeutic areas is limited.
In summary, we have described the discovery of two peptide
aldehyde inhibitors of fungal proteasome using the C. albicans
fungal test from an undescribed species of Metulocladosporiella
from Equatorial Guinea. We have demonstrated for the first time
that the chemical inhibition of fungal proteasome leads to
inhibition of fungal growth.
1727
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Journal of Natural Products
ARTICLE
’ EXPERIMENTAL SECTION
Flourescent Microscopy. PC-3 cells were seeded in eight-chamber
glass slides and incubated overnight. Cells were treated with either 400 or
2000 nM fellutamide C/D (0.05% final DMSO) for 24 or 48 h. Following
compound treatment, cells were double stained with Annexin-V (Roche
Applied Science, Indianapolis, IN) and propidium iodide or with Pan
Caspases (Millipore, Temecula, CA) and Hoechst nuclear dye and imaged
on a fluorescent microscope.
General Experimental Procedures. Optical rotation was re-
corded with a Perkin-Elmer 241 polarimeter. UV spectra were recorded
on a Perkin-Elmer Lambda 35 spectrometer. IR spectra were recorded
with a Perkin-Elmer Spectrum One FT-IR spectrophotometer. All NMR
spectra were recorded with a Varian Unity 500 (1H, 500 MHz; 13C,
125 MHz) spectrometer in DMSO-d6 or a mixture of DMSO-d6 and
CD3CO2D. All shifts were reported in δ (ppm) using solvent signals of
Extraction and Purification of Fellutamides C and D. The
scale-up fermentation broth (2.4 L) was extracted with 2.4 L of methyl
ethyl ketone by shaking on a platform shaker for 2 h. The resulting
extract was concentrated under reduced pressure to remove most of the
MEK, leaving behind mostly aqueous extract (∼0.7 L), which was extracted
twice with EtOAc (∼0.5 L each). The EtOAc extract was concentrated
under reduced pressure to yield 1 g of gum. This material was dissolved
in 15 mL of 1:1 MeOH/CH2Cl2 and charged to a 500 cm3 Sephadex
LH20 column. The column was eluted with MeOH at a flow rate of
5 mL/min. Fractions eluting from 250 to 300 mL elution volume contained
all antifungal activity (of both compounds). They were pooled and
concentrated to give 190 mg of residue. The dry material was dissolved
in MeOH/H2O and filtered, and the solution was charged to a 10 cm3
Amberchrome column at 40% aqueous MeOH and eluted with a 40À
100% aqueous MeOH with a 10% step gradient. All the activity was
found in the 90% and 100% aqueous MeOH fractions. A small portion
was tested in the fitness test. The pooled fractions together with the recovered
solid from the filtration of Amberchrome charge were chromatographed
by an Amberchrome Profile HPLC column (HPR10, 250 Â 22 mm)
using isocratic 40% aqueous CH3CN with 0.1% TFA at a flow rate of
10 mL/min. The chromatographic loading (2À5 mg/run) of these
compounds was very poor and required a number of repeated chroma-
tographies. Pooled fractions were immediately lyophilized to give 10.4 mg of
fellutamides C (4.3 mg/L) and 7.5 mg of D (3.1 mg/L).
DMSO-d6 (1H, 2.53; 13C, 39.51 ppm) as internal standards. H, 13C,
1
COSY, DEPT, HMQC, and HMBC spectra were measured using standard
Varian pulse sequences. LRMS data were recorded on an Agilent 1100
MSD with ES ionization, and HRESIFTMS was obtained on a Thermo
Finnigan LTQ-FTMS spectrometer. An Agilent HP 1100 instrument
was used for analytical HPLC.
