Antifungal spectrum, in vivo efficacy, and structure-activity relationship of ilicicolin H

Article abstract of DOI:10.1021/ml300173e

Ilicicolin H is a polyketide - nonribosomal peptide synthase (NRPS) - natural product isolated from Gliocadium roseum, which exhibits potent and broad spectrum antifungal activity, with sub-μg/mL MICs against Candida spp., Aspergillus fumigatus, and Cryptococcus spp. It showed a novel mode of action, potent inhibition (IC₅₀ = 2-3 ng/mL) of the mitochondrial cytochrome bc1 reductase, and over 1000-fold selectivity relative to rat liver cytochrome bc1 reductase. Ilicicolin H exhibited in vivo efficacy in murine models of Candida albicans and Cryptococcus neoformans infections, but efficacy may have been limited by high plasma protein binding. Systematic structural modification of ilicicolin H was undertaken to understand the structural requirement for the antifungal activity. The details of the biological activity of ilicicolin H and structural modification of some of the key parts of the molecule and resulting activity of the derivatives are discussed. These data suggest that the β-keto group is critical for the antifungal activity.

Full text of DOI:10.1021/ml300173e

Letter
pubs.acs.org/acsmedchemlett
Antifungal Spectrum, In Vivo Efficacy, and Structure−Activity
Relationship of Ilicicolin H
Sheo B. Singh,* Weiguo Liu, Xiaohua Li, Tom Chen, Ali Shafiee, Deborah Card, George Abruzzo,
Amy Flattery, Charles Gill, John R. Thompson, Mark Rosenbach, Sarah Dreikorn, Viktor Hornak,
Maria Meinz, Myra Kurtz,† Rosemarie Kelly, and Janet C. Onishi*
Departments Medicinal Chemistry and Infectious Diseases, Merck Research Laboratories, Rahway, New Jersey 07065, United States
S
* Supporting Information
ABSTRACT: Ilicicolin H is a polyketidenonribosomal
peptide synthase (NRPS)natural product isolated from
Gliocadium roseum, which exhibits potent and broad spectrum
antifungal activity, with sub-μg/mL MICs against Candida
spp., Aspergillus fumigatus, and Cryptococcus spp. It showed a
novel mode of action, potent inhibition (IC50 = 2−3 ng/mL)
of the mitochondrial cytochrome bc1 reductase, and over
1000-fold selectivity relative to rat liver cytochrome bc1 reductase. Ilicicolin H exhibited in vivo efficacy in murine models of
Candida albicans and Cryptococcus neoformans infections, but efficacy may have been limited by high plasma protein binding.
Systematic structural modification of ilicicolin H was undertaken to understand the structural requirement for the antifungal
activity. The details of the biological activity of ilicicolin H and structural modification of some of the key parts of the molecule
and resulting activity of the derivatives are discussed. These data suggest that the β-keto group is critical for the antifungal
activity.
KEYWORDS: ilicicolin H, Gliocadium roseum, antifungal activity, broad spectrum, cytochrome bc1 reductase inhibitor, in vivo efficacy
nfections caused by pathogenic fungi (e.g., Candida albicans
and Aspergillus fumigatus) are life-threatening, particularly to
5.0 μg/mL. Fluconazole-resistant C. albicans strain, MY2301,
was sensitive to iliciolin H. Ilicicolin H also inhibited the
growth of Cryptococcus species with minimum inhibitory
concentration (MIC) values of 0.1−1.56 μg/mL, which is
generally superior to comparators. When evaluated in a limited
panel of Aspergillus species, ilicicolin H was shown to inhibit A.
fumigatus (MIC 0.08 μg/mL), while A. flavus was resistant.
I
immunocompromised populations.1 Three main therapeutic
options exist for the treatment of such infections, including
azoles (e.g., fluconazole),2 macrocyclic polyenes (e.g., ampho-
tericin),3 and candins (e.g., caspofungin, micafungin, and
anidulafungin).4 Each treatment option has limitations to its
utility, thus creating a need for new antifungal agents.
