antibiotics
Article
Semisynthetic Amides of Amphotericin B and Nystatin A1:
A Comparative Study of In Vitro Activity/Toxicity Ratio in
Relation to Selectivity to Ergosterol Membranes
Anna Tevyashova 1, * , Svetlana Efimova 2 , Alexander Alexandrov 3 , Olga Omelchuk 1 , Eslam Ghazy 3,4,5 ,
Elena Bychkova 1 , Georgy Zatonsky 1 , Natalia Grammatikova 1 , Lyubov Dezhenkova 1 , Svetlana Solovieva 1 ,
Olga Ostroumova 2 and Andrey Shchekotikhin 1
1
2
3
4
5
*
Citation: Tevyashova, A.; Efimova, S.;
Alexandrov, A.; Omelchuk, O.;
Ghazy, E.; Bychkova, E.; Zatonsky, G.;
Grammatikova, N.; Dezhenkova, L.;
Solovieva, S.; et al. Semisynthetic
Amides of Amphotericin B and
Nystatin A1 : A Comparative Study of
In Vitro Activity/Toxicity Ratio in
Relation to Selectivity to Ergosterol
Gause Institute of New Antibiotics, 11 B. Pirogovskaya, 119021 Moscow, Russia
Institute of Cytology of the Russian Academy of Sciences, 4 Tikhoretsky, 194064 St. Petersburg, Russia
Federal Research Center “Fundamentals of Biotechnology” of the Russian Academy of Sciences,
Bach Institute of Biochemistry, 33 Leninsky Ave., bld. 2, 119071 Moscow, Russia
Institute of Biochemical Technology and Nanotechnology, Peoples’ Friendship University of Russia (RUDN),
6 Miklukho-Maklaya Street, 117198 Moscow, Russia
Department of Microbiology, Faculty of Pharmacy, Tanta University, Tanta 31111, Egypt
Correspondence: chulis@mail.ru; Tel.: +7-499-246-06-36
Abstract: Polyene antifungal amphotericin B (AmB) has been used for over 60 years, and remains
a valuable clinical treatment for systemic mycoses, due to its broad antifungal activity and low
rate of emerging resistance. There is no consensus on how exactly it kills fungal cells but it is
certain that AmB and the closely-related nystatin (Nys) can form pores in membranes and have a
higher affinity towards ergosterol than cholesterol. Notably, the high nephro- and hemolytic toxicity
of polyenes and their low solubility in water have led to efforts to improve their properties. We
present the synthesis of new amphotericin and nystatin amides and a comparative study of the
effects of identical modifications of AmB and Nys on the relationship between their structure and
properties. Generally, increases in the activity/toxicity ratio were in good agreement with increasing
ratios of selective permeabilization of ergosterol- vs. cholesterol-containing membranes. We also
show that the introduced modifications had an effect on the sensitivity of mutant yeast strains
with alterations in ergosterol biosynthesis to the studied polyenes, suggesting a varying affinity
towards intermediate ergosterol precursors. Three new water-soluble nystatin derivatives showed a
prominent improvement in safety and were selected as promising candidates for drug development.
Membranes. Antibiotics 2023, 12, 151.
https://doi.org/10.3390/
antibiotics12010151
Keywords: polyene antibiotics; amphotericin b; nystatin; semisynthetic derivatives; amidation;
water-soluble; antifungal activity; ergosterol; membrane permeabilization; cytotoxicity; hemolysis
Academic Editors: Ines Primožič,
Renata Odžak and Matilda Šprung
Received: 1 December 2022
1. Introduction
Revised: 24 December 2022
Systemic fungal infections, which are a common comorbidity in immunocompromised
patients, are characterized by severe symptoms and high mortality rates [1,2]. The first-line
antifungals used for chemotherapy of invasive mycoses include azoles and echinocandins;
however, their long-term use results in the widespread emergence of resistant pathogenic
fungi [3]. The coronavirus pandemic, COVID-19, announced in 2020, led to a dramatic
increase in the prescription of antibiotics to treat or prevent the development of bacterial
and fungal complications in immunocompromised patients [4]. As a result, a significant
increase in antimicrobial resistance both to antibacterial and antifungal agents is being
reported [5–7]. Thus, polyene macrolide amphotericin B (Figure 1, AmB, 1), which has a
low rate of resistance emergence, is still the most widely used antifungal in intensive care,
though its use is limited by nephrotoxicity and very low water solubility [8]. Amphotericin
B is also the drug of choice for mucormycosis treatment [9]. This condition has become
more frequent, mostly due to patients hospitalized with coronavirus infections [10].
Accepted: 5 January 2023
Published: 11 January 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
Antibiotics 2023, 12, 151. https://doi.org/10.3390/antibiotics12010151
https://www.mdpi.com/journal/antibiotics
Antibiotics 2023, 12, 151
photericin B is also the drug of choice for mucormycosis treatment [9]. This condition has
become more frequent, mostly due to patients hospitalized with coronavirus infections
[10].
The other significant polyene macrolides in clinical use are nystatin A1 (Figure 1, Nys,
2) and natamycin (Figure 1, Nat, 3) (Figure 1); however, these are not used systemically.
2 of 20
Common structural features shared by AmB, nystatin and natamycin include a conjugated double-bond system and the carbohydrate residue mycosamine.
Figure 1.
1. Structure
Structure of
of polyene
polyene antibiotics
antibiotics amphotericin
amphotericin B,
B, nystatin
nystatin A
A11 and natamycin.
Figure
The
other significant
polyene macrolides
clinicalantibiotics
use are nystatin
A1 (Figure
1, Nys,
2)
Research
on the mechanism
of action ofin
polyene
has been
conducted
since
and
natamycin
(Figure
1,
Nat,
3)
(Figure
1);
however,
these
are
not
used
systemically.
the 1970s and remains relevant nowadays [11]. Initial studies in this area revealed that
Common
structural
features
shared by AmB,
andwith
natamycin
conjugated
AmB causes
membrane
permeabilization
vianystatin
interaction
sterols include
[12–15] aand,
in 1974,
double-bond
system
and
the
carbohydrate
residue
mycosamine.
the barrel-stave model was proposed [16]. For decades, pore-formation activity was conResearch
on the
mechanism
of actionactivity
of polyene
antibiotics
hasand
been
conducted
since
sidered
the main
mode
of the fungicidal
of AmB
and Nys,
the
higher affinity
the
1970s andtoremains
relevant
nowadays
[11]. Initial
studies
in considered
this area revealed
AmB
of polyenes
ergosterol
(ERG)
vs. cholesterol
(CHOL)
was
to be that
the likely
causes
membrane
permeabilization
via
interaction
with
sterols
[12–15]
and,
in
1974,
the
explanation for their selectivity against fungal cells [17-19]. As for Nat, a smaller member
barrel-stave
model
was
proposed
[16].
For
decades,
pore-formation
activity
was
considered
of the polyene antifungal family, widely used in medicine and agriculture, the mode of
the
main
of the
fungicidal
of AmB
Nys, and
thefound
higherthat
affinity
of
action
hasmode
remained
unclear
up to activity
2008, when
Y.M. and
te Welscher
et al.
Nat did
polyenes
to
ergosterol
(ERG)
vs.
cholesterol
(CHOL)
was
considered
to
be
the
likely
not permeabilize membranes, and its antifungal action was realized through binding to
explanation
for Subsequent
their selectivity
against fungal
cells that
[17–19].
As for Nat,
a smaller
member
ergosterol [20].
investigations
showed
natamycin,
via its
interaction
with
of
the
polyene
antifungal
family,
widely
used
in
medicine
and
agriculture,
the
mode
of
ergosterol, inhibits amino acid and glucose transport across the plasma membrane [21];
action
has
remained
unclear
up
to
2008,
when
Y.M.
te
Welscher
et
al.
found
that
Nat
did
moreover, other polyenes (AmB and Nys) are thought to share this mode of action, which
not permeabilize membranes, and its antifungal action was realized through binding to
is masked by their ability to form pores. In recent years, M. Burke’s group has claimed
ergosterol [20]. Subsequent investigations showed that natamycin, via its interaction with
that pore formation is not necessary for the antifungal action of AmB [22] and that it kills
ergosterol, inhibits amino acid and glucose transport across the plasma membrane [21];
fungal cells by forming extramembranous aggregates, thereby extracting ergosterol from
moreover, other polyenes (AmB and Nys) are thought to share this mode of action, which
lipid bilayers [23]. In 2021, they expanded this sterol-sponge mode of antifungal action to
is masked by their ability to form pores. In recent years, M. Burke’s group has claimed
other glycosylated polyene macrolides [24]. According to this model, the separation of the
that pore formation is not necessary for the antifungal action of AmB [22] and that it kills
pore-forming activity and the ability to extract ergosterol through the modification of polfungal cells by forming extramembranous aggregates, thereby extracting ergosterol from
yene’s structure is a promising way to obtain less toxic antifungals [25]. However, there
lipid bilayers [23]. In 2021, they expanded this sterol-sponge mode of antifungal action
are contrary opinions about this theory. Delhom et al. proved the ability of AmB to extract
to other glycosylated polyene macrolides [24]. According to this model, the separation of
ERG from lipid bilayers, but this was not observed for CHOL-containing bilayers. This
the pore-forming activity and the ability to extract ergosterol through the modification of
observation
partly supports
the sterol-sponge
[26].antifungals
Daniel M. Kaminski
cast doubt
polyene’s
structure
is a promising
way to obtainmodel
less toxic
[25]. However,
there
on
the
relevance
of
this
model
in
living
organisms
[27].
Yamamoto
et
al.
demonstrated
are contrary opinions about this theory. Delhom et al. proved the ability of AmB to extract
that AmB
molecules
are but
located
parallel
to the lipid bilayer
[28], which
ERG
from lipid
bilayers,
this predominantly
was not observed
for CHOL-containing
bilayers.
This
observation partly supports the sterol-sponge model [26]. Daniel M. Kaminski cast doubt
on the relevance of this model in living organisms [27]. Yamamoto et al. demonstrated
that AmB molecules are located predominantly parallel to the lipid bilayer [28], which
supports the barrel stave model. Subsequent work resolved the entire structure of the
AmB channel assembly in ergosterol-containing membranes by NMR, as well as molecular
dynamics calculations, thus claiming that “other possible structures, such as the sterol
sponge model, are considered highly unlikely” [29]. Among the secondary effects of AmB,
the most prominent is the induction of oxidative stress, leading to fungal cell death [30,31].
