Synthesis of Fusidic Acid Derivatives Yields a Potent Antibiotic with an Improved Resistance Profile

Article abstract of DOI:10.1021/acsinfecdis.0c00869

Fusidic acid (FA) is a potent steroidal antibiotic that has been used in Europe for more than 60 years to treat a variety of infections caused by Gram-positive pathogens. Despite its clinical success, FA requires significantly elevated dosing (3 g on the first day, 1.2 g on subsequent days) to minimize resistance, as FA displays a high resistance frequency, and a large shift in minimum inhibitory concentration is observed for resistant bacteria. Despite efforts to improve on these aspects, all previously constructed derivatives of FA have worse antibacterial activity against Gram-positive bacteria than the parent natural product. Here, we report the creation of a novel FA analogue that has equivalent potency against clinical isolates of Staphylococcus aureus (S. aureus) and Enterococcus faecium (E. faecium) as well as an improved resistance profile in vitro when compared to FA. Importantly, this new compound displays efficacy against an FA-resistant strain of S. aureus in a soft-tissue murine infection model. This work delineates the structural features of FA necessary for potent antibiotic activity and demonstrates that the resistance profile can be improved for this scaffold and target.

Full text of DOI:10.1021/acsinfecdis.0c00869

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Article
Synthesis of Fusidic Acid Derivatives Yields a Potent Antibiotic with
an Improved Resistance Profile
Martin Garcia Chavez, Alfredo Garcia, Hyang Yeon Lee, Gee W. Lau, Erica N. Parker,
Kailey E. Komnick, and Paul J. Hergenrother*
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ABSTRACT: Fusidic acid (FA) is a potent steroidal antibiotic
that has been used in Europe for more than 60 years to treat a
variety of infections caused by Gram-positive pathogens. Despite
its clinical success, FA requires significantly elevated dosing (3 g on
the first day, 1.2 g on subsequent days) to minimize resistance, as
FA displays a high resistance frequency, and a large shift in
minimum inhibitory concentration is observed for resistant
bacteria. Despite efforts to improve on these aspects, all previously
constructed derivatives of FA have worse antibacterial activity
against Gram-positive bacteria than the parent natural product.
Here, we report the creation of a novel FA analogue that has
equivalent potency against clinical isolates of Staphylococcus aureus
(S. aureus) and Enterococcus faecium (E. faecium) as well as an
improved resistance profile in vitro when compared to FA. Importantly, this new compound displays efficacy against an FA-resistant
strain of S. aureus in a soft-tissue murine infection model. This work delineates the structural features of FA necessary for potent
antibiotic activity and demonstrates that the resistance profile can be improved for this scaffold and target.
KEYWORDS: antibiotics, fusidic acid analogues, resistance profile, in vivo efficacy, EF-G inhibitor
ecent reports have confirmed that the worrisome rise of
1,2
Rantibiotic-resistant bacterial infections is continuing.
Some of the most problematic Gram-positive pathogens are
methicillin-resistant Staphylococcus aureus (MRSA) and
vancomycin-resistant Enterococci (VRE), and accordingly,
these have been classified as serious threats by the Centers
for Disease Control and Prevention (CDC).3 The emergence
of bacteria resistant to vancomycin,4 a drug of last resort
against Gram-positive infections, highlights the need to
develop or re-engineer drugs that could be efficacious against
these problematic pathogens.
Fusidic acid (1, Figure 1) is a steroidal antibiotic that is
produced by the fungus Fusidium coccineum.5 Its mechanism of
action involves inhibition of protein synthesis by stabilizing the
elongation factor G (EF-G) complex, resulting in the
truncation of peptide elongation.6 EF-G being an unusual
Figure 1. Structure of fusidic acid (FA), with some of its properties.
antibacterial target likely minimizes the cross-resistance of FA
with other antibiotics.7 FA is a Gram-positive-only antibiotic
that has been used since the 1960s in Europe to treat Gram-
Received: December 11, 2020
Published: February 1, 2021
positive infections and has been particularly effective in
treating MRSA.7,8 To date, FA is approved in 23 countries
and is administered via oral, intravenous, and topical routes.7
In the United States, FA has achieved its primary and
secondary efficacy end points in phase 3 clinical trials for the
treatment of patients with acute bacterial skin and skin
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Figure 2. (A) Structure−activity relationship investigation of the carboxylic acid and alcohols of FA. (B) Synthesis of the C11 ketone of FA. MICs
determined using CLSI guidelines against S. aureus ATCC 29213, n = 3. Asterisk indicates solubility limit.
structure infections (ABSSSI).9 FA has also received Orphan
Drug Designation from the Food and Drug Administration
(FDA) for the treatment of prosthetic joint infections and
gained Qualified Infectious Disease Product Designation under
the Generating Antibiotic Incentives Now (GAIN) Act.10
Strikingly, an analysis conducted in 2011 showed that virtually
all (99.7%) of S. aureus strains in the United States were
susceptible to FA.7
Despite the clinical success of FA, the high resistance
frequency in the range of 1 X 10−6 to 1 X 10−8 at 2−16X the
minimum inhibitory concentration (MIC) coupled with drastic
MIC shifts upon resistance remain a major concern and are a
hindrance to its wider usage.7 FA-resistant bacterial strains
display a varied profile, illustrated by the heterogeneity in
mutations observed in the laboratory and in the clinic.11 Some
of the most prevalent mutations in the fusA gene (encoding
EF-G) observed in the clinic are V90L, H457Q, L461K, and
A655V.7 Especially concerning is the L461K mutation,
observed clinically in S. aureus strains resistant to FA, which
leads to MIC values of >256 μg/mL, a >2048-fold increase in
MIC upon resistance.7 This large shift in MIC upon resistance
observed from a single amino acid mutation is consistent with
the hypothesis that single enzyme inhibitors are more prone to
display elevated shifts in MIC upon resistance relative to
antibiotics with multiple targets.12,13 In addition to mutations
in the fusA gene, another source of clinical resistance for FA is
the overexpression of the FusB family of proteins encoded by
the fusB gene usually carried on a 21-kb plasmid (pUB101);
FusB binds to EF-G and drives the dissociation of FA from its
target.14 More recently, fusC and fusD genes have also been
discovered in clinical isolates of S. aureus.15 The fusC gene has
been associated with driving resistance in S. aureus and
coagulase-negative staphylococci, while the fusD gene has been
linked to conferring resistance among Staphylococcus saprophy-
ticus.15
The aforementioned resistance challenges necessitate a
front-loading dosing regimen, with 3 g of FA taken on the
first day of treatment and 1.2 g taken daily thereafter, allowing
for considerable drug plasma levels to be reached (Cmax of
greater than 130 μg/mL in patients).7 Despite multiple efforts
to construct FA analogues,16−32 none of these derivatives are
as active as FA against Gram-positive pathogens;7 in addition,
none of them improve on the resistance issues noted above.