Screening of natural product extracts against C. albicans
whole cell screen led to the identification of ilicicolin H (1)
produced by Gliocadium roseum.5 The structure of 1 was
elucidated by 2D NMR and mass spectral studies.6,7 This
tetracyclic polyketide was originally reported in 1971 from the
mycelium of an imperfect fungus, Cylindrocladium ilicicola, with
only weak antibiotic activity against Bacillus anthracis (6 μg/
mL).8 The structure elucidation by chemical degradation was
reported in 19769 followed by NMR assignment and antifungal
activity against C. albicans.10 The structure was confirmed by a
total synthesis of racemic ilicicolin H.11 The precursor feeding
experiments helped elucidate biosynthesis as a polyketide, and
the two methyl groups originate from S-adenosylmethionine.12
We describe, now, the antifungal spectrum, in vivo efficacy,
mechanism of action, and structure−activity relationship (SAR)
from the modification of the core structure of ilicicolin H (1).
The antifungal spectrum of ilicicolin H is comparable to
comparators including caspofungin, amphotercin B, and
fluconazole (Table S1 in the Supporting Information). The
activity against C. albicans ranged from 0.04 to 0.31 μg/mL,
while in other Candida species, sensitivities ranged from 0.01 to
The in vivo efficacy of ilicicolin H was assessed in a
disseminated C. albicans (MY1055) mouse infection model.13
Briefly, DBA/2 mice were infected intravenously with 5.4 × 104
colony-forming units (cfu) of C. albicans (MY1055). Ilicicolin
H was administered orally twice daily in 10% aqueous DMSO
for 2 days at doses of 50, 25, 12.5, and 6.25 mg/kg. Four days
after infection, kidney Candida burden was determined.
Significant reduction (p < 0.05; Excel t test) in kidney burden
relative to vehicle-treated controls was achieved at 50 mg/kg,
Received: June 28, 2012
Accepted: August 30, 2012
Published: September 7, 2012
© 2012 American Chemical Society
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ACS Medicinal Chemistry Letters
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with an effective dose 90 (ED90, dose leading to 90% reduction
of C. albicans in kidney) of 15.45 mg/kg/dose. Caspofungin
treatment (intraperitoneally) resulted in an ED90 of 0.01 mg/
kg. Ilicicolin H also showed significant activity (p < 0.05; Excel t
test) in an abbreviated 24 h version of the candidiasis model
when dosed intravenously thrice daily at a total dose of 50 or 25
mg/kg/day, with an ED90 of 22.3 mg/kg/day. In a similar
mouse model of disseminated Cryptococcus neoformans
(MY2061) infection (8 × 105 cfu/mouse), ilicicolin H reduced
spleen burden by over 1 log cfu/g of spleen (p < 0.05; Excel t
test) when administered orally at either 100 or 50 mg/kg
intraperitoneally twice daily in 5% aqueous DMSO.
The antifungal activity of ilicicolin H was dependent on the
carbon source used in the test media. MICs of the compound
in glucose-based media were >50 μg/mL against Saccharomyy-
ces cerevisiae MY 2141 and C. albicans MY 1055, but in media
containing a nonfermentable carbon source, such as glycerol,
the MICs against these yeast strains were 0.012 and 0.025 μg/
mL, respectively. The effect of carbon source on antifungal
activity was accounted for by inhibition of respiration. Whole
cell oxygen consumption was inhibited 100% in S. cerevisae and
in C. albicans at 0.003 μg/mL. Measurements of the effect of
ilicicolin H on substrate-dependent rates of oxygen con-
sumption by coupled mitochondria from S. cerevisiae MY 2141
revealed that complex I−III was the most sensitive complex in
the respiratory chain (Figure 1). The IC50 values for inhibition
mutants were detected in both yeast strains with mutations at
position G37. The MICs to ilicicolin H in the C. albicans
resistant mutants were 250−1000-fold higher than the wild-
type strain, and resistance was accounted for by resistance in
both NADH:cytochrome c oxidoreductase and the ubiquinol:-
cytochrome c reductase (Table S3 in the Supporting
Information). Interestingly, none of the ilicicolin H-resistant
mutants in S. cerevisiae or in C. albicans displayed cross-
resistance with any of the known cytochrome bc1 inhibitors
such as antimycin or myxothiazol.
The NADH:cytochrome c oxidoreductase from C. albicans
MY1055 was orders of magnitude more sensitive than the
enzyme from rat or rhesus liver. Ilicicolin H showed IC50 values
of 0.8, 1500, and 500 ng/mL, respectively. In contrast,
antimycin showed no selectivity in this assay with IC50 values
of 0.5, 0.5, and 0.1 ng/mL, respectively, against C. albicans
MY1055 and rat and rhesus NADH:cytochrome c oxidor-
eductase. Independent studies by Gutierrez et al.14 showed
>70-fold selectivity for the yeast ubiquinol: cytochrome c
reductase (S. cerevisiae) as compared to the bovine reductase
(IC50 = 87−108 ng/mL, 200−250 nM).