Due to their amphiphilic structure, polyenes tend to form aggregates, and the influence
of aggregation on activity/toxicity ratio for AmB has been investigated. AmB can permeabilize ERG-containing phosphatidylcholine membranes in a monomeric state as well as in an
Antibiotics 2023, 12, 151
3 of 20
aggregated form; while in the case of CHOL-containing and sterol-free membranes, AmB in
its monomeric form lacks pore-forming activity [32]. Furthermore, monomeric AmB solutions are less toxic than the solutions of Fungizone, a form in which the polyene is in almost
completely self-aggregated form [33]. Finally, a series of papers [34–36] presented less toxic
semisynthetic derivatives of AmB with decreased dimerization in water solutions due to a
lack of zwitterionic properties. This supports the idea that the reduction of the dimerization
properties of AmB is a promising approach to obtain less toxic antifungal agents.
The aim of our research was to create new, less toxic semisynthetic polyenes with
increased solubility in water, suitable for the further development of next generation
antifungal drugs for the treatment of systemic mycoses. The presented data shows that two
factors mainly lead to a decrease in toxicity: an increase in specific affinity to ergosterol and
a decrease in the tendency to aggregate.
Thus, we present a new series of nystatin amides and compare these with new and
previously published amides of AmB in terms of their activity/toxicity ratio in vitro on cells
and on model Erg- and Chol-containing membranes. We decided to focus on the amidation
of the carboxylic group, because this modification is both simple to introduce and has a
high impact on the activity/toxicity ratio, as first reported in 1989 [37] and later supported
by a series of papers [34,35,38–40], describing safer and more highly active water-soluble
polyene amides, which in most cases, contain an additional basic residue. We also obtained
indications that polyene amides may differ in their affinity to various ergosterol precursors,
which is relevant to drug-resistant fungal strains.
2. Results
2.1. Synthesis of Polyene Amides
Chemical modification involved the direct amidation of the C16-carboxylic group of
the
antibiotic
by the corresponding diamine in the presence of benzotriazol-1-yl-oxytripyrrAntibiotics 2023, 12, x FOR PEER REVIEW
4 of 22
olidinophosphonium hexafluorophosphate (PyBOP) and the subsequent purification of the
obtained carboxamide by column chromatography (Scheme 1).
Scheme 1. Synthesis of polyene antibiotic amides.
The
1c–1e, 2a–2e
from moderate
to good
good (20–60%)
(20–60%)
The yields
yields of
of the
the target
target amides
amides 1c–1e,
2a–2e varied
varied from
moderate to
due
well-known lability
lability of
of polyene
polyene antibiotics,
antibiotics, their
their sensitivity
sensitivity to
to light
light and
and oxygen,
oxygen,
due to
to the
the well-known
and basic/acid conditions. AmB derivatives 1a,b were described earlier [35]. All the new
amides had considerably higher solubility in water compared to their parent antibiotics.
For several of them, the determination of the solubility was performed using UV spectroscopy of standard and saturated solutions according to the method described previously
Antibiotics 2023, 12, 151
4 of 20
and basic/acid conditions. AmB derivatives 1a,b were described earlier [35]. All the new
amides had considerably higher solubility in water compared to their parent antibiotics. For
several of them, the determination of the solubility was performed using UV spectroscopy
of standard and saturated solutions according to the method described previously [41,42]
with minor modifications. Saturation of the solutions at a concentration of 100 g/L was
not achieved for the compounds 1a,c,e, and 2a,c,e, thus indicating a solubility of more than
100 g/L (solubility of AmB–0.75 g/L, Nys–0.36 g/L [43]).
The purity of new amides 1a–1e, 2a–2e was determined by TLC and HPLC, and the
structure was confirmed by HR-ESI mass-spectrometry and NMR spectra. To fully assign
signals in both the 1 H and 13 C spectra, a set of 2D experiments was performed: 1 H-1 H
COSY, TOCSY, NOESY (ROESY), HSQC, HMBC, and H2BC. The latter experiment was
especially useful for assigning resonances from the polyene part of the molecules. In
addition, a set of selective TOCSY experiments was performed in order to assign the signals
of the H-19, H-20, H-33, and H-34 atoms of the polyene part. The assignment of 1 H and
13 C signals for compounds 1c–1e, 2a–2e is shown in Table S1 (Supporting information).
2.2. In Vitro Antifungal Activity and Toxicity toward Mammalian Cells
The antifungal activity of polyene derivatives 1a–1e, 2a–2e was tested against strains
of Candida spp. (reference strains C. parapsilosis ATCC 22019, C. albicans ATCC 10231
and clinical isolates C. krusei 432M, C. albicans 604M, C. albicans 8R, C. glabrata 61L, C.
tropicalis 3010, C. parapsilosis 58L) and filamentous fungi (Aspergillus fumigatus ATCC 46645,
Aspergillus niger 37a and Trichophyton rubrum 2002) and compared to that of AmB (1) and
Nys (2) using the broth microdilution method, as described in the EUCAST definitive
documents [44,45] (Table 1). Reference strain C. parapsilosis ATCC 22019 was used as
the control in each experiment. In all experiments, the in vitro microdilution technique
in 96-microwell plates was applied. The minimum inhibitory concentration (MIC) was
defined as the lowest concentration that resulted in complete growth inhibition after
incubation for 24 and 48 h for Candida spp. and 48–72 h for dermatophytes.
Table 1. The antifungal activity of the semisynthetic polyene antibiotic derivatives 1a–1e, 2a–2e in
comparison with AmB (1) and Nys (2).
Minimum Inhibitory Concentration (MIC, µg/mL)
Tested Strains
C. parapsilosis ATCC
22019
C. albicans ATCC 10231
A. fumigatus ATCC
46645
A. niger 37a
T. rubrum 2002
C. krusei 432M
C. albicans 604M
C. albicans 8R
C. glabrata 61L
C. tropicalis 3010
C. parapsilosis 58L
Compound
1a
1
2a
0.03/0.06 2
0.5/1
1
2b
1c
2c
1d
2d
1e
2e
1
2
0.06/0.125
2/4
1/1
1/2
2/2
2/4
1/2
1/2
1/2
2/2
1b
n/t
1/2
n/t
2/4
1/1
2/4
2/2
2/4
1/1
4/8
0.5/0.5
1/2
n/t
2
n/t
4
1
2
4
2
2
8
2
4
0.125
0.5
0.25/0.5
0.05/0.1
0.05/0.1
0.03/0.06
n/t
n/t
8
8
4/4
1/1
1/2
1/1
1/1
1/2
0.5
0.25
0.25/0.5
n/t
n/t
n/t
n/t
n/t
16
32
8/8
2/4
4/4
2/2
2/2
2/4
4
2
2/2
1/1
1/1
1/1
0.5/1
1/1
8
8
16/16
2/2
2/4
1/2
2/2
1/2
1
1
2/2
2/2
2/2
2/4
2/2
2/2
32
8
8/8
2/2
4/4
2/2
2/2
2/4
2
2
2/2
1/1
1/1
2/2
1/1
1/1
16
8
8/16
2/4
2/4
2/2
2/4
2/2
2
1
0.5/1.0
1/1
1/1
1/2
0.5/1
0.5/1
8
8
4/4
1/2
1/2
1/2
1/2
1/2
1
Published earlier in [35]; 2 24/48 h.
All of the new Nys derivatives showed good antifungal activity, close to that of parent
antibiotic (with the exception of C. krusei 432M); in the AmB group, the most prominent results
were obtained for amides with ethylenediamine and N-(2-hydroxyethyl)ethylenediamine
moieties, 1a and 1b, respectively, published earlier.
A preliminary estimation of the toxicity of the new polyene amides was performed
using the MTT test on human embryonic kidney cells (HEK293) (Table 2). The reference
drugs used were AmB (1), Nys (2), and the antitumor antibiotic doxorubicin. For the new
Antibiotics 2023, 12, 151
5 of 20
compounds with the highest activity/toxicity ratios, we also determined the hemolytic
activity (Figure 2).
Table 2. The antiproliferative activity of the semisynthetic polyene antibiotic derivatives 1a–1e, 2a–2e
in comparison with AmB (1), Nys (2) and Doxorubicin.
Compound
35.0 ± 4.0
4.0 ± 0.4
3.9 ± 0.5
20.0 ± 2.4
6.0 ± 0.7
47.0 ± 5.2
47.0 ± 4.7
>50.0
15.0 ± 1.7
>50.0
18.0 ± 4.0
15.0 ± 3.0
0.20 ± 0.02
1a
1b
1c
1d
1e
2a
2b
2c
2d
2e
1 (AmB)
2 (Nys)
Doxorubicin
Antibiotics 2023, 12, x FOR PEER REVIEW
60
10 μМ
20 μМ
50
50 μМ
80
Hemolysis, %%
Hemolysis, %%
IC50 , µM (HEK293)
40
30
20
10
0
1 (AmB)
6 of 22
2 (Nys)
60
40
20
0
0
6
12
18
24
t, hours
(a)
(b)
Figure
2. Hemolytic
activity
of selected
amidesamides
of Nysof
and
AmB:
hemolysis
ratio after
1 h after
incu- 1 h
Figure
2. Hemolytic
activity
of selected
Nys
and(a)
AmB:
(a) hemolysis
ratio
bation
of
red
blood
cells
with
10,
20
and
50
μM
of
tested
compounds;
(b)
hemolysis
ratio
during
24
incubation of red blood cells with 10, 20 and 50 µM of tested compounds; (b) hemolysis ratio during
h incubation
of
red
blood
cells
with
20
μM
of
tested
compounds.
24 h incubation of red blood cells with 20 µM of tested compounds.
TheThe
most
active
NysNys
derivative
2a was
3-fold
lessless
toxic
against
kidney
cells
andand
most
active
derivative
2a was
3-fold
toxic
against
kidney
cells
showed
similar
hemolytic
activity
at 10
20 μM
with
notably
lower
activity
at 50atμM,
showed
similar
hemolytic
activity
atand
10 and
20 µM
with
notably
lower
activity
50 µM,
as compared
to
Nys.
Derivatives
2c
and
2e
did
not
display
toxicity
against
kidney
cells
as compared to Nys. Derivatives 2c and 2e did not display toxicity against kidney cells
andand
possessed
weak
hemolytic
activity
up to
After
24 h,
hemolysis
ratio
of 2c
possessed
weak
hemolytic
activity
up50
toμM.
50 µM.
After
24the
h, the
hemolysis
ratio
of 2c
diddid
notnot
exceed
5%,5%,
while
NysNys
demonstrated
a ratio
of 59%.
AmB
derivatives
were
quite
exceed
while
demonstrated
a ratio
of 59%.