Producing Organism and Its Characterization. The produ-
cing organism (F-192,783) was isolated from a soil sample collected near
Rio Campo, Equatorial Guinea, using a method for plating washed soil
particles.30 Frozen stock cultures in 10% glycerol (À80 ꢀC) are maintained
in the collection of Fundaciꢀon MEDINA. Total genomic DNA was
extracted from mycelia grown on YM agar. The 28S rDNA fragments,
containing D1-D231 and ITS regions, were PCR amplified; sequence
alignments32 and phylogenetic analyses were previously described.9 Results
from morphological and phylogenetic analyses are illustrated in Supple-
mental Figures S1ÀS3.
Fermentations in Nutritional Arrays and Scale-up for
Isolation. The strategy and protocols for fermentation of fungi on
nutritional arrays have been described previously.2,33 The antifungal
activity, determined by the zone of inhibition of wild-type C. albicans, of
F-192,783 in the fermentation extract of YES medium (yeast extract
20 g; sucrose 150 g; MgSO4 7H2O 0.5 g; FeSO4 7H2O 0.01 g;
3
3
Fellutamide C (1): [R]23 À10.0 (c 0.4, MeOH); UV(MeOH)
CuSO4 5H2O 5 mg; ZnSO4 7H2O 10 mg; distilled H2O 1000 mL).
3
3
D
The scale-up fermentation employed static cultures of 200 mL of YES
medium in 2.8 L Fernbach flasks at 22 ꢀC for 14 days. The resulting
fermentation broth was extracted with an equal volume of acetone, and
aliquots were used in the fitness test and chemical fractionation.
C. albicans Fitness Test and Biological Assays. The
C. albicans fitness test and strains were described previously.5 Liquid
assays were performed using media indicated in the figure legends, with
the starting OD600 of 0.02 in 96-well plates. The growth was determined
by OD600 after 20 h growth at 30 ꢀC and normalized by that of the same
strains in the absence of drug treatment (mock). The chymotrypsin-like
activity of the proteasome (Proteasome-Glo, Promega) was assessed
according to the manufacturer’s instruction with modifications using
whole-cell extracts prepared from C. albicans grown in the exponential
phase. Each assay contained extracts equivalent to ∼106 cells. The
antifungal MIC reported in Table 2 was measured as previously described.9
Human Cancer Cell Proliferation Assay. ATP Vialight (Lonza,
Rockland ME) detection of viable cell mass was used to measure cell
proliferation for PC-3 (ATCC #) and A549 (ATCC #) cells following
the method described by Nagashima et al.,34 with the exception that the
experiments employed 8-point dose curves and final test compound
concentrations of 50 000, 12 500, 3125, 781.3, 195.3, 48.8, 12.2, and
3.1 nM. IC50 values were calculated using a four parameter fit logistic
equation of the % inhibition data plotted as a function of the log of
concentration.
λmax end absorption; IR (ZnSe) νmax 3271, 2920, 2850, 1624, 1559,
1372, 1200, 1015 cmÀ1; ESIMS m/z 586 [M + H]+; HRESIFTMS m/z
586.3807 (calcd for C28H51N5O8 + H, 586.3818), 568.3700 (M À
H2O), 485.30 (M À valinal), 341.24 (M À HyGln-valinal); 1H and 13
C
NMR data, see Table 1.
Fellutamide D (2): [R]23D À22.5 (c 0.4, MeOH); UV (MeOH) λmax
end absorption; IR (ZnSe) νmax 3284, 2956, 2920, 2851, 1663, 1624, 1547,
1408, 1203, 1182, 1134, 1051, 1025 cmÀ1; ESIMS m/z 600 [M + H]+;
HRESIFTMS m/z 600.3960 (calcd for C29H53N5O8 + H, 600.3967), 582
(M À H2O), 485.2965 (M À leucinal), 341.2433 (M À HyGln-leucinal);
1H and 13C NMR data, see Table 1.