There are several possible explanations that may account for
the selective action of ilicicolin H against NADH:cytochrome c
reductase prepared from lower and higher eucaryotes. First, it is
postulated that the binding interactions for the substrate,
ubiquinone, differ. The yeast ubiquinol possesses a shorter
length isoprene side chain (n = 4−6) as compared to
mammalian ubiquinol (n = 9−10). Second, a sequence
alignment of cytochrome b, focusing on segments containing
the Qi site amino acids, points out the differences between two
lower (S. cerevisiae and C. albicans) and two higher (bovine and
rat) species (Figure S1 in the Supporting Information). S.
cerevisiae and C. albicans possess high overall sequence
homology (76% identity and 90% similarity) with even higher
homology of residues forming the Qi site (91% identity and
95% similarity). Homology to higher species decreases (e.g., S.
cerevisiae vs bovine: 51/72% for overall identity/similarity and
54/73% for Qi site) and may provide a possible explanation for
ilicicolin's selectivity. For example, bulky and aromatic tyrosine
at position 16 in lower eukaryotes is replaced by smaller amino
acid (alanine or serine) in higher eukaryotes. Other examples of
dramatic differences in Qi site amino acids between lower and
higher eukaryotes occur at positions 17, 22, 27, 221, and 225
(Figure S1 in the Supporting Information).
The modest efficacy of ilicicolin H was surprising given the
excellent in vitro MICs against target yeast strains and relatively
good pharmacokinetics properties in mice, which showed low
clearance of 16 mL/min/kg, reasonable half-life (2.5 h), and
excellent oral bioavailability (F = 72%). It is postulated that low
in vivo activity as compared to in vitro activity was attributed to
high plasma protein binding as shown by progressive loss of
activity in the presence of varying % of plasma in the
cytochrome c reductase assay. A complete loss of activity was
observed in the presence of 50% mouse plasma (Figure S2 in
the Supporting Information). Similarly, the C. albicans activity
(MIC) of ilicicolin H was shifted to >1000 ng/mL when tested
in the presence of 10% mouse serum.
Figure 1. Effect of ilicicolin H on substrate-dependent rates of oxygen
consumption by coupled mitochondria from S. cerevisiae MY 2141
substrates used to measure oxygen consumption. Complex I−III:
●
○
▼
■
NADH ( ), ethanol ( ), and glycerol-3-phosphate ( ). Complex
△
II−III: succinate ( ). Complex IV: ascorbate/TMPD ( ).
of complex I−III by ethanol, glycerol-3-phosphate, and NADH
were 0.008, 0.02, and 0.08 μg/mL, respectively. Inhibition of
complex II−III and IV was orders of magnitude less sensitive
with IC50 values of 1.0 and >10.0 μg/mL, respectively.
Inhibition of complex I−III was accounted for by inhibition
of NADH:cytochrome c oxidoreductase; IC50 values of 0.8 and
1.0 ng/mL (1.85 and 2.31 nM) were determined for C. albicans
and S. cerevisiae, respectively. Ilicicolin H was shown to inhibit
the center N (Qn site, also called Qi site) of the cytochrome bc1
complex with an IC50 value of 3−5 nM in the ubiquinol:cy-
tochrome c reductase of S. cerevisiae.14
Isolation of ilicicolin H-resistant mutants in S. cerevisiae
showed that resistance was accounted for by seven types of
single amino acid changes at four sites in the cytochrome b
gene involved in ubiquinone binding.15,16 In an independent
study, ilicicolin H-resistant mutants in C. albicans were isolated
with a frequency of 10−7. Five single amino acid changes at
three sites in the cytochrome b gene, many of which were
identical to those described in S. cerevisiae, were identified
(Table S2 in the Supporting Information). Multiple resistant
With broad antifungal spectrum, a novel mode of action, and
in vivo efficacy, ilicicolin H (1) represented a great starting
point for structural modification to define SAR and improve in
vitro potency and efficacy. Because the modest in vivo efficacy
is likely directly related to very high serum binding, one of the
goals of the program was to explore the feasibility of reducing
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ACS Medicinal Chemistry Letters
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protein binding to increase in vitro and in vivo antifungal
activity. To accomplish this goal, structural modifications to
ilicicolin H were performed using a chemical, biotransforma-
tion, and enzymatic approaches. For initial SAR, the semi-
synthetic compounds were tested for whole cell MIC for C.
albicans (MY1055), IC50 of C. albicans, and rat liver
NADH:cytochrome c1 reductases. The effect of 50% serum
on enzymatic activity was assessed as well.
model based on the crystal structure of yeast (S. cerevisiae)
cytochrome bc1 complex with ubiquinol in Qi site.18
As mentioned before, the residues forming cytochrome b Qi
site in both proteins are practically identical, with binding site
similarity ∼95% (Figure S1 in the Supporting Information).