AmB
derivatives
were
quite
toxic
to HEK293,
andand
the the
least
toxic
derivative
1d caused
rapid
hemolysis
at all
tested
toxic
to HEK293,
least
toxic
derivative
1d caused
rapid
hemolysis
at the
all the
tested
concentrations
to 20–30%,
after
thehemolysis
hemolysisratio
ratiodid
didnot
notchange,
change,while
while nanative
concentrations
up up
to 20–30%,
butbut
after
2424
hh
the
with
tiveantibiotics
antibiotics(Nys
(Nysand
andAmB)
AmB)showed
showeda agradual
gradualincrease
increaseininmembrane
membranepermeabilization
permeabilization
time
up
to
60%.
with time up to 60%.
Electrophysiological
Experiments
2.3.2.3.
Electrophysiological
Experiments
Figure
3 demonstrates
theability
abilityofofthe
the parent
parent polyene
their
Figure
3 demonstrates
the
polyene macrolide
macrolideantibiotics
antibioticsand
and
derivatives
at
5
µM
to
disengage
calcein
from
large
unilamellar
vesicles
prepared
from
their derivatives at 5 μM to disengage calcein from large unilamellar vesicles prepared
67 67
mol%
palmitoyloleoylphosphocholine
(POPC)
and and
33 mol%
CHOL
(Figure
3a,b) or
from
mol%
palmitoyloleoylphosphocholine
(POPC)
33 mol%
CHOL
(Figure
ERG (Figure 3c,d). POPC is a hybrid monounsaturated lipid that is made up of one fully
3a,b) or ERG (Figure 3c,d). POPC is a hybrid monounsaturated lipid that is made up of
saturated 16C chain and one monounsaturated 18C (D9-Cis) fatty acid chain, a variant of
one fully saturated 16C chain and one monounsaturated 18C (D9-Cis) fatty acid chain, a
phosphatidylcholine, the major abundant phospholipid of eukaryotic cell membranes. The
variant of phosphatidylcholine, the major abundant phospholipid of eukaryotic cell memfirst lipid mixture (POPC/CHOL) imitates the antibiotic action on mammalian cell membranes. The first lipid mixture (POPC/CHOL) imitates the antibiotic action on mammalian
cell membranes and the second one (POPC/ERG) mimics fungal membranes. The twoexponential dependences were used to fit the time dependences of calcein release induced
by different polyenes (AmB and Nys) and their derivatives from POPC/CHOL- and
POPC/ERG-liposomes as a first-order approximation. The characteristic parameters of de-
Antibiotics 2023, 12, 151
6 of 20
branes and the second one (POPC/ERG) mimics fungal membranes. The two-exponential
dependences were used to fit the time dependences of calcein release induced by different
polyenes (AmB and Nys) and their derivatives from POPC/CHOL- and POPC/ERGliposomes as a first-order approximation. The characteristic parameters of dependences,
Antibiotics 2023, 12, x FOR PEER REVIEW
7 of 22
the maximal leakage, RFmax , and the times related to fast and slow components, t1 and t2 ,
respectively, are presented in Table 3.
NyS
(a)
(b)
(c)
(d)
POPC/ERG
POPC/CHOL
AmB
Figure
of the
thepolyene-induced
polyene-inducedrelative
relative
fluorescence
of calcein
%) leaked
Figure3.3. The
The dependence of
fluorescence
of calcein
(RF,(RF,
%) leaked
from
from
POPC/CHOL
(67/33
mol
(upper
panel)
and
POPC/ERG(67/33
(67/33 mol
POPC/CHOL
(67/33
mol
%)%)
(upper
panel)
and
POPC/ERG
mol %)
%) (lower
(lowerpanel)
panel)vesicles
vesicles
on
ontime.
time.The
Themoment
momentofofaddition
additionofof55μM
µMofofAmB
AmB(a,c)
(a,c)and
andNyS
NyS(b,d)
(b,d)derivatives
derivativesinto
intoliposomal
liposomal
suspension
is
referred
to
as
zero
point.
The
relation
between
the
color
symbol
and
the
suspension is referred to as zero point. The relation between the color symbol and thecompound
compoundisis
given on the figure.
given on the figure.
Table 3. The characteristic parameters of the dependence of calcein release from large unilamellar
The efficacy of the tested AmB derivatives (1d, 1c and 1e) to disengage calcein from
vesicles of indicated composition induced by 5 μM of antibiotic on time.
POPC/CHOL vesicles decreased in the following order: 1d (RFmax is about 40%) > 1c ≈ 1e
(about 15%)
> AmB (1) (about
6%) %)
(Figure 3a
and Table (67/33
3). In the
POPC/CHOL
(67/33 mol
POPC/ERG
molcase
%) of the POPC/ERG
EI 3
liposomes,
RF t1 2decreased
the same
the effects
Polyene
RFthe
max 1, %max
, min t2 2,inmin
RFmaxorder,
, % but
t1, min
t2, were
min 1.5-times greater
for AmB,
and Table
case of
in the
AmB
(1) 1d,
6 ± 1c
1 and
0.61e± (Figure
0.3 12.43c
± 8.1
9 ± 13). In0.3the
± 0.1
5.8Nys
± 1.1derivatives
1.5 ± 0.4
POPC/CHOL vesicles, the RFmax decreased in the following order: 2b (about 70%) > 2c ≈
1c
13 ± 1
0.6 ± 0.1 7.9 ± 0.4
16 ± 2
0.5 ± 0.2
6.9 ± 1.1
1.2 ± 0.2
2d ≈ 2a (about 40%) > 2e (about 20%) > Nys (2) (about 10%) and in POPC/ERG liposomes
1d
39in
± 3the series:
0.5 ± 0.1
13.1
3
0.4
± 0.1
11.9 ± 1.2
± 0.2
it decreased
2c ≈
2b ±≈4.8
2d ≈ 57
2e ±(about
80%)
> 2a (about
45%) >1.6
Nys
(about
1e
16
±
1
0.3
±
0.1
4.7
±
2.6
21
±
3
0.1
±
0.1
9.7
±
2.4
1.3
±
0.2
20%) (Figure 3b,d and Table 3).
Nys (2)
10 ± 2 we
0.9estimated
± 0.3 5.7 the
± 1.9efficiency
19 ± 3index,
0.7EI,
± 0.1
5.4 ±of
0.5the maximal
1.9 ± 0.8leakAdditionally,
as a ratio
2aof calcein
33 ± from
5
0.5
± 0.2 4.2 vesicles
± 0.2
44 ± 2
0.5
0.1
4.8 ± 0.6
1.3 ± %)
0.3 and
age
unilamellar
composed
of ±POPC/ERG
(67/33 mol
POPC/CHOL
%) (Table
3). The
corresponds
higher safety.
It was
2b
71 ± (67/33
7
0.5mol
± 0.1
4.6 ± 0.3
82higher
± 5 EI0.5
± 0.1
4.3to
± 0.4
1.3 ± 0.3
found
this
decreased
in the 86
following
orders
derivatives:
2c that 37
± 9parameter
0.2 ± 0.1
2.4 ± 0.2
±8
0.3
± 0.1for AmB
6.6 ± and
2.4 NyS2.3
± 0.8
1d2d
≈ AmB 39
≥ ±1e9 ≈ 1c0.3
and
2e >3.9
2c ≥
Nys ≈70
2d±>6 2b =0.5
2a.± 0.1
± 0.1
± 0.3
7.9 ± 2.4
1.8 ± 0.6
2e
22 ± 4
0.9 ± 0.4 6.7 ± 0.4
75 ± 7
0.4 ± 0.1
2.9 ± 0.7
3.4 ± 0.9
1 RFmax—maximal leakage of calcein from unilamellar vesicles composed of POPC/CHOL and
POPC/ERG respectively; 2 t1, t2—characteristic times referring to fast and slow components, respectively, of two exponential dependence fitting the time dependence of calcein release 3 EI—the efficiency index determined as a ratio of the RFmax from unilamellar vesicles composed of POPC/ERG
Antibiotics 2023, 12, 151
7 of 20
Antibiotics 2023, 12, x FOR PEER REVIEW
8 of 22
Table 3. The characteristic parameters of the dependence of calcein release from large unilamellar
vesicles of indicated composition induced by 5 µM of antibiotic on time.
The efficacy
of the tested
AmB
derivatives
(1d,POPC/ERG
1c and 1e)(67/33
to disengage
POPC/CHOL
(67/33
mol
%)
mol %) calcein from
3
POPC/CHOL
vesicles
decreased
in
the
following
order:
1d
(RF
max is about 40%) > 1cEI
≈ 1e
1
2
2
RFmax , %
t1 , min
t2 , min
Polyene
RFmax , %
t1 , min
t2 , min
(about 15%) > AmB (1) (about 6%) (Figure 3a and Table 3). In the case of the POPC/ERG
6±1
0.6 ± 0.3
12.4 ± 8.1
9±1
0.3 ± 0.1
5.8 ± 1.1
1.5 ± 0.4
AmB (1)
liposomes, the RFmax decreased in the same order, but the effects were 1.5-times greater
1c
13 ± 1
0.6 ± 0.1
7.9 ± 0.4
16 ± 2
0.5 ± 0.2
6.9 ± 1.1
1.2 ± 0.2
for AmB, 1d, 1c and 1e (Figure 3c and Table 3). In the case of Nys derivatives in the
39 ± 3
0.5 ± 0.1
13.1 ± 4.8
±3
0.4 ± 0.1
11.9 ± 1.2 1.6 ± 0.2
1d
in the57following
order: 2b (about
70%) > 2c ≈
POPC/CHOL
vesicles, the
RFmax decreased
± 1 > 2e (about
0.3 ± 0.120%)4.7
± 2.6(2) (about
21 ± 310%) 0.1
0.1POPC/ERG
9.7 ± 2.4 liposomes
1.3 ± 0.2
2d ≈1e2a (about1640%)
> Nys
and±in
10
±
2
0.9
±
0.3
5.7
±
1.9
19
±
3
0.7
±
0.1
5.4
±
0.5
1.920%)
± 0.8
Nys
(2)
it decreased in the series: 2c ≈ 2b ≈ 2d ≈ 2e (about 80%) > 2a (about 45%) > Nys (about
2a
33
±
5
0.5
±
0.2
4.2
±
0.2
44
±
2
0.5
±
0.1
4.8
±
0.6
1.3
± 0.3
(Figure 3b,d and Table 3).
71 ± 7we estimated
0.5 ± 0.1the efficiency
4.6 ± 0.3 index,
82 ±EI,
5 as a0.5
± 0.1
± 0.4 leakage
1.3 ± 0.3
2b
Additionally,
ratio
of the4.3
maximal
of calcein
from
vesicles
composed
of8 POPC/ERG
and
2c
37 ± unilamellar
9
0.2 ± 0.1
2.4 ±
0.2
86 ±
0.3 ± 0.1 (67/33
6.6 ± mol
2.4 %)
2.3 ±
0.8
POPC/CHOL
(67/33
mol
%)
(Table
3).