Hydrolysis and Absolute Configuration Determination. A
10.5 mg sample of the mixture of the two compounds was dissolved in
0.5 mL of MeOH, 4À5 mg of NaBH4 was added at room temperature,
and the mixture was stirred for 1 h. The reaction was complete, and
slightly more polar products were formed. This material was diluted by
addition of 1 mL of H2O and passed through a 1 mL Amberchrome
column packed in water and washed with water. The product alcohols
were eluted with 100% MeOH, and the solution was concentrated to
dryness, yielding 10 mg of the mixture of two peptidic alcohols. The
peptidic alcohols were dissolved in 0.5 mL of 6 N HCl in a sealed tube
and heated at 105 ꢀC overnight. The solution was first diluted with 1 mL
of H2O, and 1/10 aliquot was saved for amino acid analysis using
Marfey’s reagent. The remaining 9/10 portion was extracted with 1 mL
of CHCl3, and the organic extract was concentrated to dryness, dissolved
in MeOH, and purified by semipreparative HPLC (Zorbax RX C8, 9.6 Â
250 mm, 35 ꢀC at 4 mL/min). The major peak observed in a light-
scattering detector was collected and lyophilized to give 0.4 mg of
powder: [R]23D À10 (c 0.2, CHCl3), [(3R)-hydroxytetradecanoic acid
Flow Cytometry. Briefly, PC-3 cells were plated in 10 mm cell
culture treated dishes and incubated overnight. Final concentrations
of 400 or 2000 nM of fellutamide C/D (0.05% final DMSO) were
added to the cells followed by incubation for 24, 48, and 72 h. After
compound treatment, both floating and attached cells were collected,
stained for DNA content, subjected to cell cycle analysis on a BD FACS
Calibur (Becton Dickinson, San Jose, CA), and further analyzed using
FlowJo7.
25
lit.35,36], [R]D À16 (c 1.0 CHCl3); 1H NMR (CDCl3) δ 0.88 (3H, t,
7 Hz), 1.25À1.6 (16H, m), 1.43 (2H, m), 4.09 (1H, m), 2.55 (2H, m),
2.45 (2H, m).
1728
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Journal of Natural Products
ARTICLE
Cbz-(S)-Asn-(S)-Gln-O-t-Bu (5). To a solution of CBZ-(S)-
asparagine (266 mg, 1 mmol) in DMF (2 mL) were added H-gluta-
mine-tert-butyl ester (202 mg, 1 mmol) and PyBop reagent (327 mg,
1.1 mmol) followed by DIPEA (561 mg, 2.2 mmol) at room tempera-
ture, and the reaction mixture was stirred. A precipitate appeared after
overnight stirring, and the reaction was complete. Water (20 mL) was
added, the solution was stirred for 10 min, and the product was collected
by filtration through a sintered glass funnel. The product was air-dried
followed by drying under vacuum to give 430 mg (yield 93%) of 5 as a
1.50À1.01 (22 H, m), 0.85 (3H, d, J = 6.5 Hz), 0.84 (3H, t, J = 7 Hz),
0.80 (3H, d, J = 6.5 Hz); ESIMS m/z 570 (M + H).