The homology model therefore provides a reliable representa-
tion of the C. albicans Qi binding pocket that could provide
insights into ilicicolin H SAR. Because antimycin is a potent Qi
site inhibitor, we used antimycin's binding mode in the crystal
structure of bovine bc1 complex19 as an initial template for
ilicicolin H binding (Figure S3 in the Supporting Information).
A refined model of ilicicolin H binding pose was subsequently
obtained by minimization of a complex with protein heavy
atoms fixed.20
The initial overlay of ilicicolin H to antimycin presents
several notable features. The hydrophobic decalin group of
ilicicolin H is pointing out of the binding pocket toward the
lipid bilayer. The allyl group is positioned similarly to the
pentyl group of antimycin, interacting with hydrophobic
residues at the exposed end of the binding pocket (V13 and
L17 in Figure 2A). The hydroxy phenyl group of 1 is roughly
The highly acidic and metal chelating β-diketo group (C4−
C3−C7) is likely playing a significant role in the serum binding;
therefore, this became a first focal point for chemical
modification. Sodium borohydride reduction of 1 produced
>70% yield of a diastereomeric mixture of two alcohols 2 and 3
(ratio 4:1), which underwent facile elimination under acidic
condition to afford the exocyclic olefins 4 and 5. To eliminate
the chelating properties of the β-diketone while conserving the
overall conformation and electronic properties of ilicicolin H, a
series of oxime, hydroazone, pyrazole, isoxazole, and novel
1,4,5-oxadiazocines analogues were synthesized. The syntheses
of hydrazones (6−8), pyrazole (9−11), and oxadiazocine (12)
were recently reported.17 The oxime (13) and isoxazole (14)
were prepared by reaction of 1 with hydroxyl amine at room
temperature followed by heating with acetic acid. Both
geometrical isomers of oxime and hydrazones were formed
but did not show any difference in their activity; therefore, the
data for only one of the isomer are shown. Activities of these
compounds are reported in Table S4 in the Supporting
Information. In general, chemical modifications of the β-diketo
group led to significant loss of the whole cell C. albicans activity
with concomitant attenuation of fungal cytochrome bc1
reductase activity. Surprisingly, these compounds became less
selective with respect to rat liver cytochrome c1 reductase
activity. However, compounds that retained the general
molecular shape of ilicicolin H, for example, hydrazones 6−8
and corresponding pyrazoles 9−11, lost only 10−20-fold
antifungal activity as compared to compounds with significant
change in the molecular shape (e.g., 2−5), which lost >250-fold
activity (Table S4 in the Supporting Information). It appears
that the retention of the shape is not sufficient for the retention
of the activity, for example, the oxadiazocine (12), oxime (13),
and isoxazole (14) did not show any activity at 10000 ng/mL.
The whole cell activity SAR and the fungal reductase activity
SAR did not track well. The pyrazole 9 showed about 10-fold
loss in the antifungal activity (MIC 250 ng/mL) while
maintaining the same fungal reductase activity (IC50 3 ng/
mL) and selectivity.
Figure 2. (A) Binding mode of ilicicolin in the C. albicans model.
Several key residues are shown as thin sticks. G37 is shown in thicker
sticks. (B) To better appreciate the steric constraints of the binding
pocket, ilicicolin H is shown in CPK representation. The necessity for
a perpendicular orientation of the left- and right-hand side of ilicicolin
H is obvious. G37 Cα carbon with hydrogens shown is also displayed
in CPK.