No FA derivatives have gained traction translationally, and
only FA itself has moved forward to the clinic from this
antibiotic class. While no FA derivatives with improved activity
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Figure 3. (A) Synthesis of aliphatic aldehyde derivative of FA. (B) Construction of halogenated side chain analogues of FA. (C) Generation of
fluorinated side chain analogue of FA. (D) Synthesis of an FA analogue with a terminal olefin side chain. (E) Synthesis of cyclic side chain
derivatives of FA. (F) Formation of saturated side chain derivative of FA. MICs determined using CLSI guidelines, against S. aureus ATCC 29213,
n = 3.
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against Gram-positive bacteria have been reported, derivatives
with improved activity relative to FA have been reported
against the malaria parasite Plasmodium falciparum.21,26,30
Notable challenges in constructing FA variants through
direct modification of this natural product include the presence
of reactive functional groups such as the olefinic side chain, an
α,β-unsaturated carboxylic acid, an acetate, and two sterically
hindered alcohols (Figure 1). Furthermore, the unique chair−
boat−chair core conformation of FA makes the total synthesis
of this antibiotic challenging, and such routes are not yet
suitable for the rapid generation of derivatives.24,25,33 Intrigued
by the translational potential of FA, here we report the
development of synthetic routes from FA that provide rapid
access to novel variants with modified side chains, leading to
the discovery of a novel FA analogue with an improved
resistance profile.
alcohols. Oxidation of the secondary alcohols separately is
more tolerated, with the monoketones at C3 (compound 5)
and at C11 (compound 10) having MIC values only 4−8-fold
worse than FA (Figure 2).
Synthesis and Antibacterial Evaluation of Side Chain
Analogues. As modifications to FA shown in Figure 2 were
all deleterious to antibacterial activity, the focus shifted to
modifications of the gem-dimethyl alkene side chain. This work
was informed by the crystal structure of the ribosome trapped
with EF-G in a post-translocational state with FA bound;36 this
complex indicates that the side chain of FA is positioned in a
large, hydrophobic pocket of EF-G.36
Several FA analogues with modifications to the side chain
have been previously reported, such as derivatives where the
C24 and C25 or the C17 and C20 double bonds have been
reduced.17,22 Furthermore, truncation of the C26 and C27
methyl groups has also been reported as well as analogues
where the C26 and C27 methyl groups were modified via
microbial oxidation.37,38 Heteroatoms have also been intro-
duced via ozonolysis of the C24 and C25 double bond.18 Aryl
side chain analogues of FA have also been synthesized and
used to conduct photolabeling studies.23 Notably, all of the
previously reported side chain analogues of FA have reduced
antibacterial activity relative to FA.
To provide access to FA derivatives with novel side chains, a
modular synthetic route for rapid generation of a variety of
compounds with modified side chains was envisioned.
Aldehyde 11, generated previously by ozonolysis23 and herein
via dihydroxylation followed by oxidative cleavage (Figure 3A),
was selected as the pluripotent intermediate that could be
transformed via olefinations, one-carbon homologations, and
cross-metathesis reactions to the various analogues. Using 11,
halogens were introduced via a one-carbon homologation
reaction with carbon tetrachloride, carbon tetrabromide, and
carbon tetraiodide, respectively, to generate intermediates 12−
14 (Figure 3B). Subsequent deprotection with potassium
carbonate in methanol afforded novel halogenated compounds
15−17 (Figure 3B). Construction of the analogous fluorinated
compound was accomplished by treating aldehyde 11 with
chlorodifluoroacetic acid and triphenylphosphine at elevated
temperatures to generate 18 and deprotection to generate
difluorinated derivative 19 (Figure 3C). Aldehyde 11 was also
converted to olefin 20 and fully deprotected to generate 21 as
previously reported (Figure 3D).37
Olefin 20 was used as another key point of diversity and
divergence to generate compounds containing symmetrical
side chains with the C24 and C25 double bond intact. Thus, to
form cyclic derivatives, olefin 20 was coupled via a cross-
metathesis reaction with methylenecyclopentane, methylene-
cyclohexane, and 4-methylenetetrahydropyran using Grubbs’
catalyst 2 to generate analogues 22−24. Deprotection
generated novel cyclic side chain derivatives 25−27 (Figure
3E). Lastly, compound 28 with a fully saturated side chain was
synthesized as previously reported using Lindlar’s catalyst
(Figure 3F).22
With this collection of FA derivatives in hand, the MIC of
each compound against S. aureus was determined (Figure 3).
Compounds 15, 17, 19, 21, and 28 displayed modest decreases
in potency relative to FA. Notably, analogues 16, 25 (hereafter
referred to as FA-CP), 26, and 27 displayed equipotent activity
to FA, suggesting the antibiotic potential of FA derivatives with
modified side chains (Figure 3).
RESULTS
■
Synthesis and Antibacterial Assessment of FA
Derivatives Where the Carboxylic Acid and Alcohols
Are Modified. Key to this study was to first determine the
important structural features necessary to retain the potent
antibacterial activity of FA. Although FA derivatives have been
reported in the literature,16−32 a systematic antimicrobial
activity assessment of FA derivatives with MIC values against
key Gram-positive pathogens does not exist. Thus, as a prelude
to our work, we first synthesized several known FA analogues
and assessed their antibacterial activity, with the specific goals
of probing the importance of the carboxylic acid and alcohols.
The role of the carboxylic acid was investigated via
construction of ester 2 and amide 3, using previously published
routes (Figure 2A).26,27 Diketone 4 was synthesized previously
using the Jones oxidation reaction.34 Herein, diketone 4 was
synthesized using pyridinium chlorochromate (Figure 2A).
Oxidation solely at C3 was effected using recently published
conditions to provide compound 5 (Figure 2A).28 The
monoketone at C11 was previously synthesized by treating
FA with Kiliani’s reagent in a low yielding process.29 Herein, a
five-step sequence was developed to generate the C11 ketone
(Figure 2B). This route required the protection of the
carboxylic acid to generate intermediate 6.21,23 The steric
hindrance at the C11 position induced by 1,3-diaxial
interactions was leveraged, and the C3 alcohol was selectively
protected with TBS to generate 7. The C11 alcohol was then
oxidized with pyridinium chlorochromate to generate 8.