The
higher
EI
corresponds
to
higher
safety.
It
was
39 ± 9
0.3 ± 0.1
3.9 ± 0.3
70 ± 6
0.5 ± 0.1
7.9 ± 2.4
1.8 ± 0.6
2d
found
decreased
in
following
for
AmB
derivatives:
2e that this22parameter
±4
0.9
± 0.4
6.7the
± 0.4
75 ±orders
7
0.4 ±
0.1 and
2.9NyS
± 0.7
3.4 ± 0.9
AmB
≥
1e
≈
1c
and
2e
>
2c
≥
Nys
≈
2d
>
2b
=
2a.
11d
RF≈
—maximal
leakage
of
calcein
from
unilamellar
vesicles
composed
of
POPC/CHOL
and
POPC/ERG
max
2 t mentioned
As was
above,
different
macrolides
were respectively,
shown to have
respectively;
times
referringpolyene
to fast and
slow components,
of twodifferent
exponential
1 , t2 —characteristic
3
dependence
fitting
timethe
dependence
of calcein release
efficiency index
a ratio
of the
modes of actionthe
with
same target—the
fungalEI—the
cell membrane.
It isdetermined
believedasthat
AmB
RFmax from unilamellar vesicles composed of POPC/ERG and POPC/CHOL.
and Nys form pores via interaction with sterols (especially ERG) in the membrane, which
thenAs
leads
uncontrolled
leakage
of thepolyene
intracellular
contents
andshown
cell death
[46]. different
wastomentioned
above,
different
macrolides
were
to have
Diphytanoyl
phospholipids,
and
especially,
diphytanoylphosphocholine
modes of action with the same target—the fungal cell membrane. It is believed(DPhPC),
that AmB
are commonly
used via
as model
membranes
when(especially
conductingERG)
electrophysiological
experiand
Nys form pores
interaction
with sterols
in the membrane,
which
ments.
DPhPC
has
two
fully
saturated
16C
fatty
acid
chains
with
four
methyl
groups
then leads to uncontrolled leakage of the intracellular contents and cell death [46]. attached
to each chain.
It is believedand
thatespecially,
synthetic phytanyl-chained
glycolipid bilayers
areare
Diphytanoyl
phospholipids,
diphytanoylphosphocholine
(DPhPC),
promising used
materials
for the
reconstitution
transportelectrophysiological
studies of membrane
proteins
commonly
as model
membranes
whenand
conducting
experiments.
[47].
This
relates
to
their
unique
properties,
such
as
high
chemical/physical
stability
(in- to
DPhPC has two fully saturated 16C fatty acid chains with four methyl groups attached
cluding
under
mechanical
stress),
low
water
permeability,
and
no
gel-to-fluid
phase
traneach chain. It is believed that synthetic phytanyl-chained glycolipid bilayers are promising
sition at ambient
temperature [48].
high chemical
mechanical proteins
stability of
phytmaterials
for the reconstitution
andThe
transport
studiesand
of membrane
[47].
This
anoyl
lipids
is
attributed
to
the
entirely
saturated
alkyl
chains,
which
are
much
less
sensirelates to their unique properties, such as high chemical/physical stability (including
tive to mechanical
chemical degradation
bywater
air or light
than unsaturated
ones, including
POPC.
How- at
under
stress), low
permeability,
and no gel-to-fluid
phase
transition
ever,
the
negative
spontaneous
curvature
of
diphytanoyl
lipids
does
not
allow
their
use
ambient temperature [48]. The high chemical and mechanical stability of phytanoyl lipids
in
leakage
techniques.
is attributed to the entirely saturated alkyl chains, which are much less sensitive to chemical
Figure 4by
presents
the electrical
current fluctuations
corresponding
to the opening
and
degradation
air or light
than unsaturated
ones, including
POPC. However,
the negative
closure
of
single
channels
formed
by
AmB
and
its
tested
conjugates,
1c,
1d
and
1e,
in
the
spontaneous curvature of diphytanoyl lipids does not allow their use in leakage techniques.
lipidFigure
bilayers
composed
DPhPC current
and ERG
(67/33 molcorresponding
%) and bathedtointhe
2M
KCl (pH
4 presents
theofelectrical
fluctuations
opening
and
7.4)
at
transmembrane
voltage
200
mV.
One
can
see
that
all
tested
derivatives
(1c,
1d and
closure of single channels formed by AmB and its tested conjugates, 1c, 1d and 1e,
in the
1e) asbilayers
well ascomposed
1a [35] produced
pores
smaller
amplitude
those ofinAmB
under
the
lipid
of DPhPC
andofERG
(67/33
mol %) than
and bathed
2 M KCl
(pH
7.4)
same
conditions.
The
single
channel
amplitude
decreased
in
the
series
of
AmB
>
1d
>
1c
at transmembrane voltage 200 mV. One can see that all tested derivatives (1c, 1d and 1e)> as
1e. as 1a [35] produced pores of smaller amplitude than those of AmB under the same
well
conditions. The single channel amplitude decreased in the series of AmB > 1d > 1c > 1e.
Figure 4. Current fluctuations corresponding to openings and closures of single channels induced
by AmB, 1d, 1c and 1e in the lipid bilayers composed of DPhPC/ERG (67/33 mol %) and bathed in
Figure 4. Current fluctuations corresponding to openings and closures of single channels induced
2.0 M KCl (pH 7.4). The transmembrane voltage was equal to 200 mV.
by AmB, 1d, 1c and 1e in the lipid bilayers composed of DPhPC/ERG (67/33 mol %) and bathed in
2.0 M KCl (pH 7.4). The transmembrane voltage was equal to 200 mV.
Figure 5 shows G(V) diagrams of the pores produced by AmB derivatives. The chemical
modification of the natural polyene molecule did not practically affect the shape of the
Figure 5 shows G(V) diagrams of the pores produced by AmB derivatives. The chemnonlinear G(V)-dependence of the channels formed by the parent antibiotic (Figure 5). Table 4
ical modification of the natural polyene molecule did not practically affect the shape of
shows the mean dwell times, τ, and the probability of polyene channels opening, Pop .
the nonlinear G(V)-dependence of the channels formed by the parent antibiotic (Figure 5).
Antibiotics 2023, 12, x FOR PEER REVIEW
Antibiotics 2023, 12, 151
9 of 22
Table 4 shows the mean dwell times, τ, and the probability of polyene channels opening,
8 of 20
Pop.
Figure 5. G–V curves of single channels produced by AmB, 1c, 1d and 1e. Membranes were made
Figure
5. G–V curves(67/33
of single
produced
byMAmB,
1c, 1d
and 1e. Membranes were made
from DPhPC/ERG
molchannels
%) and bathed
in 2.0
KCl (pH
7.4).
from DPhPC/ERG (67/33 mol %) and bathed in 2.0 M KCl (pH 7.4).
Table 4. Characteristic parameters of the single ion-permeable pores induced by different AmB
Table 4. Characteristic parameters of the single ion-permeable pores induced by different AmB dederivatives in DPhPC/Erg (67/33 mol %) bilayers.
rivatives in DPhPC/Erg (67/33 mol %) bilayers.
2
3
G200 mV 1 , pS
τ 2, msτ , ms
PopP3op
G200 mV 1, pS
0.49
± 0.09
31.7 ± 2.1 31.7 ± 2.1
23 ± 4 23 ± 4
0.49
± 0.09
20.8 ± 1.8 20.8 ± 1.8
6±2 6±2
0.23
±
0.07
0.23 ± 0.07
21.3 ± 3.2
10 ± 1
0.24 ± 0.11
21.3 ± 3.2
10 ± 1
0.24 ± 0.11
21.2 ± 2.6
11 ± 1
0.14 ± 0.08
0.14
± 0.08
16.4 ± 2.2 21.2 ± 2.6
17 ± 2 11 ± 1
0.29
± 0.12
1 G200 mV—the mean conductance of polyene channels at 200 mV. 2 τ—the mean dwell time of polyene
1e
16.4 ± 2.2
17 ± 2
0.29 ± 0.12
3 Pop—the probability of polyene channels being open. 24—according to [35].
1 G
pores.
τ—the
—the
mean
conductance
of
polyene
channels
at
200
mV.
mean
dwell
time
of polyene pores.
200 mV
Polyene
Polyene
4
4
1 (AmB)
1 (AmB)
4
1a
1a 4
1c
1c
1d
1d
1e
3
Pop —the probability of polyene channels being open. 4 —according to [35].
The lifetime decreased in the order of AmB ≥ 1e > 1d > 1c > 1a. Moreover, the Pop of
pores The
induced
by 1c,
1d and in
1ethe
is two
toofthree
op of
AmB channels.
lifetime
decreased
order
AmBtimes
≥ 1elower
> 1d >than
1c >P1a.
Moreover,
the Pop
These
data
are
in
a
good
agreement
with
previously
published
results
[35,49].
of pores induced by 1c, 1d and 1e is two to three times lower than Pop of AmB channels.
Atdata
neutral
Nys channels
are
characterized
by a low
conductance
These
are pH,
in a single
good agreement
with
previously
published
results
[35,49]. that does
not exceed
the
level
of
current
noise
of
about
0.5
pA
[50,51];
therefore,
the registration
of
At neutral pH, single Nys channels are characterized by a low conductance
that does
the
step-like
transmembrane
current
fluctuations
that might
be related
to single
notsingle
exceed
the level
of current noise
of about
0.5 pA [50,51];
therefore,
the registration
channels
produced
by Nys
or their derivatives
not be that
performed
at neutral
pH.
of the single
step-like
transmembrane
current could
fluctuations
might be
related to
single
The one-sided
of their
AmBderivatives
or Nys to the
membrane-bathing
produced
channels
producedaddition
by Nys or
could
not be performedsolution
at neutral
pH.
an increase
in macroscopic
conductance
a dose-dependent
manner. Figure
6 shows
the
The one-sided
addition
of AmB or in
Nys
to the membrane-bathing
solution
produced
bilogarithmic
of the dependences
transmembrane
current
flowing
an increase inplots
macroscopic
conductanceof
in steady-state
a dose-dependent
manner. Figure
6 shows
the
bilogarithmic
plots composed
of the dependences
steady-state
transmembrane
flowing
through
membranes
of DPhPC of
and
CHOL or ERG
at 100 mV oncurrent
the concentrathrough
of DPhPC
andderivatives
CHOL or ERG
at 100
mVThe
on the
concentration
of themembranes
tested AmBcomposed
(Figure 6a,b)
and Nys
(Figure
6c,d).
slopes
of the
tion of
the testedofAmB
(Figuresection
6a,b) and
Nyspresented
derivatives
(Figure
6c,d). and
The NyS
slopes
of the
linear
regression
the growth
of the
curves
for AmB
derivalinear
regression
growth
of the presented
curves
forthe
AmB
and NyS derivatives
tives
are
close to 5of÷the
6 and
2 ÷ section
3, respectively.