Myristyl-(S)-Asn-(S)-Gln-(S)-Leucinal (10). To a solution of
leucinol peptide 9 (8 mg, 0.014 mmol) in DMSO (1 mL) was added
TEA (0.01 mL, 0.07 mmol), and the solution was stirred at room
temperature under N2 for 20 min. It was cooled to 0 ꢀC, and then
SO3ÀPy complex (11.2 mg, 0.070 mmol) was added. The reaction
mixture was stirred at 0À5 ꢀC for 30 min. The reaction was quenched
with 100 uL of water, diluted with 1 mL of MeOH, and filtered through a
4.5 μm filter, and 50% was purified by reversed-phase HPLC (Zorbax
RX C18 (21.2 Â 250 mm), 37 min gradient of 10À95% MeOH(aq) at
12 mL/min). Fractions 35À37 (35À37 min) contained the desired
product and were pooled. These pooled fractions were concentrated to
give 2.4 mg (60%) of the aldehyde 10 as a colorless powder: 1H NMR
(DMSO-d6) δ 9.33 (1 H, s), 8.28 (1 H, d, J = 7.32 Hz), 8.09 (1 H, d,
J = 7.65 Hz), 8.02 (2 H, d, J = 7.40 Hz), 7.39 (1 H, s), 7.19 (1 H, s), 6.90
(1 H, s), 6.73 (1 H, s), 4.48 (1 H, q, J = 7 Hz), 4.17 (1 H, m), 4.03 (1 H, m),
2.62 (1 H, m), 2.50 (1 H, dd, J = 15, 6 Hz), 2.39 (1 H, dd, J = 15, 7 Hz), 2.34
(1 H, m), 2.08 (4 H, m), 1.94 (1 H, m), 1.75 (1 H, m), 1.61
(1 H, m), 1.46 (2 H, m), 1.22 (22 H, s), 0.87 (3 H, d, J = 6.5 Hz), 0.84
(3H, t, J = 6.5 Hz), 0.82 (3H, d, J = 7 Hz); ESIMS m/z 568 (M + H).
1
colorless powder: H NMR (DMSO-d6) δ 8.16 (1 H, d, J = 7.4 Hz),
7.38À7.29 (6 H, m), 7.23 (1 H, brs), 6.88 (1 H, brs), 6.75 (1 H, brs),
4.99 (2 H, s), 4.40À4.34 (1 H, m), 4.06À4.01 (1 H, m), 2.46 (1 H, dd,
J = 15, 4.4 Hz), 2.37 (1 H, dd, J = 15, 9.4 Hz), 2.10 (2 H, t, J = 8 Hz),
1.91À1.84 (1 H, m), 1.79À1.74 (1 H, m), 1.37 (9 H, s); ESIMS m/z 451
(M + H).
H2N-(S)-Asn-(S)-Gln-O-t-Bu (6). A solution of the sparingly
soluble Cbz-dipeptide 5 (230 mg) in MeOH (20 mL) was hydrogenated
overnight using 10% Pd/C (10 mg) using a balloon filled with hydrogen.
The catalyst was removed by filtration through a 0.45 μm filter and
concentrated to give 160 mg of 6 as a colorless powder (yield 99%): 1H
NMR (DMSO-d6) δ 8.12 (1H, d, J = 7.8 Hz), 7.38 (1 H, s), 7.24 (1 H, s),
6.84 (1 H, s), 6.75 (1 H, s), 4.08 (1 H, m), 3.48 (1 H, dd, J = 9.30,
3.7 Hz), 2.39 (1 H, dd, J = 15, 3.9 Hz), 2.22À2.04 (3 H, m), 1.94À1.86
(1H, m), 1.80À1.72 (1 H, m), 1.38 (9H, s); ESIMS m/z 317, (M + H).
Myristyl-(S)-Asn-(S)-Gln-O-t-Bu (7). To a solution of myristic
acid (113 mg, 0.05 mmol) and H2N-dipeptide 6 (142 mg, 0.45 mmol) in
DMF (2 mL) was added PyBop reagent (219 mg, 0.5 mmol) followed by
DIPEA (0.173 mL, 0.99 mmol). The solution was stirred at room
temperrature overnight, leading to the formation of a precipitate, which
was diluted with 20 mL of water and collected by filtration through a
sintered funnel. The precipitate was thoroughly washed with water,
dried under air followed by in vacuo to give the myristylated dipeptide 7
’ ASSOCIATED CONTENT
S
Supporting Information. Photograph of the producing
b
strain (Figure S1) and phylogenetic tree (Figures S2 and S3).
This material is available free of charge via the Internet at http://
pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*Corresponding Author: email: sheo.singh@merck.com.