Removal of the TBS protecting group in compound 8 with
aqueous hydrofluoric acid provided 9, and subsequent
deprotection of the pivaloyloxymethyl protecting group
under mild basic conditions afforded the target monoketone,
compound 10 (Figure 2B). Key to this work was utilizing a
suitable protecting group for the carboxylic acid that could be
removed under mild basic conditions, as lactonization between
the C16 acetyl and the C21 carboxylic acid occurs under
strongly basic conditions.21,23 Accordingly, POM-Cl, used
previously to protect the FA carboxylic acid,21,23 was utilized to
protect the carboxylic acid as a pivaloyloxy-methyl ester based
on its ability to be removed under mild conditions.21,23
Antibacterial activity of FA analogues 2−5 and 10 was
assessed against S. aureus ATCC 29213 using standard MIC
assays following Clinical and Laboratory Standards Institute
(CLSI) guidelines.35 As shown in Figure 2, FA analogues 2−4
display significantly reduced antibacterial activity, reinforcing
the importance of the free carboxylic acid and both of the
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Figure 4. (A) Resistance frequency of FA-CP. (B) Resistance frequency of compound 26. (C) Resistance frequency of compound 16. (D)
Resistance frequency of compound 27. Resistance frequency of FA and equipotent analogues was determined in S. aureus ATCC 29213. FA data is
the same in all four experiments. Error is SEM, n = 3. NS = not significant, **p < 0.01, *** p < 0.001, and **** p < 0.0001.
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Evaluation of Resistance Frequency of Equipotent
Analogues of FA. The resistance frequency of the equipotent
analogues (16, 25 (FA-CP), 26, and 27) was assessed in head-
to-head experiments with FA. The large inoculum method was
used in order to generate resistance mutants and derive the
resistance frequency of FA and the equipotent analogues.39
The resistance frequency was determined at 2, 4, 8, 16, and
32X the MIC, which is 0.125 μg/mL for all compounds. The
resistance frequency at the listed MICs was then compared
head-to-head against FA, and statistical significance was then
determined using one-way ANOVA with Tukey’s multiple
comparisons. As shown in Figure 4A,B, compounds FA-CP
and 26 displayed an improvement in resistance frequency
relative to FA. FA-CP displayed a modest improvement in
resistance frequency at 2 and 32X the MIC, while analogue 26
only displayed an improvement at 32X the MIC (Figure 4A,B).
This is in contrast with analogues 16 and 27, which displayed a
worse resistance frequency relative to FA (Figure 4C,D).
Effect of Human Serum Binding, Assessment of
Metabolic Stability, and Mammalian Cell Toxicity. The
translational potential of the novel derivatives was further
investigated by assessing parameters important in antibacterial
development, such as the effect of human serum binding,
metabolic stability, and mammalian cell toxicity. FA is reported
to be highly protein bound, with human plasma protein
binding of 97%.40 Additionally, the MIC for FA when 50%
human serum is included in the media shifts to 16−256X
higher.40 In this work, the shift in MIC for S. aureus in 50%
human serum was determined to be very similar for FA as
compared to compounds 16, FA-CP, 26, and 27 (Table S1). In
addition, the stability of the novel derivatives to mouse liver
microsomes was similar to FA (Table S2). FA, 16, FA-CP, 26,
and 27 exhibited low toxicity toward human foreskin
fibroblast-1 cells (HFF-1), with all compounds giving IC50
values greater than 50 μM (Table S3).
Mode-of-Action of Novel FA Derivative. FA-CP was
selected for additional studies investigating the mode-of-action.
For these studies, resistance mutants were generated at 32X
the MIC of FA-CP and FA, and sequencing of the fusA gene
(coding for EF-G, the target of FA) revealed mutations in fusA,
suggesting that FA-CP engages the same target as FA.
Specifically, for FA and FA-CP, the fusA gene was sequenced
for 40 different resistant colonies generated at 32X the MIC,
20 colonies for each compound. As shown in Table 1, the
resistance mutants observed for FA are in accord with those
seen previously in analogous studies. Specifically, mutations
F88L,11,41 T436I,11,41 H457N,41 and D434N11 have been
identified in bacterial cell culture studies with S. aureus and FA.
Additionally, H457Y11,41−44 has been observed in bacterial cell
culture studies as well as clinical isolates of S. aureus that are
resistant to FA. Consistent with the notion that FA-CP engages
the same target as FA, single amino acid substitutions (H457Y,
D434N, and F88L) within EF-G were found in the 20 different
FA-CP colonies (Table 1).
Table 1. Observed Mutations in the EF-G Protein (Encoded
by fusA) and MIC Values for FA and FA-CP at 32X the MIC
in S. aureus ATCC 29213
strains
resistant to
FA
EF-G
MIC strains resistant
EF-G
mutation
MIC for
FA-CP
mutation for FA
to FA-CP
FA-32X-1
FA-32X-2
FA-32X-3
FA-32X-4
FA-32X-5
FA-32X-6
FA-32X-7
FA-32X-8
FA-32X-9
FA-32X-10
FA-32X-11
FA-32X-12
FA-32X-13
FA-32X-14
FA-32X-15
FA-32X-16
FA-32X-17
FA-32X-18
FA-32X-19
FA-32X-20
H457Y
T436I
H457Y
H457N
F88L
T436I
H457Y
H457Y
F88L
128
8
128
256
256
16
128
128
256
128
256
128
128
128
128
256
256
256
128
128
FA-CP-32X-1
FA-CP-32X-2
FA-CP-32X-3
FA-CP-32X-4
FA-CP-32X-5
FA-CP-32X-6
FA-CP-32X-7
FA-CP-32X-8
FA-CP-32X-9
FA-CP-32X-10 D434N
FA-CP-32X-11 H457Y
FA-CP-32X-12 D434N
FA-CP-32X-13 H457Y
FA-CP-32X-14 H457Y
FA-CP-32X-15 H457Y
FA-CP-32X-16 F88L
FA-CP-32X-17 F88L
FA-CP-32X-18 H457Y
FA-CP-32X-19 H457Y
FA-CP-32X-20 H457Y
H457Y
H457Y
H457Y
H457Y
H457Y
H457Y
H457Y
H457Y
H457Y
32
64
64
32
64
64
64
64
32
32
32
32
64
32
64
64
64
64
32
32
H457Y
F88L
H457Y
H457Y
H457Y
H457Y
F88L
H457N
D434N
H457Y
H457Y
In a separate experiment, S. aureus strains resistant to FA
were generated at 4, 8, 16, and 32X the MIC. Mutations found
in the fusA gene of the resistant colonies are consistent with
what has been observed in previous studies conducted with
FA. Specifically, observed mutations in the fusA gene causing
single amino acid substitutions, T385N,45 R464L,11 F88L,11,41
H457L,41 T436I,11,41 H457N,41 and D434N,11 have been
identified in bacterial cell culture studies with S. aureus and FA.