This means
that
steady-state
polyeneare closeconductance
to 5 ÷ 6 andis2 proportional
÷ 3, respectively.
Thisthe
means
that
steady-state
induced
to about
fourth
tothe
fifth
and secondpolyene-induced
to third power
conductance
is
proportional
to
about
the
fourth
to
fifth
and
second
to third power of the
of the AmB and Nys derivative concentrations, respectively.
AmB
and
Nys derivative
The
measurements
of concentrations,
the macroscopicrespectively.
ionic conductance of polyene-modified planar
The measurements
the macroscopic
ionic
of polyene-modified
planar
lipid bilayers
also made itofpossible
to estimate
theconductance
EI value in the
terms of the ratio of
the
CHOL
CHOL
lipid
bilayers
also
made
it
possible
to
estimate
the
EI
value
in
the
terms
of
the
ratio
threshold concentrations (
) (Table 5). The magnitude of
decreased in the follow-of
ERG CCHOL
ERG
the threshold concentrations
( CERG ) (Table 5). The magnitude
of CCCHOL
decreased in the
ERGto the order of EI
ing series, 1d ≥ AmB ≥ 1e ≈ 1c and 2c ≈ 2e ≥ Nys ≈ 2b ≈ 2a ≈ 2d, similar
following series, 1d ≥ AmB ≥ 1e ≈ 1c and 2c ≈ 2e ≥ Nys ≈ 2b ≈ 2a ≈ 2d, similar to the
reduction in the AmB and Nys groups presented in Table 3.
order of EI reduction in the AmB and Nys groups presented in Table 3.
Antibiotics
Antibiotics2023,
2023,12,
12, 151
x FOR PEER REVIEW
10 of 229 of 20
NyS
(a)
(b)
(c)
(d)
DPhPC/ERG
DPhPC/CHOL
AmB
Figure
dependence
of the
steady-state
polyene-induced
transmembrane
current
on on
Figure6.6.Bilogarithmic
Bilogarithmic
dependence
of the
steady-state
polyene-induced
transmembrane
current
the
concentration
of
AmB
(a,c)
and
Nys
(b,d)
derivatives
in
the
bilayer
bathing
solution.
The
transthe concentration of AmB (a,c) and Nys (b,d) derivatives in the bilayer bathing solution. The transmembrane voltage was equal to 100 mV. Membranes were composed of DPhPC/CHOL (67/33 mol
membrane voltage was equal to 100 mV. Membranes were composed of DPhPC/CHOL (67/33 mol %)
%) (upper panel) and DPhPC/ERG (67/33 mol %) (lower panel) and bathed in 2.0 M KCl (pH 7.4).
(upper
panel)
and DPhPC/ERG
(67/33
(lower panel)
and
in 2.0 M KCl (pH 7.4). The
The relation
between
the color symbol
andmol
the %)
compound
is given
onbathed
the figure.
relation between the color symbol and the compound is given on the figure.
Table 5. The characteristic parameters of the dependences of macroscopic ionic conductance induced
different
AmB andparameters
Nys derivatives
CHOL- andofERGenrichedionic
bilayers
on the concencharacteristic
of the in
dependences
macroscopic
conductance
induced by
Table by
5. The
tration of polyenes.
different AmB and Nys derivatives in CHOL- and ERG- enriched bilayers on the concentration of polyenes.
Polyene CCHOL 1, 10−7 M mCHOL 2 CERG 1, 10−7 M mERG 2
CCHOL/CERG
1
2
1
2
−7
−7
Polyene
CCHOL /CERG
AmB
(1)
0.98C±CHOL
0.17 , 105–6M 0.67m±CHOL
0.16
6–6CERG , 10 M 1.5m
± ERG
0.6
AmB
0.98 ± 0.17
1.5 ± 0.6
1c (1) 0.88 ± 0.08
5–5
0.98 ± 5–6
0.07
4–5 0.67 ± 0.16
0.9 ± 6–6
0.1
1d1c
0.94 ± 0.11
4–5
0.51
±
0.09
5–6
1.8
±
0.5
0.88 ± 0.08
5–5
0.98 ± 0.07
4–5
0.9 ± 0.1
1e
1.05 ± 0.05
4–5
0.92 ± 0.09
5–6
1.1 ± 0.2
1d
0.94 ± 0.11
4–5
0.51 ± 0.09
5–6
1.8 ± 0.5
Nys (2)
1.9 ± 0.2
2–3
1.8 ± 0.1
2–3
1.1 ± 0.2
1e
1.05
±
0.05
4–5
0.92
±
0.09
5–6
1.1 ± 0.2
2a
2.2 ± 0.3
2–2
2.1 ± 0.6
2–2
1.0 ± 0.4
2b (2)
1.7 ± 0.1
3–3
1.6 ± 2–3
0.2
2–3
1.1 ± 2–3
0.2
Nys
1.9 ± 0.2
1.8 ± 0.1
1.1 ± 0.2
2c
3.1 ± 0.2
2–2
2.0 ± 0.4
2–3
1.6 ± 0.4
2a
2.2 ± 0.3
2–2
2.1 ± 0.6
2–2
1.0 ± 0.4
2d
2.9 ± 0.5
2–3
2.7 ± 0.8
2–2
1.0 ± 0.5
1.7 ± 0.1
1.6 ± 0.2
1.1 ± 0.2
2e2b
2.4 ± 0.2
2–3
1.6 ± 3–3
0.3
2–3
1.5 ± 2–3
0.4
1 CCHOL, CERG—the threshold concentration of polyenes in the solution bathing lipid bilayers com2c
3.1 ± 0.2
2–2
2.0 ± 0.4
2–3
1.6 ± 0.4
posed of DPhPC/CHOL (67/33 mol %) and DPhPC/ERG (67/33 mol %), respectively; 2 mCHOL, mERG—
2d
2.9 ± 0.5
2–3
2.7 ± 0.8
2–2
1.0 ± 0.5
the slopes of the linear regression of the growth section of lgI(lgC) dependence characterizing the
number
complexes
of the polyene
and CHOL
or 0.3
ERG, respectively.
2eof pore forming
2.4 ±
0.2
2–3
1.6 ±
2–3
1.5 ± 0.4
1
CCHOL , CERG —the threshold concentration of polyenes in the solution bathing lipid bilayers composed of
DPhPC/CHOL (67/33 mol %) and DPhPC/ERG (67/33 mol %), respectively; 2 mCHOL , mERG —the slopes of
the linear regression of the growth section of lgI(lgC) dependence characterizing the number of pore forming
complexes of the polyene and CHOL or ERG, respectively.
Antibiotics 2023, 12, 151
model with varying cell properties, we used mutants of the yeast Saccharomyces cerevisiae,
which harbor specific gene-deletions in the ergosterol biosynthesis pathway. Because
these cells lack ergosterol, but possess different precursors of this molecule, the fungal
cells are still viable; however, their sensitivity to the polyenes changes. Differences in these
mutant susceptibility patterns allow us to hypothesize that the different derivatives have
10 of 20
a distinct affinity toward different ergosterol precursors. Notably, some of the tested gene
deletions are known to cause polyene resistance in clinical fungal isolates (see legend to
Figure 7), which suggests that understanding how potential novel antifungals can interact
2.4. such
Comparison
of might
Susceptibility
of Ergosterol-Pathway
Mutantsstrategies.
to Polyene Derivatives
with
mutants
be relevant
for planning treatment
Testing
of
the
MICs
of
the
various
mutants
showed
the
following
patterns. ComThe differences identified in the susceptibility of various
fungal pathogens
to the
pared
to
wild
type
yeast,
deletion
of
ERG6
showed
the
highest
increase
in relative
presented semisynthetic derivatives are highly relevant for their potential
clinicalreuse
sistance
to 2,
as well they
as to are
all of
the tested
derivatives
of 1 and 2,sense,
with 1because
showing
the
lowest
(Table 1);
however,
difficult
to interpret
in a molecular
the
properties
increase
in resistance.
Amides 1a that
and drive
1d retained
this property;
1c order
showed
inof the cell
wall and membrane
these differences
are however,
unclear. In
to gain
creased
resistance.
of the tested
derivatives
of 2 had somewhat
relative
rean insight
into theAll
differences
between
the synthesized
derivativeslowered
in a more
controlled
sistance.
Note,varying
this property
might bewe
clinically
relevant,
because
mutations
have
model with
cell properties,
used mutants
of the
yeast ERG6
Saccharomyces
cerevisiae,
been
implicated
in clinical
polyene resistance
[52]. Deletion
of ERG3
causes resistance
to
which
harbor specific
gene-deletions
in the ergosterol
biosynthesis
pathway.
Because these
2 cells
and lack
its derivatives
and
this
resistance
does
not
change
depending
on
the
introduced
ergosterol, but possess different precursors of this molecule, the fungal cells are
modifications.
However,
forsensitivity
1 and its to
derivatives,
thechanges.
situationDifferences
is more complex.
still viable; however,
their
the polyenes
in these ERG3
mutant
deletion
has little
effect allow
on theus
resistance
to 1 orthat
1a, but
causes some
resistance
to a1d
and
susceptibility
patterns
to hypothesize
the different
derivatives
have
distinct
affinity
toward
different ergosterol precursors. Notably, some of the tested gene deletions
even
more
so to 1c.
are Finally,
known to
polyene
resistance
in clinical
fungal isolates
(see legend
to Figure
thecause
strongest
increases
in polyene
sensitivity
were observed
for ERG4
dele-7),
which
suggeststo
that
understanding
potential
novel
antifungals
can polyenes.
interact with such
tion
in response
1, as
well as to 2a,how
but were
weak
for the
other tested
mutants might be relevant for planning treatment strategies.
Figure 7. Susceptibility of ergosterol-pathway mutants to parent and derived antibiotics. Schematic
of the7.ergosterol
biosynthesis
pathway (A) mutants
with genes
implicated
in clinical
polyeneSchematic
resistance
Figure
Susceptibility
of ergosterol-pathway
to parent
and derived
antibiotics.
ofhighlighted
the ergosterol
biosynthesis
pathway
(A)for
with
genes
implicated
clinical
in red
([52] for Erg6,
[53,54]
Erg2,
[55]
for Erg3) in
and
testedpolyene
genes inresistance
boxes. (B)highTable
lighted
in red ([52]infor
Erg6,[53,54]
forstrains,
Erg2, [55]
for Erg3)toand
in boxes.