1
(230 mg, 93%) as a colorless powder: H NMR (DMSO-d6) δ 8.01
(1 H, d, J = 7.64 Hz), 7.91 (1 H, d, J = 8.13 Hz), 7.22 (2 H, d, J = 13.03
Hz), 6.86 (1 H, s), 6.74 (1 H, s), 4.58À4.52 (1 H, m), 4.05À3.98 (1 H,
m), 2.45 (1 H, dd, J = 15, 4.5 Hz), 2.34 (1 H, dd, J = 16.12, 8.92 Hz), 2.06
(4 H, m), 1.92À1.82 (1 H, m), 1.73 (1 H, m), 1.44 (2 H, s), 1.37 (9 H, s),
1.22 (20 H, s), 0.83 (3 H, t, J = 6.9 Hz); ESIMS m/z 527 (M + H).
Myristyl-(S)-Asn-(S)-Gln-OH (8). To a solurion of myristyl-
dipeptide-tert-butyl ester 7 (130 mg, 0.247 mmol) in 5 mL of CH2Cl2
and 2 mL of MeOH was added TFA (0.95 mL), and the solution was
stirred overnight at room temperature. After completion of the reaction,
volatile materials were removed under a stream of N2 and the residue
was dried under vacuum to give 112 mg (93%) of 8 as a yellowish
powder: 1H NMR (DMSO-d6) δ 7.97 (1 H, d, J = 7.7 Hz), 7.92 (1 H, d,
J = 8 Hz), 7.24 (1 H, s), 7.19 (1 H, s), 6.85 (1 H, s), 6.73 (1 H, s), 4.54
(1 H, td, J = 8, 5 Hz), 4.12 (1 H, td, J = 8.2, 5 Hz), 2.42 (1 H, dd, J = 15,
4.5 Hz), 2.33 (1 H, dd, J = 15.5, 8.4 Hz), 2.18À2.02 (4 H, m), 1.96À1.87
(1 H, m), 1.79À1.69 (1 H, m), 1.44 (2 H, s), 1.22 (20 H, s), 0.84 (3 H, t,
J = 6.8 Hz); ESIMS m/z 471 (M + H).
Myristyl-(S)-Asn-(S)-Gln-(S)-Leucinol (9). To a solution of
myristylated dipeptide 8 (14.1 mg, 0.03 mmol) and (S)-leucinol (3.86 mg,
0.033 mmol) in DMF (1 mL) was added PyBop reagent (14.6 mg,
0.033 mmol) followed by DIPEA (0.011 mL, 0.060 mmol). The reaction
mixture was stirred at room temperature for 4 h and diluted with water
(20 mL). The precipitate was collected by filtration through a sintered
glass funnel, washed with water, and dried under air and in vacuo to give
13 mg (76%) of 9 as a colorless powder: 1H NMR (DMSO-d6) δ 8.02
(1 H, d, J = 7.5 Hz), 7.97 (1 H, d, J = 8 Hz), 7.43 (1 H, d, J = 8.5 Hz), 7.37
(1 H, s), 7.17 (1 H, s), 6.91 (1 H, s), 6.71 (1 H, s), 4.52 (1 H, t, J = 5.5 Hz),
4.45 (1 H, q, J = 7 Hz), 4.08 (1 H, dt, J = 8, 5 Hz), 3.75 (1 H, m) 3.25
(1 H, m), 3.16 (1H, m), 2.50 (1H, dd, J = 15.5, 6.5 Hz), 2.37 (1 H, dd, J =
15.53, 7.1 Hz), 2.27À2.20 (1 H, m), 2.10À1.97 (4 H, m), 1.92À1.84
(1 H, m), 1.74À1.65 (1 H, m), 1.59À1.49 (1 H, m), 1.45 (2 H, s),
’ ACKNOWLEDGMENT
The authors thank M. Arocho, K. Calati, and K. Ferguson for
the preliminary isolation and assay support, and members of
the Center of Fungal Genetics for their contributions to CaFT
screening of natural products.
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