Furthermore, H457Y11,41−44 and P406L11,41,42,45 have been
observed in bacterial cell culture studies as well as clinical
isolates of S. aureus that are resistant to FA. FA-CP was
evaluated against these 11 FA-resistant strains for antimicrobial
activity. Although FA-CP displays cross-resistance with FA,
expected as the target is conserved between the two
compounds, FA-CP displays a 2−8-fold improvement in
activity against these FA-resistant strains relative to FA
(Table 2).
Activity of FA-CP and FA against Multidrug-Resistant
Clinical Isolates of S. aureus, E. faecium, and an S.
aureus Strain Harboring the fusC Gene. The activity of
FA, FA-CP, and vancomycin was assessed against 29 different
clinical isolates of S. aureus. FA and FA-CP displayed no cross-
resistance with vancomycin and retained potent activity against
the 29 different clinical isolates assessed (Figure 5A).
Noteworthy is that within this collection of clinical isolates,
there are strains with resistance to a wide variety of antibiotics,
including cefoxitin, clindamycin, doxycycline, erythromycin,
gentamicin, levofloxacin, oxacillin, penicillin, tetracycline, and
trimethoprim/sulfamethoxazole (see Tables S4 and S5 for a
full list of resistance profiles).
The MICs of FA and FA-CP against these resistance
mutants were also determined, and it was found that the
mutants generated from FA-CP typically displayed a reduced
shift in MIC upon resistance relative to the mutants generated
from FA (Table 1). Specifically, the highest MIC observed for
colonies generated from FA-CP was 64 μg/mL (fold increase
from wild-type (WT) S. aureus: 512) (Table 1). In our in-
house studies, the highest MIC observed for FA was 256 μg/
mL (fold increase from WT S. aureus: 2048) (Table 1).
The activity of FA and FA-CP was also assessed against a S.
aureus strain that possesses the fusC gene, strain S. aureus
ATCC BAA-1721.46 FA-CP displayed cross-resistance with
FA, as both compounds displayed an MIC of 8 μg/mL against
this strain.
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against a sensitive strain of E. faecium ATCC 19434 is 2 μg/mL
for both antibacterial agents. FA and FA-CP displayed no
cross-resistance with vancomycin in the E. faecium clinical
isolates. Excitingly, FA and FA-CP retained potent activity
against VRE with high levels of vancomycin resistance
(vancomycin MIC = 512 μg/mL). See Table S6 for a full
list of resistance profiles.
Pharmacokinetic Studies and in vivo Efficacy of FA
and FA-CP. As a prelude to the mouse infection studies, the
tolerability of FA (sodium fusidate) and FA-CP was assessed in
mice at 50 mg/kg (intraperitoneal injection, once-a-day for 4
days), and both compounds were well tolerated by mice at this
dose. Noteworthy is the rapid metabolism of FA in rodents,
which is a major hurdle in the preclinical evaluation of FA
analogues in mouse models.28,32 Head-to-head assessment of
the pharmacokinetics of FA and FA-CP in mice revealed that
these compounds have similar PK profiles (Figure 6A). The
mouse and human PK profiles for FA are considerably
different, as FA has a long half-life in humans of around 10−14
h,7,47 with predominant metabolism by the liver;48 the rapid
clearance observed in mice could be driven by the formation of
Table 2. MIC Values for FA and FA-CP against 11 Different
FA-Resistant Strains of S. aureus ATCC 29213
a
strains resistant to FA EF-G mutation MIC for FA MIC for FA-CP
FA-4X-A
FA-4X-B
FA-8X-A
FA-8X-B
FA-16X-A
FA-32X-A
FA-32X-B
FA-32X-C
FA-32X-D
FA-32X-E
FA-32X-F
T385N
T385N
T385N
R464L
H457Y
F88L
P406L
H457L
T436I
8
8
8
16
128
256
32
256
8
2
2
2
8
64
128
4
128
2
H457N
D434N
256
256
128
32
a
FA-resistant strains were generated at 4X (n = 2), 8X (n = 2), 16X (n
= 1), or 32X (n = 6) the MIC as indicated. MICs determined using
CLSI guidelines, n = 3.
The activity of FA, FA-CP, and vancomycin was also
assessed against six different clinical isolates of E. faecium
(Figure 5B). As a reference point, the MIC of FA and FA-CP
Figure 5. (A) Activity against 29 clinical isolates of S. aureus. (B) Activity against six different clinical isolates of E. faecium. MICs determined using
CLSI guidelines, n = 3. See Tables S4−S6 for a list of resistance genes in clinical isolate panels. A full listing of this MIC data is in Tables S5 and S6.
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Figure 6. (A) Pharmacokinetic analysis of fusidic acid and FA-CP. C57BL/6 mice were treated with 50 mg/kg of fusidic acid or FA-CP via
intraperitoneal injection, with three mice per time point (0, 15, 30, 45, 60, 120, and 240 min). After the stated time points, mice were sacrificed, and
the serum concentrations of fusidic acid or FA-CP were determined by liquid chromatography−tandem mass spectrometry (LC−MS/MS). The
data are illustrated as the mean, and error bars represent the standard error. The pharmacokinetic parameters illustrated above were calculated with
a one-compartment model using a nonlinear regression program (Phoenix WinNonlin Version 8.1, Certara USA Inc., Princeton, NJ, USA). (B) S.
aureus ATCC 29213 neutropenic thigh infection burden study. CD-1 mice were first rendered neutropenic with cyclophosphamide. Mice were
injected on Day -4 to Day -2 with 150 mg/kg and on Day -1 with 100 mg/kg of cyclophosphamide, respectively. Acute thigh infections initiated in
CD-1 mice with S. aureus ATCC 29213 (8.9 X 105 colony forming units (CFU) mouse−1 via intramuscular injection) were treated with vehicle,
fusidic acid, and FA-CP (eight mice per group) at 1, 2, and 3 h postinfection (50 mg/kg via intraperitoneal injection), and the bacterial burden was
evaluated at 24 h postinfection. (C) S. aureus (FA-32X-B) neutropenic thigh infection study. CD-1 mice were first rendered neutropenic with
cyclophosphamide as described above. Acute thigh infections initiated in CD-1 mice with S. aureus (FA-32X-B) (1.08 X 106 CFU mouse−1 via
intramuscular injection) were treated with vehicle, fusidic acid, and FA-CP (eight mice per group) at 1, 2, and 3 h postinfection (50 mg/kg via
intraperitoneal injection), and the bacterial burden was evaluated at 8 h postinfection. Drugs were formulated with 5% DMSO, 10% Tween 20, 85%
PBS immediately before injection. The data is shown as means s.d., and statistical significance was determined by one-way ANOVA with Tukey’s
multiple comparisons. NS indicates not significant (P > 0.05), **P < 0.01, ****P < 0.0001.