(B) Table
of
of fold-changes
MIC
of different
normalized
thetested
MIC ofgenes
the wild
type strain
of each
fold-changes
MIC
of differentwas
strains,
normalized
to the3 MIC
thethe
wild
type strain
comcompound. in
Each
experiment
repeated
no less than
timesofand
average
resultofiseach
presented.
pound. Each experiment was repeated no less than 3 times and the average result is presented.
Testing of the MICs of the various mutants showed the following patterns. Compared
3.to
Discussion
wild type yeast, deletion of ERG6 showed the highest increase in relative resistance to 2,
as well
as to all of theoftested
derivatives
of 1 and of
2, with
1 showing
the lowest
increase
The development
semisynthetic
derivatives
amphotericin
B with
an improved
in
resistance.
Amides
1a
and
1d
retained
this
property;
however,
1c
showed
increased
pharmacological profile has been underway for several decades. Among all the tested
resistance. Allthe
of the
tested derivatives
of 2distinguished
had somewhatby
lowered
relative
resistance. Note,
modifications,
following
changes were
a significant
improvement
in
this property might be clinically relevant, because ERG6 mutations have been implicated in
clinical polyene resistance [52]. Deletion of ERG3 causes resistance to 2 and its derivatives
and this resistance does not change depending on the introduced modifications. However,
for 1 and its derivatives, the situation is more complex. ERG3 deletion has little effect on
the resistance to 1 or 1a, but causes some resistance to 1d and even more so to 1c.
Finally, the strongest increases in polyene sensitivity were observed for ERG4 deletion
in response to 1, as well as to 2a, but were weak for the other tested polyenes.
3. Discussion
The development of semisynthetic derivatives of amphotericin B with an improved
pharmacological profile has been underway for several decades. Among all the tested
modifications, the following changes were distinguished by a significant improvement
in the pharmacological properties of the polyenes: (1) the modification of the carboxylic
group by amidation, esterification, reduction [17,18]; (2) the introduction of additional
Antibiotics 2023, 12, 151
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positive charge [17,18]; (3) the removal of the C2’-OH [56] and C35-OH groups [57] (for
AmB); (4) the introduction of bulk substituents into the mycosamine group [58] and (5) the
synthesis of urea [59]. The first two observations were made before the 2010s on the basis
of experimental data when comparing the antifungal and hemolytic activity of a polyene
antibiotic (in most cases, amphotericin) and its derivatives. The first attempt to explain
the increase in the selectivity of the semisynthetic polyene derivatives was implemented
using molecular dynamics methods, simulating the interaction of antibiotics with ERGand CHOL-containing membranes. Two bis-modified AmB derivatives (esterification of
carboxyl group and N-alkylation) penetrated more deeply into the bilayer with an alteration
of their wobble dynamics, which in turn led to facilitated self-association in the ordered
Erg-containing membranes; the ability of additional basic groups to form hydrogen bonds
with phospholipids was more pronounced in ERG-containing bilayers [60]. It should
be pointed out that this model might not accurately reproduce a real membrane since
it was performed using dimyristoylphosphatidylcholine (DMPC) with shorter and fully
saturated alkyl chains than those of phosphatidylcholines naturally occurring in biological
membranes. Later, it was assumed that the decrease in toxicity in the case of AmB amides
was the result of decreasing of aggregation properties [34,35]. The decrease in toxicity
observed when the OH group was removed from the mycosamine was explained by the M.
Burke group as a change in the native conformation of the AmB molecule, in which it is
able to bind only to ergosterol. The removal of the 35-OH group from the macrocycle led to
a loss of channel-forming activity, while the affinity for sterols, as well as antifungal activity,
remained, so the authors proposed the “sterol-sponge model” according to which the most
promising approach for obtaining non-toxic polyenes would involve diminishing pore
formation while maintaining a high affinity to ergosterol. Finally, a possible explanation
for the decrease in the toxicity of polyenes by modifications (4) and (5) is the destruction of
the "salt bridge" between the amino group of mycosamine and the side carboxyl group of
the macrocycle, stabilizing the conformation of the amphotericin molecule, providing an
affinity to both cholesterol and ergosterol. It should be noted that the described studies of
the causes of reducing the toxicity of semisynthetic derivatives were mainly carried out for
AmB, while in the studies on the modification of nystatin, the authors of [38,58] did not
perform such analyses.
The preparation of a new series of nystatin amides 2a-2e and AmB amides 1c-1e and
their investigation in comparison to the previously described 1a and 1b allowed us to
compare the structure–property relationships within the two major polyenes cores, Nys
and AmB. Studies have shown that despite the similarity of the structures of amphotericin
and nystatin, similar modifications lead to different effects. Although all modifications
led to decreased zwitterionic character, the insertion of an additional positive charge and
disruption of the “salt-bridge” between the C16 carboxylic group and the amino group of
mycosamine, which would be expected to decrease toxicity according to known data, the
observed effect on the safety of the compounds depended on both the polyene core and the
diamine nature. Thus, the insertion of a short ethylenediamine moiety into the AmB and
Nys cores led to more active and safe derivatives 1a [35] and 2a, respectively (for the last
one this was shown in in vitro experiments only). However, unlike the situation with 1a,
in the case of nystatin, this effect could not be explained by the increased selectivity to
ERG-containing membranes vs. CHOL-containing ones. We are more prone to attribute
the result to the alteration of polyene–sterol interactions by membrane phospholipids and
sphingolipids [61], although decreased aggregation properties due to the loss of amphiphilic
structure might be considered as well. Insertion of the short aminoethanol group into the
nystatin structure also decreased the toxicity of Nys [38]. Modification with longer diamines
such as N,N-dimethylethylenediamine and N-methyl-1,3-propanediamine have differential
effects on the two types of polyene core—the corresponding Nys amides 2c, 2e with
antifungal activity similar to the parent antibiotic, were not toxic to human embryo kidney
cells HEK293 and showed low hemolytic activity, which was well-correlated with increasing
selectivity towards ERG-containing vs. CHOL-containing membranes. In contrast, AmB
Antibiotics 2023, 12, 151
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amides bearing the same moieties 1c and 1e proved to be more toxic to kidney and red
blood cells, which coincided with a lower ratio of selectivity towards ERG-containing
membranes. Long amide chains, both lipophilic (2-((2-fluorobenzyl)amino)ethyl)amine
and hydrophilic 2-((2-aminoethyl)amino)ethanol (compounds 1b,d and 2b,d), led to the
most pronounced increase in the permeability of CHOL- and ERG-membranes without
changing the selectivity for both tested polyene cores, which might be the result of currently
unidentified interactions with the lipid environment. Investigations of the single ionpermeable pores formed by AmB and its amides in ERG-containing bilayers indicated
that a loss of the zwitterionic properties of the AmB molecule leads to decrease in the
lifetime of the transmembrane pores and their probability to be open due to electrostatic
repulsion between positively charged derivative molecules destabilizing the channel. Most
likely, the introduction of a radical also affects the polarization of bonds between oxygen
and hydrogen in hydroxyls in the polar chain of the lactone ring, which determines the
conductance of single channels, as was shown in the case of other AmB derivatives [50].
Thus, the design of new structures should take into account not only the affinity for sterols,
but also changes in the interaction of polyenes with the lipid environment, as well as
the physicochemical and physicobiological properties of polyenes that affect their selfassociation in membranes. However, electrophysiological experiments showed that the
number of polyene–sterol complexes forming the channel does not depend on the type of
chemical modification of the parent molecules.
Cases of polyene resistance are recorded quite rarely. Using the example of a clinical
isolate of C. albicans ATCC 200955, it was shown that resistance to the action of polyenes
was associated with a mutation at the ERG2 locus, which caused induction in cells of a
diverse array of stresses. To survive, the mutant strain requires a high level of expression
of the heat shock protein Hsp90, which makes it highly sensitive to oxidative stress and
temperature rise, thus making it an easier target for the immune system of an infected
organism. Moreover, AmB-resistant strains have defects in the filamentation and invasion
of tissues and organs. Such fungi turn out to be nonvirulent in tests on mice in vivo. [62] The
main mechanism of resistance to polyenes of Candida auris strains, known for their resistance
to all classes of antifungal drugs, is considered to be a change in ergosterol biosynthesis
(mutations in the ERG2, ERG3, and ERG6 genes, overexpression of the ERG1, ERG2, ERG3,
EGR5, ERG6, and ERG13 genes) [63]. To the best of our knowledge, no one has previously
described the effect of chemical modifications of polyenes on the activity against mutant
fungal strains that have alterations in the pathway of ergosterol biosynthesis. In this paper,
it was shown that changes to the C16-carboxylic group can have considerable effects on the
putative substrate specificity of polyenes toward ergosterol-pathway intermediates that
are: (1) specific to the parent antifungal and (2) important for the membrane homeostasis of
polyene-resistant fungal isolates. It is also possible that the observed effects are caused by
general changes in the membrane properties and a complete molecular-level understanding
requires further study.
Among the AmB amides obtained by our group, 1a remains the most promising
candidate for future drug-development. From the Nys series, two water-soluble amides—
2c and 2e—showed excellent results in tests for cell toxicity, while in the antifungal testing,
2c was somewhat more active than 2e. The most active Nys derivative 2a also proved to be
less toxic than the parent antibiotic, but the reason for the decreased toxicity was not clear.
Nevertheless, we consider all three amides as candidates for further in vivo study.
4. Materials and Methods
4.1. General
All chemicals were purchased from commercial suppliers and used as received. Amphotericin (AmB), Nystatin (Nys), benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP), dimethylsulfoxide (DMSO), calcein, KCl, 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid (HEPES), KOH, triton X-100, ethylenediamine, N,N-dimethylethylenediamine were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany).
Antibiotics 2023, 12, 151
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N-(2-hydroxyethyl)ethylenediamine, N-(2-fluorobenzyl)ethane-1,2-diamine, N-methyl1,3-propanediamine were purchased from abcr GmbH (Karlsruhe, Germany). Synthetic
1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC), 1-palmitoyl-2-oleoyl-sn-glycero-3phosphocholine (POPC), ergosterol (ERG), and cholesterol (CHOL) were obtained from
Avanti Polar Lipids (Alabaster, AL, USA). Water was distilled twice and deionized. Solutions of 2.0 or 0.1 M KCl were buffered using 5 mM HEPES-KOH at pH 7.4. Thin-layer
chromatography (TLC) analysis was performed on the 0.20 mm silica gel 60 F254 plates (aluminum sheets 20 × 20 cm) Macherey-Nagel (Duren, Germany) in n-PrOH–EtOAc–NH4 OH,
3:3:2. All final compounds were purified to 90%+ by reverse phase flash chromatography on
C18 silica gel cartridges (Biotage SNAP) or by normal phase flash chromatography on silica
gel cartridges (SNAP Ultra). The purity was assessed by reverse phase high performance
liquid chromatography (HPLC) which was performed on a Shimadzu HPLC instrument
of the LC 20AD series (Japan) on a Kromasil-100 C18 column (4.6 × 250 mm, particle size
5 µm, Ekzo Nobel, Sweden) with an injection volume of 20 µL (concentration of substances
0.025–0.05 mg/mL) at a flow rate of 1.0 mL/min and monitored by a diode array ultraviolet
detector at 408 nm for amphotericin B derivatives and at 320 nm for nystatin derivatives.