aligned with antimycin's N-formylamino-salicyl group pointing
deep into the pocket and likely being responsible for some of
the binding affinity and specificity. The shape of the binding
pocket necessitates a conformation where two sides of the
molecule fall roughly into two perpendicular planes. While
antimycin served as a good template for initial positioning of
gross hydrophobic and polar features of ilicicolin H, the two
inhibitors represent distinct chemical classes and differ in the
details of their interactions in the binding pocket. Specifically,
the key interaction of D229 with antimycin's phenolic OH and
the formylamido NH is absent in ilicicolin, which instead
engages hydroxyl phenyl in the interaction with N31. The
amide linker in antimycin also presents very different donor/
acceptor pattern than the pyridinone moiety in ilicicolin H,
even though both functional groups are assumed to occupy the
same space. Those and other differences in chemical nature and
interaction patterns of the two inhibitors unsurprisingly lead to
differences in their biophysical characterization as discussed by
Trumpower.21
To facilitate the understanding of the structural aspects of
ilicicolin H enzyme inhibition and SAR, a binding model of
ilicicolin H in C. albicans cytochrome bc1 was constructed.
Because no crystal structure of ilicicolin H and C. albicans
cytochrome bc1 complex is available, we used a homology
The ilicicolin H bioactive conformation (Figure 2) has left-
hand side of the molecule buried deep in the binding pocket
and lipophilic right-hand side aligned with the pocket opening
and rotated ∼90° relative to hydroxyl-pyridinone central ring.
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ACS Medicinal Chemistry Letters
Letter
(2) Zonios, D. I.; Bennett, J. E. Update on azole antifungals. Semin.
Respir. Crit. Care Med. 2008, 29, 198.
The adjacent carbonyl linker is forming an intramolecular
hydrogen bond with the hydroxyl group of the pyridinone ring.
The presence of this internal hydrogen bond is supported by
two observations. In solution, ilicicolin H forms six-center
hydrogen bond as confirmed by the NMR spectrum (data not
shown). More importantly, compound 9, the pyrazole
analogue, with locked conformation similar to those with
intramolecular hydrogen bond, is active in the fungal
cytochrome c1 reductase assay (Table S4 in the Supporting
Information).
The highly twisted nature of the proposed bioactive
conformation has important implications for SAR. The binding
mode further suggests that the phenolic group of the hydroxy
phenyl makes a hydrogen bond interaction with N31 side chain,
which is likely to be responsible for the specificity of ilicicolin's
binding. The crystal structure of antimycin complex suggests
that D229 makes critical hydrogen bonds. As already
mentioned above, we do not observe this interaction since it
would only be accessible to ilicicolin H analogues with hydroxy
substitution in the ortho or meta position. The model also
allows us to speculate about the loss of activity of ilicicolin H in
G37 mutants. Mutations of that residue will increase the bulk in
that region and cause a steric clash with ilicicolin H. The tight
packing of ilicicolin H relative to G37 in the pocket is illustrated
in Figure 2B.
Ilicicolin H is a natural product produced by an imperfect
fungus, C. ilicicola and G. roseum, which showed broad spectrum
antifungal activity. It imparts its activity by selectively inhibiting
fungal cytochrome c1 oxidoreductase activity and respiration. It
demonstrated modest in vivo activity in a Candida and
Cryptococcus infection mouse model. The in vivo activity was
limited by high plasma protein binding. Preliminary medicinal
chemistry efforts pointed out the criticality of the β-diketone
feature of the molecule and lead to mostly less active or inactive
compounds. The homology model suggests that its binding
mode has some similarities but also differences relative to
antimycin binding and provides valuable insight to SAR and
fungal specificity. These studies open the window for future
work on ilicicolin H and the development of new modes of
action antifungal agents.
(3) Gallis, H. A.; Drew, R. H.; Pickard, W. W.; Amphotericin, B 30
years of clinical experience. Rev. Infect. Dis. 1990, 12, 308.
(4) Georgopapadakou, N. H. Update on antifungals targeted to the
cell wall: Focus on beta-1,3-glucan synthase inhibitors. Expert Opin.
Invest. Drugs 2001, 10, 269.
(5) Junker, B.; Zhang, J.; Mann, Z.; Reddy, J.; Greasham, R. Scale-Up
Studies on a Defined Medium Process for Pilot Plant Production of
Illicicolin by Gliocladium roseum. Biotechnol. Prog. 2001, 17 (2), 278−
286.
(6) Singh, S. B.; Li, X.; Chen, T. Biotransformation of antifungal
ilicicolin H. Tetrahedron Lett. 2011, 52, 6190.
(7) Guan, Z.; Liesch, J. M. Solvation of acylium fragment ions in
electrospray ionization quadrupole ion trap and Fourier transform ion
cyclotron resonance mass spectrometry. J. Mass. Spectrom. 2001, 36,
264.