3-epiFA, a metabolite that has not been observed in
humans.28,32
Encouraged by the efficacy of FA-CP against the panel of
clinical isolates, the lack of toxicity to mammalian cells, and the
promising tolerability and pharmacokinetic data, FA-CP was
compared head-to-head to FA in in vivo efficacy studies; the
design of these efficacy studies was guided by mouse models
previously conducted with FA.49 First, a sensitive strain of S.
aureus was assessed in a neutropenic thigh infection burden
model utilizing S. aureus ATCC 29213, in which FA-CP and
FA display the same MIC of 0.125 μg/mL. After infection,
three doses of FA-CP and FA were administered (50 mg/kg,
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intraperitoneal injection) 1, 2, and 3 h postinfection to
separate groups of mice. Mice were sacrificed 24 h post-
infection, and bacterial burden in the thigh muscle tissue
homogenates was determined by serial dilution plating onto
tryptic soy agar. Significant reductions in bacterial burden were
observed with FA and FA-CP (Figure 6B).
The improved potency of FA-CP against FA-resistant strains
motivated and guided the second neutropenic thigh infection
burden model. An FA-resistant S. aureus strain, FA-32X-B,
generated at 32X the MIC of FA, was used for this study. FA
has an MIC of 32 μg/mL, while FA-CP displays an MIC of 4
μg/mL (Table 2). After infection, three doses of FA-CP and
FA were administered (50 mg/kg, intraperitoneal injection) 1,
2, and 3 h postinfection to separate groups of mice. Mice were
sacrificed 8 h postinfection, and bacterial burden in the thigh
muscle tissue homogenates was determined by serial dilution
plating onto tryptic soy agar. This resulted in reductions in
bacterial burden with FA-CP and no efficacy with FA (Figure
6C). This study is, to our knowledge, the first mouse bacterial
infection model in which a novel derivative of FA displays
improved efficacy relative to FA.
not only been observed in bacterial cell culture studies but also
been identified in clinical isolates of S. aureus,11,42 highlighting
the potential of FA-CP to retain efficacy in vivo against some
clinical isolates of S. aureus that are resistant to FA.
Sequencing studies also revealed some interesting differ-
ences between the mutational profile of bacteria arising from
FA versus FA-CP treatment. In our in-house studies, five
different amino acid changes were observed in EF-G for FA-
resistant S. aureus, while only three different amino acid
mutations in EF-G were observed for FA-CP-resistant S.
aureus. Mutations in EF-G have been classified into four
different groups based on their role in resistance to FA: Group
A (mutations affecting FA binding), Group B (mutations
affecting EF-G ribosome interactions), Group C (mutations
affecting EF-G conformation), and Group D (mutations
affecting EF-G stability).50 Moreover, Group A mutations
involve amino acids that are in direct contact with FA and with
residues that can shape the drug pocket.50 Additionally, it has
been postulated that Group C mutations are related to the
interdomain orientation of EF-G, which can affect the FA-
binding pocket along with the conformation dynamics and FA
locking of EF-G.50 The mutations in FA-resistant strains found
in our studies have been previously classified and belong to
Group A and Group C, whereas all the mutations observed for
FA-CP-resistant strains belong to Group A, indicating that
these mutations are in regions that can affect FA binding.
While mutation to fusA, the gene encoding for EF-G, is a
major mechanism of resistance for FA, it is important to note
that other resistance mechanisms are operational in clinical
isolates, specifically, the horizontal acquisition of plasmids
harboring fusB or fusC.15 These resistance genes encode
protective proteins that drive the dissociation of the EF-G
ribosome complex from FA.51 Analogous to the shifts in MIC
observed for FA upon mutations to the fusA gene, an increase
in MIC has also been reported in clinical isolates that possess
the fusB and/or fusC genes. For example, MIC ranges of 2−64
μg/mL in staphylococcal clinical isolates have been reported
for clinical isolates that carry the fusC gene, and MIC ranges of
4−1024 μg/mL have been reported for strains that possess the
fusB gene.15,44,46 Due to the clinical relevance of strains that
have acquired the fusC gene, the MIC of FA-CP was assessed
against a strain that possesses this gene. FA-CP displays the
same MIC as FA against S. aureus ATCC BAA-1721, with an
MIC of 8 μg/mL. While resistance driven by the fusB and fusC
genes is worrisome, the primary mechanism of clinical
resistance to FA is still resistance mediated by mutations in
the fusA gene.7,42 Specifically, mutation L461K found in
clinical isolates of S. aureus is of high concern due its drastic
shift in MIC (MIC > 256 μg/mL) upon a single amino acid
mutation.7 Perhaps even more problematic are clinical isolates
of S. aureus that are resistant to FA and possess four different
EF-G amino acid alterations within the same strain. Five such
clinical isolates have been isolated, and they all showed the
following alterations in EF-G: V90I, H457Q, L461K, and
A655V.42 All of these strains have an MIC greater than 256
μg/mL, highlighting the prominent role that mutations to the
fusA gene play in the development of resistance to FA in the
clinic.42 It may be possible to design FA derivatives that retain
strong binding to these mutant versions of EF-G, and FA
derivatives with a further improved resistance profile can also
be envisioned.
DISCUSSION
■
Despite FA being approved in Europe since the 1960s to treat
problematic S. aureus infections, there have been no follow-on
drugs, and FA remains the only member of the fusidane class
to be used clinically.7 The high resistance frequency, drastic
shifts in MIC upon bacterial resistance to FA, and lack of
effective derivatives have narrowed the development path for
this antibiotic class.