The system consisted of buffer—0.2% HCOONH4 at pH 4.5, 7.8, 8.4—and organic phase
acetonitrile. The proportion of acetonitrile was varied from 30 to 70% for 30 min (pH 4.5,
system A), 40 to 60% for 15 min (pH 8.4, system B), 20 to 60% for 30 min (pH 4.5, system C)
and 35 to 60% for 15 min (pH 7.8, system D). NMR spectra were recorded on a Bruker
Avance III 500 MHz NMR spectrometer with 500.23 and 125.78 MHz resonance frequencies for 1 H and 13 C, respectively. Spectra were recorded in DMSO-d6 solutions at 303 K
and were referenced against residual solvent signals: 2.50 ppm for DMSO-d5 for 1 H and
39.50 ppm for DMSO-d6 for 13 C, respectively. Mixing times in the range of 10–180 ms were
used to mimic stepwise magnetization propagation. For 2D TOCSY, a mixing time of 80 ms
was used. For 2D ROESY and NOESY experiments, mixing times of 300 and 500 ms were
used, respectively. ESI MS spectra were recorded on a Bruker microTOF-Q II instrument
(BrukerDaltonics GmbH, Bremen, Germany).
4.2. Carboxamides of Amphotericin B and Nystatin (1c,d,e, 2a–e) (General Method)
AmB or Nys (200 mg, 0.22 mmol) were dissolved in DMSO (5 mL) under argon flow,
and then PyBOP (172 mg, 0.33 mmol) and the corresponding diamine (1.1 mmol) were
added. The reaction mixture was stirred for 1.5 h, and then Et2 O (15 mL) was added. The
mixture was stirred vigorously, then the Et2 O layer was removed and the procedure was
repeated several times until viscous oil was formed. Viscous oil was dissolved in methanol
(15 mL) and then Et2 O (100 mL) was added; the formed precipitate was filtered off, washed
with Et2 O and dried under vacuum. The progress of the reactions, chromatography purification and the purity of final compounds were monitored by TLC and HPLC analysis.
The crude amide was purified by reverse-phase or normal-phase flash chromatography on
Biotage SNAP C18 (10 g) and SNAP Ultra (10 g) cartridges, respectively. Reverse-phase
chromatography was performed as follows: the amide was dissolved in 0.5% aq. AcOH
(2 mL) or in the MeCN/0.5% aq. AcOH mixture (1:1) (2 mL) and put on a column preequilibrated with 0.5% aq. NH4 OH. The elution was carried out with (A) 0.5% aq. NH4 OH
then H2 O then 0.5% aq. AcOH and (B) acetonitrile (0→50% B). Normal-phase chromatography was performed as follows: the amide was dissolved in CH2 Cl2 /MeOH mixture
(1:1) and put on a column pre-equilibrated with CH2 Cl2 :MeOH:H2 O:CH3 COOH mixture
(64:6:1:0.1). The elution was carried out with (A) CH2 Cl2 and (B) MeOH:H2 O:CH3 COOH
(6:1:0.1) (10→35% B). Fractions containing the target compound were combined and evaporated to a small volume. The addition of Et2 O to the solution gave the precipitate of an
acetate of a targeted compound that was filtered off and dried.
Diacetate of N-(2-(N’,N’-dimethyl)aminoethyl)amide of AmB (1c): purified by reversephase chromatography; yield: 89 mg (37%); yellow powder; mp. 160–163 ◦ C (decomp.);
Rf 0.55; Rt (system A) 10.49 min; purity 93.5%; HRMS (ESI) m/z: [M + H]+ Calc. for
C51 H84 N3 O16 + 994.5846. Found 994.5904.
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Diacetate of N-((2-(2-fluorophenyl-1-methyl)amino)ethyl)amide of AmB (1d): purified
by normal-phase chromatography; yield: 60 mg (23.5%); orange powder; mp. 168–170 ◦ C
(decomp.); Rf 0.56; Rt (system A) 12.21 min; purity 91.0%; HRMS (ESI) m/z: [M + H]+ Calc.
for C56 H85 FN3 O16 + 1074.5908. Found 1074.5863.
Diacetate of N-(3-(N’-methyl)aminopropyl)amide of AmB (1e): purified by reversephase chromatography; yield: 145 mg (60%); yellow powder; mp. 155–158 ◦ C (decomp.);
Rf 0.45; Rt (system A) 10.72 min; purity 90%; HRMS (ESI) m/z: [M + H]+ Calc. for
C51 H84 N3 O16 + 994.5846. Found 994.5882.
Diacetate of N-(2-aminoethyl)amide of Nys (2a): purified by reverse-phase chromatography; yield: 78 mg (33%); beige powder; mp. 146–149 ◦ C (decomp.); Rf 0.47; Rt (system B)
4.82 min; purity 96%; HRMS (ESI) m/z: [M + H]+ Calc. for C49 H82 N3 O16 + 968,5690. Found
968,5723.
Diacetate of N-(2-((2-Hydroxyethyl)amino)ethyl)amide of Nys (2b): purified by reversephase chromatography; yield: 48 mg (22%); beige powder; mp. 138–140 ◦ C (decomp.);
Rf 0.50; Rt (system D) 8.44 min, purity 95%; HRMS (ESI) m/z: [M + H]+ Calc. for
C51 H86 N3 O17 + 1012.5952. Found 1012.6009.
Diacetate of N-(2-(N’,N’-dimethyl)aminoethyl)amide of Nys (2c): purified by reversephase chromatography; yield: 109 mg (45%); beige powder; mp. 140–143 ◦ C (decomp.);
Rf 0.53; Rt (system C) 17.76 min, purity 92%; HRMS (ESI) m/z: [M + H]+ Calc. for
C51 H86 N3 O16 + 996.6003. Found 996.5966.
Diacetate of N-((2-(2-fluorophenyl-1-methyl)amino)ethyl)amide of Nys (2d): purified
by normal-phase chromatography; yield: 151 mg (70%); beige powder; mp. 138–140 ◦ C
(decomp.); Rf 0.53; Rt (system C) 18.68 min; purity 90%; HRMS (ESI) m/z: [M + H]+ Calc.
for C56 H87 FN3 O16 + 1076.6065. Found 1076.6062.
Diacetate of N-(3-(N’-methyl)aminopropyl)amide of Nys (2e): purified by reversephase chromatography; yield: 86 mg (36%); beige powder; mp. 134–137 ◦ C (decomp.);
Rf 0.41; Rt (system A) 10.90 min; purity 95%; HRMS (ESI) m/z: [M + H]+ Calc. for
C51 H86 N3 O16 + 996.6003. Found 996.6058.
4.3. Solubility Testing
For UV spectrometry, a stock solution of 4 mg/mL was prepared in pure methanol
after which this solution was diluted 20-fold with water to obtain 0.2 mg/mL solutions
in 5% methanol. These solutions and their sequential dilutions were used to build a
calibration curve, which was then used to determine the concentration of compound in a
saturated solution. These were prepared by incubating 10 mg of each sample of compound
with 100 µL of deionized water for 10 min with shaking, after which the suspension was
centrifuged for 1 min at 16,000× g, and then the supernatant was collected and centrifuged
at the same speed for 20 min. The resulting supernatant was used for determining the UV
absorbance. For the solutions of compounds 1a,c,e and 2a,c,e, no sediments were detected,
and the concentration of the corresponding supernatants, determined by measuring the
absorption of a sequentially diluted solutions, was 100 g/L. Addition of methanol to 5% did
not affect the results. Calibration curve data are presented in the Supplemental Materials
(Figures S16–S23).
4.4. Antifungal Susceptibility Testing
4.4.1. Organisms
Strains of Candida spp. (C. parapsilosis 58L, C. albicans 604M, C. albicans 8R, C. glabrata
61L, C. tropicalis 3010, C. krusei 432M) and Aspergillus niger 37a, Trichophyton rubrum
2002 analyzed in this study were obtained from the Medical Microbiology Laboratory
of the State Research Center for Antibiotics (Moscow, Russia). Cells of Candida spp. and
spores of filamentous fungi were stored in medium supplemented with 10% (v/v) glycerol
at −80 ◦ C.
The reference strain C. parapsilopsis ATCC 22019 and AmB was used as control in
each experiment.
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The Saccharomyces cerevisiae strain BY4742 (MATα his3∆1 leu2∆0 lys2∆0 ura3∆0) and its
derivatives from yeast deletion collection (EUROSCARF) were used for determining the
susceptibility of ergosterol pathway mutants to the chemical samples.
4.4.2. Preparation of Chemical Samples (for Pathogen Exposure)
Substances of antifungal antibiotics were dissolved in DMSO (Merck, Germany), stock
solutions were prepared taking into account the activity of the antifungal pharmaceutical
substance specified by the developer. The calculation of the mass of the substance or the
volume of the solvent required for the preparation of the stock solution was calculated
using the formulas recommended by EUCAST (European Committee on Antimicrobial
Susceptibility Testing). Dilutions were prepared according to ISO 20776-1:2006. Working solutions were prepared with a final concentration of 0.125–64 mg/mL. All of the
test compounds were dissolved to concentrations of 10 mg/mL in 100% DMSO (Merck,
Darmstad, Germany). The dissolved stock concentration chemical compounds were then
diluted in the growth medium RPMI1640 (Merck) to obtain an initial concentration of
64 µg/mL (L+), which was then directly serially diluted before testing of MIC. The final
range concentrations of the test compounds was 0.015–32 µg/mL.
The 24 h-old cultures of Candida spp. and ~3 week culture of Trichophyton and Aspergilli
grown in Sabourau dextrose agar (Sigma-Aldrich, St. Louis, MO, USA) were used to
evaluate sensitivity to antimicrobial agents. For inoculum preparation of filamentous
fungi, part of the colony was transferred to sterile distilled water, containing 0.1% (v/v)
Tween 80 and shaken vigorously. The suspension was filtered through sterile gauze and the
number of spores/conidia was counted using a hemocytometer, as a stock spore solution for
inoculum. Antifungal susceptibility testing was performed by EUCAST broth microdilution
according to the E.DEF 7.3.2 and E.DEF 9.3.2 methods in the liquid nutrient medium PRMI
1640 with 2% (m/v) glucose, with L-glutamine and without bicarbonate (Merck, Germany).