(8) Hayakawa, S.; Minato, H.; Katagiri, K. Ilicicolins, antibiotics from
Cylindrocladium ilicicola. J. Antibiot. 1971, 24, 653.
(9) Matsumoto, M.; Minato, H. Structure of ilicicolin H, an
antifungal antibiotic. Tetrahedron Lett. 1976, 3827.
(10) Matsumoto, M. Structure of Ilicicolin H, an antifungal antibiotic
from Cylindrocladium ilicicola. J. Sci. Hiroshima Univ., Ser. A: Phys.
Chem. 1979, 41, 47.
(11) Williams, D. R.; Bremmer, M. L.; Brown, D. L.; D'Antuono, J.
Total synthesis of ( )-ilicicolin H. J. Org. Chem. 1985, 50, 2807.
(12) Tanabe, M.; Urano, S. Biosynthetic studies with carbon-13. The
antifungal antibiotic ilicicolin H. Tetrahedron 1983, 39, 3569.
(13) Abruzzo, G. K.; Flattery, A. M.; Gill, C. J.; Kong, L.; Smith, J. G.;
Pikounis, V. B.; Balkovec, J. M.; Bouffard, A. F.; Dropinski, J. F.;
Rosen, H.; Kropp, H.; Bartizal, K. Evaluation of the echinocandin
antifungal MK-0991 (L-743,872): Efficacies in mouse models of
disseminated aspergillosis, candidiasis, and cryptococcosis. Antimicrob.
Agents Chemother. 1997, 41, 2333.
(14) Gutierrez-Cirlos, E. B.; Merbitz-Zahradnik, T.; Trumpower, B.
L. Inhibition of the Yeast Cytochrome bc1 Complex by Ilicicolin H, a
Novel Inhibitor That Acts at the Qn Site of the bc1 Complex. J. Biol.
Chem. 2004, 279, 8708.
(15) Ding, M. G.; di Rago, J. P.; Trumpower, B. L. Investigating the
Qn site of the cytochrome bc1 complex in Saccharomyces cerevisiae
with mutants resistant to ilicicolin H, a novel Qn site inhibitor. J. Biol.
Chem. 2006, 281, 36036.
(16) Rotsaert, F. A.; Ding, M. G.; Trumpower, B. L. Differential
efficacy of inhibition of mitochondrial and bacterial cytochrome bc1
complexes by center N inhibitors antimycin, ilicicolin H and
funiculosin. Biochim. Biophys. Acta 2008, 1777, 211.
(17) Liu, W. G.; Guan, Z. Q.; Singh, S. B. A new method for the
synthesis of 1,4,5-oxadiazocines and its application in the structure
modification of natural products. Tetrahedron Lett. 2005, 46, 8009.
(18) Lancaster, C. R. D.; Hunte, C.; Kelley, J.; Trumpower, B. L.;
Ditchfield, R. A comparison of stigmatellin conformations, free and
bound to the photosynthetic reaction center and the cytochrome
bc(1) complex. J. Mol. Biol. 2007, 368, 197.
(19) Huang, L. S.; Cobessi, D.; Tung, E. Y.; Berry, E. A. Binding of
the respiratory chain inhibitor antimycin to the mitochondrial bc(1)
complex: A new crystal structure reveals an altered intramolecular
hydrogen-bonding pattern. J. Mol. Biol. 2005, 351, 573.
(20) (MOE), M. O. E. 1010 Sherbooke St. West, Suite #910,
Montreal, QC, Canada, H3A 2R7, 2010.10.
(21) Covian, R.; Trumpower, B. L. Ilicicolin Inhibition and Binding
at Center N of the Dimeric Cytochrome bc1 Complex Reveal Electron
Transfer and Regulatory Interactions between Monomers. J. Biol.
Chem. 2009, 284, 8614.
ASSOCIATED CONTENT
* Supporting Information
■
S
Experimental procedure for bioassays and mutation data. This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*E-mail: sheo.singh@merck.com (S.B.S.) or jconishi@verizon.
net (J.C.O.).
■
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Notes
The authors declare no competing financial interest.
†Deceased.
REFERENCES
■
(1) Hof, H. Will resistance in fungi emerge on a scale similar to that
seen in bacteria? Eur. J. Clin. Microbiol. Infect. Dis. 2008, 27, 327.
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