A considerable challenge in the creation of new and effective
derivatives of FA has been a clouded structure−activity
relationship for this compound, due to heterogeneity in the
reported biological data for derivatives, with many compounds
evaluated against the malaria parasite Plasmodium falcipa-
rum21,26,30 and Mycobacterium species28,31,32 but not against
Gram-positive bacteria. In this work, we have constructed and
assessed both previously synthesized derivatives and novel
derivatives, allowing for a clearer structure−activity relation-
ship (SAR) to emerge. Most notably, while previous
investigations suggested that changes in the hydrophobic side
chain of FA diminished antibacterial activity,17,18,22,23,37,38 here
we show that certain changes at this position are not only
tolerable but lead to derivatives with favorable resistance
profiles compared to FA.
Specifically, the reduced shift (relative to that observed for
FA) in MIC upon resistance of S. aureus to FA-CP is an
important feature of this compound. The highest MIC for the
resistance mutants generated to FA-CP was 64 μg/mL, versus
256 μg/mL for resistance mutants generated to FA. In
principle, this improvement could allow for administration of
less FA-CP or a similar compound. Although FA is considered
a safe antibiotic, gastrointestinal side effects have been
observed in 30−58% of patients treated with FA, and up to
30% of patients have elevated bilirubin.47
Another interesting and potentially useful aspect of FA-CP is
the ability of this compound to retain some activity against
strains of S. aureus that are resistant to FA, including in a
mouse model of infection. Specifically, FA-CP displayed in vivo
efficacy against an FA-resistant S. aureus strain generated at
32X the MIC of FA (strain FA-32X-B). Our sequencing
studies reveal that FA-32X-B possesses the mutation P406L in
the EF-G protein. Noteworthy is the fact that this mutation has
FA-CP is the first analogue to outperform FA in a mouse
infection model, which more broadly suggests the potential for
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certain FA derivatives. The important improvement in the
resistance profile observed for FA-CP has some precedence in
antibacterial drug discovery, for example in the dihydrofolate
reductase inhibitor iclaprim52 and leucyl-tRNA synthetase
inhibitor DS86760016,53 with improvement in each case likely
due to subtly different modes of target engagement relative to
the parent antibiotics (trimethoprim and GSK2251052,
respectively). With access to derivatives now facile and the
ability to improve upon its resistance profile now demon-
strated, novel antibiotics of the fusidane class can be developed
for the treatment of a variety of bacterial infections. Finally, as
demonstrated by its outstanding activity against permeability
defect Escherichia coli,54 FA has potential against Gram-
negative bacteria but is accumulation limited; the detailed SAR
reported herein should assist in exploiting this untapped
potential of FA to combat drug-resistant Gram-negative
infections.
Analyst 1.6.2 was used for data acquisition and analysis. The
1200 series HPLC system (Agilent Technologies, Santa Clara,
CA) includes a degasser, an autosampler, and a binary pump.
The LC separation was performed on an Agilent Sb-Aq
column (4.6 X 50 mm, 5 μm) with mobile phase A (0.1%
formic acid in water) and mobile phase B (0.1% formic acid in
acetonitrile). The flow rate was 0.3 mL/min. The linear
gradient was as follows: 0−3 min, 100% A; 10−16 min, 5% A;
16.5−22 min, 100% A. The autosampler was set at 10 °C. The
injection volume was 1 μL. Mass spectra were acquired under
both positive (ion spray voltage +5500 V) and negative (ion
spray voltage −4500 V) electrospray ionization (ESI). The
source temperature was 450 °C. The curtain gas, ion source
gas 1, and ion source gas 2 were 33, 65, and 60 psi,
respectively. Multiple reaction monitoring (MRM) was used
for quantitation.
Selection of Resistant Mutants. Resistant mutants were
selected via the large inoculum method. Briefly, S. aureus
ATCC 29213 (1 X 109 CFU) was plated on 100 mm plates of
LB agar containing 4, 2, 1, 0.5, and 0.25 μg/mL. Colonies were
visible after incubation at 37 °C for 24 h. Resistant colonies
were confirmed by streaking on selective media with the same
concentration of fusidic acid and compounds 16, FA-CP (25),
26, 27.
Sequencing of fusA. FusA was amplified by colony
polymerase chain reaction (PCR). Colonies were picked and
diluted in 100 μL of sterile H2O. PCR reactions are set up by
combining MiFi Mix (Bioline, London, UK), 20 μM primer
mix [fusA-F2, fusA_seq1, forward EF-G2, and reverse EF-G]
(S. aureus ATCC 29213), template DNA, and H2O. Reactions
were performed on a C1000 Thermal Cycler (Bio-Rad,
Hercules, CA) with the following conditions: 5 min of
denaturation at 95 °C, followed by 30 cycles of 20 s at 95
°C, 20 s at 50 °C, and either 1 min (fusA1) or 1.5 min (fusA2)
at 72 °C. A 10 μL portion of the PCR reaction mixture was
analyzed by agarose gel to confirm the product. PCR reactions
were purified using GeneJET PCR Purification Kit (Thermo
Scientific). PCR amplicons were submitted to the Core DNA
Sequencing Facility at the University of Illinois at Urbana−
Champaign for Sanger sequencing with the following primers
to sequence the fusA [fusA2, fusA_seq1, forward EF-G2,
forward EF-G3, forward EF-G4]. See the Supporting
Information to find the sequence of the primers used in this
study.
Mouse Maximum Tolerated Dose (MTD) of Sodium
Fusidate and FA-CP (25). The protocol was approved by the
Institutional Animal Care and Use Committee (IACUC) at the
University of Illinois at Urbana−Champaign (protocol
number: 16144 and 19181). In these studies, 10- to 12-
week-old female C57BL/6 mice purchased from Charles River
were used. The maximum tolerated dose (MTD) of a single
compound was determined first. Sodium fusidate and FA-CP
(25) were formulated in 5% DMSO, 10% Tween 20, and 85%
PBS. Sodium fusidate and FA-CP (25) were given by
intraperitoneal (IP) injection. All the mice were monitored
for signs of toxicity for 2 weeks. For multiple doses, the
compound was given by daily IP injection for 4 consecutive
days, and mice were monitored for signs of toxicity for 1
month. The MTD was the highest dosage with acceptable
toxicity (e.g., < 20% weight loss). Sodium fusidate and FA-CP
(25) were well tolerated as a single dose of 50 mg/kg. Further
analysis showed that sodium fusidate and FA-CP (25) were
well tolerated with daily dosing of 50 mg/kg for 4 consecutive
METHODS
■
Bacterial Strains. S. aureus ATCC 29213, E. faecium
ATCC 19434, and S. aureus ATCC BAA-1721 were obtained
from the American Type Culture Collection (ATCC).