MOPS (Sigma-Aldrich, USA) was used as a buffer to this medium at a final concentration
of 0.165 mol/L at pH 7.0. Minimum inhibitory concentration (MIC) was defined as the
lowest concentration that resulted in complete growth inhibition after incubation for 24,
48 h for Candida spp., 48–72 h for dermatophytes.
For routine internal quality control when assessing sensitivity to antifungal drugs
(MIC), a control test strain of Candida parapsilosis ATCC 22019, recommended by EUCAST, was used. Target values of the MIC for amphotericin: 0.25–0.5; allowable values:
0.125–1.0 mg/mL. To determine the sensitivity of pathogenic yeasts, an 18–48 h yeast
culture grown at a temperature of 35 ± 2 ◦ C under aerobic conditions on non-selective
nutrient agar (Sabouraud agar) was used. The final concentration of the inoculum was
0.5–2.5 × 105 CFU/mL. Microdilution plates were incubated at 35 ± 2 ◦ C under aerobic
conditions for 24 ± 2 h.
The study was carried out by the method of serial microdilutions in a liquid nutrient
medium. For the fungal pathogens, to set up the experiment, we used liquid nutrient
medium RPMI 1640 with 2% (m/v) glucose, with L-glutamine and without bicarbonate
(Merck, Germany). MOPS (Sigma-Aldrich, USA) was used as a buffer to this medium
at a final concentration of 0.165 mol/L at pH 7.0. The studies were performed in sterile
disposable 96-well plates with flat-bottomed wells with a capacity of 300 µL (Corning,
Somerville, MA, USA).
For S. cerevisiae, cells from a 12 h culture in YPD (1% yeast extract (w/v), 2% peptone
(w/v), 2% glucose (w/v)) medium were inoculated into fresh YPD supplemented with
2-fold differing concentrations of tested substances in DMSO, and a constant concentration
(0.05% v/v) of DMSO (Sigma) in 96-well plates (200 µL final volume), at an initial OD600
of 0.05. Cell growth was judged after 24 h of growth at 30 ◦ C with shaking, with the MIC
being the lowest concentration of drug where no growth was observed.
Antibiotics 2023, 12, 151
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4.5. Cell Culture and Antiproliferative Activity
HEK293 cells (from ATCC) were propagated in Dulbecco’s modified Eagle’s medium
supplemented with 5% fetal calf serum, 2 mM L-glutamine, 100 units/mL penicillin and
100 µg/mL streptomycin at 37 ◦ C, 5% CO2 in a humidified atmosphere. Cells in logarithmic
phase of growth were used in the experiments. Tested compounds were dissolved in
DMSO as 10 mM stock solutions followed by serial dilutions with medium immediately
before experiments. The cytotoxicity was determined in a formazan conversion assay
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test. Briefly, cells
(5 × 103 in 190 µL of culture medium) were plated into a 96-well plate (Becton Dickinson,
Franklin Lakes, NJ, USA) and treated with 0.1% DMSO (vehicle control) or with the tested
compounds (0.10–1000 µM; each concentration in duplicate) for 72 h. After completion of
drug exposure, 50 µg of MTT was added to each well for an additional 2 h. Formazan was
dissolved in DMSO, and the absorbance at 540 nm was measured. Cell viability at a given
drug concentration (% MTT conversion) was calculated as the percentage of absorbance in
wells with drug-treated cells to absorbance in wells with DMSO-treated cells (100%).
4.6. Testing of Hemolysis Activity
Briefly, erythrocytes were isolated from human blood. The blood was incubated at
4 ◦ C (2–3 h). Erythrocyte suspension (100 µL) was diluted in physiological buffer (pH 7.2)
to a total volume of 500 µL. Solutions of the compounds under investigation with an initial
concentration of 10 mM (DMSO) were diluted with phosphate-buffered saline (PBS) buffer
(1:10). The resulting solutions were added to Eppendorf tubes in the amount necessary to
create the test concentration (10, 20 and 50 µM) and a mixture of PBS with erythrocytes was
added to a total volume of 200 µL. The mixture of red blood cells with 1% triton X-100 was
used as a positive control. Intact control solution was a mixture of erythrocytes with PBS
and solvent (DMSO). Probes were incubated for 1 h (additionally for AmB (1), Nys (2), 2c
and 1d maximum 24 h) at 37 ◦ C, and centrifuged at 252 relative centrifugal force (RCF) for
1.5 min, and the supernatants were transferred to a 96-well plate. The optical density of
supernatants was measured on a microplate spectrophotometer (BioTek, ELx800, Winooski,
VT, USA) at 570 nm. The percentages of hemolysis were calculated relative to positive
control, which was taken as 100%.
4.7. Calcein Release from Large Unilamellar Vesicles
The fluorescence of calcein released from large unilamellar vesicles was used to
monitor the membrane permeabilization induced by different polyenes. Liposomes were
prepared from POPC/CHOL (67/33 mol %) and POPC/ERG (67/33 mol %) by extrusion
using Avanti Polar Lipids (Alabaster, AL, USA) as described in previous work [49]. Polyenes
(AmB (1), Nys (2) and their derivatives) from stock solution (10 mM in DMSO or H2 O) were
added to calcein-loaded liposomes. Time-dependent increase in fluorescence of released
calcein induced by 5 µM of antibiotics was measured during 30–60 min.
The degree of calcein release was determined at 25 ◦ C using a “Fluorat-02-Panorama”
spectrofluorometer (Lumex, Saint-Petersburg, Russia). The excitation wavelength was
490 nm and the emission wavelength was 520 nm. The detergent triton X-100 (at a final
concentration of 1%) was added at the end of each experiment for complete disruption of
liposomes (leading to complete release of calcein and maximal fluorescence).
The relative intensity of calcein fluorescence (RF, %) was used to describe the dependence of the permeabilization of the liposomes by various polyenes. RF was calculated
using the following Formula (1):
RF =
I − I0
· 100%
Imax /0.9 − I0
(1)
where I and I0 were the calcein fluorescence intensities in the sample in the presence and
in the absence of AmB, Nys and their derivatives, respectively, and Imax was the maximal
Antibiotics 2023, 12, 151
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fluorescence of the sample after lysis of liposomes by triton X-100. The factor of 0.9 was
introduced to calculate the dilution of the sample by triton X-100.
Time series of calcein release were fitted with two-exponential functions with characteristic times t1 and t2 related to fast and slow components of calcein release.
The values of RF, t1 and t2 were presented as mean ± s.e. (p ≤ 0.05).
4.8. Registration of Ion Channels in Planar Lipid Bilayers
Virtually solvent-free planar lipid bilayers were prepared according to the monolayeropposition technique [64] on a 50 µm diameter aperture in a 10 µm thick Teflon film
separating two (cis and trans) compartments of the Teflon chamber. The aperture was pretreated with hexadecane. The lipid bilayers were made from DPhPC/ERG (67/33 mol %).
After the membrane was completely formed and stabilized, AmB and its derivatives from
a stock solution (1 mM in DMSO or H2 O) were added to cis-compartments to obtain the
different concentrations presented in Figure 5. Ag/AgCl electrodes with agarose/2 M KCl
bridges were used to apply a transmembrane voltage and measure the transmembrane
current. “Positive voltage" refers to the case in which the cis-side compartment was positive
with respect to the trans-side. All experiments were performed at room temperature. The
final concentration of solvent in the chamber did not exceed 10−4 mg/mL and did not
produce any changes in the stability and conductance of the bilayers.
Current measurements were performed using an Axopatch 200B amplifier (Molecular
Devices, San Jose, CA, USA) in voltage clamp mode. Data were digitized by a Digidata
1440A and analyzed using a pClamp 10 (Molecular Devices, San Jose, CA, USA) and Origin
7.0 (OriginLab Corporation, Northampton, MA, USA). Data were acquired at a sampling
frequency of 5 kHz using low-pass filtering at 1 kHz, and the current tracks were processed
through an 8-pole Bessel 100-kHz filter.
Single-channel conductance (G) was defined as the ratio between the current flowing
through a single polyene channel (i) and transmembrane potential (V). The total numbers
of events used for the channel conductance fluctuation and dwell time (τ) analysis were
500 ÷ 1000 and 1500 ÷ 2000, respectively. The probability of the polyene channel to be in
an open state (Pop ) was calculated using the following Formula (2):
Pop =
τ
τ + τclose
(2)
where τ is the dwell time of the single polyene channel and τclose is the time that the channel
is in a closed state.
The values of the threshold polyene antibiotic concentrations (CCHOL/ERG ) were determined by the intersections of the straight line fitting of the initial and growth portion
sections of the bilogarithmic plots of the dependences of steady-state transmembrane current flowing through polyene-modified membranes at 100 mV on the concentration of the
tested derivatives.
The values of G were presented as mean ± s.d. (p ≤ 0.05); τ, Pop , and CCHOL/ERG were
presented as mean ± s.e. (p ≤ 0.05).
Supplementary Materials: The following supporting information can be downloaded at:
https://www.mdpi.com/article/10.3390/antibiotics12010151/s1: Table S1: 1 H and 13 C spectra
assignment for derivatives 1c–1e, 2a–2e; Figures S1–S15: 1 H, 13 C NMR spectra of the derivatives
1c–1e, 2a–2e; Figures S16–S23: Calibration curves (solubility in water).
Author Contributions: Conceptualization, A.T., S.E. and A.S.; methodology, A.T. and S.E.; validation,
S.S. and O.O. (Olga Ostroumova); formal analysis, N.G., L.D. and O.O. (Olga Ostroumova); investigation, E.B., G.Z., N.G., L.D., E.G. and O.O. (Olga Ostroumova); data curation, O.O. (Olga Omelchuk);
writing—original draft preparation, O.O. (Olga Omelchuk), A.T., A.A. and S.E.; writing—review and
editing, A.T. and A.S.; supervision, A.S.; project administration, A.T. and A.A.; funding acquisition,
A.T. and A.A. All authors have read and agreed to the published version of the manuscript.
Antibiotics 2023, 12, 151
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Funding: This research was funded by Russian Science Foundation, grant number 21-74-20102 (A.T.,
S.E., O.O., E.B., G.Z., N.G., O.O.) and 21-74-10115 (A.A., E.G. – work involving testing of S. cerevisiae
ergosterol biosynthesis mutants). E.G. was also partially funded by a joint scholarship (Executive
program) from the Arab Republic of Egypt and the Russian Federation.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or
in the decision to publish the results.
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