Resistant strains of E. faecium were obtained from the CDC.
Resistant strains of S. aureus were obtained from the Network
on Antimicrobial Resistance in Staphylococcus aureus (NARSA)
and the CDC.
Antimicrobial Susceptibility Tests. Susceptibility testing
was performed in biological triplicate, using the microdilution
broth method as outlined by CLSI. Bacteria were cultured with
cation-adjusted Muller-Hinton broth (Sigma-Aldrich, Cat#
90922) in round-bottom 96-well plates (Corning, Cat# 3788).
Human serum (ultrafiltrate, unspecified gender, 30K Dalton
membrane filtered) was purchased from BioIVT (Hicksville,
NY).
Cell Culture. HFF-1 cells were obtained from ATCC.
HFF-1 cells were grown in DMEM with 15% fetal bovine
serum (Gemini Benchmark, Cat# 100−106), 100 μg/mL of
penicillin, and 100 μg/mL of streptomycin. All cells were
cultured at 37 °C in a 5% CO2 environment. Media was
prepared by the University of Illinois School of Chemical
Sciences Cell Media Facility.
Cell Viability. Cells were harvested, seeded in a 96-well
plate, and allowed to adhere. Cells were treated with
investigational compounds in DMSO (1% final concentration).
Cells were incubated for 72 h before viability was assessed by
the Alamar Blue Assay. Raptinal (20 μM) was used as a dead
control.
Mouse Liver Microsome Stability Assay. A mixture of
PBS (pH 7.4), NADPH regenerating system solution A
(Corning Life Sciences), and NADPH regenerating system
solution B (Corning Life Sciences) was incubated at 37 °C in a
shaking incubator for 5 min. Next, compound was added in
DMSO (final concentration 50 μM, 0.5% DMSO) before ice-
cold mouse liver microsomes (Thermo Fisher, male CD-1
mice, pooled) were added (final protein concentration of 1
mg/mL). An aliquot was immediately removed, quenched with
an equal volume of 100 μM internal standard, and centrifuged
at 13 000 rcf for 3 min. The reactions were incubated at 37 °C
in a shaking incubator for 3 h. A second aliquot was removed
and quenched. Samples were analyzed with the 5500 QTRAP
LC-MS/MS system (Sciex, Framingham, MA) in the
Metabolomics Laboratory of Roy J. Carver Biotechnology
Center, University of Illinois at Urbana−Champaign. Software
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days. The MTD of FA-CP (25) was used to inform the dosing
schedule used in subsequent efficacy studies.
2, and 3 h postinfection (hpi) individually in 100 μL of
volume. Infected animals were monitored for myositis and
lameness until euthanasia. At indicated times (24 hpi for the 50
mg/kg cohorts for the S. aureus ATCC 29213 infection model
and 8 hpi for the 50 mg/kg cohorts for the S. aureus (FA-32X-
B) infection model), mice were euthanized with CO2
asphyxiation from a compressed gas source followed by
cervical dislocation. Infected thigh muscle tissues were
harvested and homogenized with a Omni Soft Tissue Tip
Homogenizer (OMNI International) in 2 mL of sterile PBS.
Bacterial burden in the tissue homogenates was determined by
serial dilution plating onto tryptic soy agar.
Pharmacokinetic Assessment of Sodium Fusidate
and FA-CP (25). The protocol was approved by the IACUC
at the University of Illinois at Urbana−Champaign (protocol
number: 16144, 19181). In these studies, 10- to 12-week-old
female C57BL/6 mice purchased from Charles River were
used. The compounds were formulated in 5% DMSO, 10%
Tween 20, and 85% PBS. Mice were treated with sodium
fusidate or FA-CP (25) (50 mg/kg) via IP injection with three
mice per time point (15, 30, 45, 60, 120, and 240 min). At
specific time points, mice were sacrificed, blood was collected
and centrifuged, and the serum was frozen at −80 °C until
analysis. The proteins in a 10 μL aliquot of serum were
precipitated by the addition of 50 μL of acetonitrile with the
addition of 10 μL of 1.6 μg/mL of internal standard (sodium
fusidate was the internal standard when measuring FA-CP
(25), and FA-CP (25) was the internal standard when
measuring sodium fusidate). The sample was then vortexed
and centrifuged to remove the proteins. Supernatants were
analyzed with the QTRAP 5500 LC-MS/MS system (Sciex) in
the Metabolomics Laboratory of the Roy J. Carver
Biotechnology Center, University of Illinois at Urbana−
Champaign. Software Analyst 1.6.2 was used for data
acquisition and analysis. The 1200 Series HPLC System
(Agilent Technologies) includes a degasser, an autosampler,
and a binary pump. The LC separation was performed on an
Agilent Zorbax SB-Aq column (4.6 X 50 mm; 5 μm) with
mobile phase A (0.1% formic acid in water) and mobile phase
B (0.1% formic acid in acetonitrile). The flow rate was 0.3 mL/
min. The linear gradient was as follows: 0−1 min, 95% A; 8−
13 min, 0% A; 8.1−18.5 min, 95% A. The autosampler was set
at 10 °C. The injection volume was 5 μL. Mass spectra were
acquired under negative electrospray ionization with a voltage
of −4500 V. The source temperature was 450 °C. The curtain
gas, ion source gas 1, and ion source gas 2 were 32, 60, and 60
psi, respectively. MRM was used for quantitation: fusidic acid:
m/z 515.3 → 393.3, FA-CP (25): m/z 541.4 → 437.2. The
limit of quantitation of (S/N = 10) was 1 nM.
Pharmacokinetic parameters were calculated with a one-
compartment model using a nonlinear regression program
(Phoenix WinNonlin Version 8.1; Certara USA).
Neutropenic Thigh Infection Burden Study with
Sodium Fusidate and FA-CP (25). Mouse studies were
carried out in strict accordance with the recommendations in
the guide for the Care and Use of Laboratory Animals of the
National Institutes of Health. The animal protocol was
approved by the IACUC at the University of Illinois at
Urbana−Champaign (protocol number: 17271). Briefly,
seven-week-old male CD-1 mice (cohorts of eight) were
rendered neutropenic by IP injection of cyclophosphamide
(150 mg/kg on Day -4 to Day -2 and 100 mg/kg on Day -1).
On Day -1, mice were anesthetized with a combination of
xylazine/ketamine, and furs on the right hind thigh were
removed by clipping with a pair of scissors followed by
application of depilating gel (Veet Aloe Vera Legs & Body Hair
Remover Gel Cream). After 24 h, mice were anesthetized with
isoflurane, and infected with S. aureus ATCC 29213 or the S.
aureus FA-resistant strain 32X-B at a concentration of ∼1 X 106
CFUs (in 50 μL) by injection into the thigh muscle (bicep
femoris) with a 25G 5/6″ needle. Infected mice were
intraperitoneally treated with vehicle (85% PBS, 10% Tween,
5% DMSO), 50 mg/kg of sodium fusidate, or FA-CP (25) at 1,
ASSOCIATED CONTENT
* Supporting Information
■
sı
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acsinfecdis.0c00869.
Tables S1−S6, MIC values for FA and equipotent
analogues in S. aureus in 50% human serum, stability to
mouse liver microsomes, IC50 in human foreskin
fibroblast-1 cells, list of antibiotics and associated
reference numbers with clinically relevant levels of
resistance in S. aureus and E. faecium, antimicrobial
assessment of FA, vancomycin, and FA-CP in panel of
multidrug-resistant clinical isolates of S. aureus, and
antimicrobial assessment of FA, vancomycin, and FA-CP
in panel of multidrug-resistant clinical isolates of E.
faecium. Synthetic procedures and characterization for
core and side chain derivatives of FA (PDF)
AUTHOR INFORMATION
Corresponding Author
■
Paul J. Hergenrother − Department of Chemistry, University
of Illinois at Urbana−Champaign, Urbana, Illinois 61801,
United States; Carl R. Woese Institute for Genomic Biology,
University of Illinois at Urbana−Champaign, Urbana, Illinois
61801, United States; orcid.org/0000-0001-9018-3581;
Email: hergenro@illinois.edu
Authors
Martin Garcia Chavez − Department of Chemistry, University
of Illinois at Urbana−Champaign, Urbana, Illinois 61801,
United States; Carl R. Woese Institute for Genomic Biology,
University of Illinois at Urbana−Champaign, Urbana, Illinois
61801, United States
Alfredo Garcia − Department of Chemistry, University of
Illinois at Urbana−Champaign, Urbana, Illinois 61801,
United States; Carl R. Woese Institute for Genomic Biology,
University of Illinois at Urbana−Champaign, Urbana, Illinois
61801, United States
Hyang Yeon Lee − Department of Chemistry, University of
Illinois at Urbana−Champaign, Urbana, Illinois 61801,
United States; Carl R. Woese Institute for Genomic Biology,
University of Illinois at Urbana−Champaign, Urbana, Illinois
61801, United States
Gee W. Lau − Department of Pathobiology, College of
Veterinary Medicine, University of Illinois at
Urbana−Champaign, Urbana, Illinois 61801, United States;
orcid.org/0000-0002-7962-3950
Erica N. Parker − Department of Chemistry, University of
Illinois at Urbana−Champaign, Urbana, Illinois 61801,
United States; Carl R. Woese Institute for Genomic Biology,
503
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(6) Tanaka, N., Kinoshita, T., and Masukawa, H. (1968) Mechanism
of protein synthesis inhibition by fusidic acid and related antibiotics.
Biochem. Biophys. Res. Commun. 30, 278−283.
(7) Fernandes, P. (2016) Fusidic Acid: A Bacterial Elongation
Factor Inhibitor for the Oral Treatment of Acute and Chronic
Staphylococcal Infections. Cold Spring Harbor Perspect. Med. 6,
a025437−a025437.
(8) Jones, R. N., Castanheira, M., Rhomberg, P. R., Woosley, L. N.,
and Pfaller, M. A. (2010) Performance of fusidic acid (CEM-102)
susceptibility testing reagents: broth microdilution, disk diffusion, and
Etest methods as applied to Staphylococcus aureus. J. Clin. Microbiol.
48, 972−976.
University of Illinois at Urbana−Champaign, Urbana, Illinois
61801, United States
Kailey E. Komnick − Department of Chemistry, University of
Illinois at Urbana−Champaign, Urbana, Illinois 61801,
United States; Carl R. Woese Institute for Genomic Biology,
University of Illinois at Urbana−Champaign, Urbana, Illinois
61801, United States
Complete contact information is available at:
https://pubs.acs.org/10.1021/acsinfecdis.0c00869
Notes
(9) Cempra. Cempra’s Fusidic Acid Achieves Primary Endpoint in
Phase 3 Study of ABSSSI, http://ir.melinta.com/static-files/d621fa2f-
d0d4-4fc3-833f-a64286cfb7ed (accessed November 11, 2020).
(10) Melinta Therapeutics. Cempra, Inc. Receives U.S. Orphan
Drug Designation for Taksta(TM) for Treatment of Prosthetic Joint
Infections, http://ir.melinta.com/news-releases/news-release-details/
cempra-inc-receives-us-orphan-drug-designation-takstatm (accessed
November 11, 2020).
The authors declare the following competing financial
interest(s): The University of Illinois has filed patents on
some of the compounds described in this manuscript.
ACKNOWLEDGMENTS
■
This work was supported by the NIH (AI136773) and the
University of Illinois at UrbanaChampaign. We thank L. Li
(Metabolomics Center, Roy J. Carver Biotechnology Center,
UIUC) for LC−MS/MS analysis and Professor Levent
Dirikolu (School of Veterinary Medicine, Louisiana State
University) for the help with PK parameter calculations.
M.G.C. and A.G. are members of the NIH Chemistry-Biology
Interface Training Grant (T32-GM136629). M.G.C. is also
supported by the NSF Graduate Research Fellowship (NSF-
GRFP). We are also grateful for the support services of the
NMR and mass spectrometry facilities of the University of
Illinois at Urbana−Champaign.
̈
(11) Nagaev, I., Bjorkman, J., Andersson, D. I., and Hughes, D.
(2001) Biological cost and compensatory evolution in fusidic acid-
resistant Staphylococcus aureus. Mol. Microbiol. 40, 433−439.
(12) Silver, L. L. (2017) The Antibiotic Future. Top. Med. Chem. 25,
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(13) Silver, L. L. (2016) Appropriate Targets for Antibacterial
Drugs. Cold Spring Harbor Perspect. Med. 6, a030239.
(14) Tomlinson, J. H., Kalverda, A. P., and Calabrese, A. N. (2020)
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