Inhibition of Isoleucyl-tRNA Synthetase by the Hybrid Antibiotic Thiomarinol

Rachel A. Johnson, Andrew N. Chan, Ryan D. Ward, Caylie A. McGlade, Breanne M. Hat ﬁ eld,

Article

Inhibition of Isoleucyl-tRNA Synthetase by the Hybrid Antibiotic

Thiomarinol

Jason M. Peters, and Bo Li*

Cite This: J. Am. Chem. Soc. 2021, 143, 12003 −12013

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sı Supporting Information

ABSTRACT: Hybrid antibiotics are an emerging antimicrobial strategy

to overcome antibiotic resistance. The natural product thiomarinol A is a

hybrid of two antibiotics: holothin, a dithiolopyrrolone (DTP), and

marinolic acid, a close analogue of the drug mupirocin that is used to

treat methicillin-resistant Staphylococcus aureus (MRSA). DTPs disrupt

metal homeostasis by chelating metal ions in cells, whereas mupirocin

targets the essential enzyme isoleucyl-tRNA synthetase (IleRS).

Thiomarinol A is over 100-fold more potent than mupirocin against

mupirocin-sensitive MRSA; however, its mode of action has been

unknown. We show that thiomarinol A targets IleRS. A knockdown of

the IleRS-encoding gene, ileS, exhibited sensitivity to a synthetic

analogue of thiomarinol A in a chemical genomics screen. Thiomarinol

A inhibits MRSA IleRS with a picomolar K and binds to IleRS with low

femtomolar aﬃnity, 1600 times more tightly than mupirocin. We ﬁnd that thiomarinol A remains eﬀective against high-level

mupirocin-resistant MRSA and provide evidence to support a dual mode of action for thiomarinol A that may include both IleRS

inhibition and metal chelation. We demonstrate that MRSA develops resistance to thiomarinol A to a substantially lesser degree than

mupirocin and the potent activity of thiomarinol A requires hybridity between DTP and mupirocin. Our ﬁndings identify a mode of

action of a natural hybrid antibiotic and demonstrate the potential of hybrid antibiotics to combat antibiotic resistance.

INTRODUCTION

example shows the promise of repurposing existing antibiotics

through fusion to an additional chemical scaﬀold to broaden

the antimicrobial spectrum and evade existing antibiotic

■

A pressing global health concern of the 21st century is the

escalating problem of antimicrobial resistance and insuﬃcient

development of the antimicrobial pipeline. Natural products

from plants and microorganisms harbor unique chemical

structures and can exhibit potent antimicrobial activities. In

fact, the majority of the antibiotics in clinical use are natural

resistance mechanisms.

A few hybrid antibiotics have been isolated from natural

sources: the simocyclinones, everninomicin P, and the

thiomarinols (Figure S1). The simocyclinones consist of an

aminocoumarin linked to an angucycline (Figure S1) and are

over 80 times more active than derivatives lacking either

products or derivatives thereof; yet, the discovery of new

antimicrobial classes from both natural and synthetic sources

has dwindled drastically since the 1960s. Combination therapy

of antibiotics or of an antibiotic and an adjuvant can reduce or

overcome antibiotic resistance, albeit formulation and

administration of the combinations can require extensive

component. Unlike standalone aminocoumarins that act as

competitive inhibitors for ATP in the GyrB subunit of DNA

gyrase, simocyclinone prevents DNA binding through

interactions with the GyrA subunit in two distinct pockets.

This example reveals that the mechanism of a hybrid antibiotic

may diﬀer from its constituents. A coumarin derivative is also

found in the hybrid natural product, isocoumarindole A, which

exhibits mild antifungal activity, but its mode of action is

optimization. An alternative to combination treatment is

covalently linking antibiotics to create a hybrid or bifunctional

antibiotic; these compounds consist of two conjugated

antibiotics (to achieve dual targeting) or an antibiotic linked

to an adjuvant (to improve drug uptake or activity). The ﬁrst

bifunctional antibiotic, ceﬁderocol, was approved for clinical

use by the FDA in 2019 (Figure S1). As a synthetic

Received: March 9, 2021

Published: August 3, 2021

siderophore-β-lactam conjugate, the drug uses a “Trojan horse”

mechanism, mimicking a siderophore to exploit the side-

rophore transport systems of Gram-negative bacteria for

eﬃcient uptake of the β-lactam antibiotic hybrid. This

2021 American Chemical Society

J. Am. Chem. Soc. 2021, 143, 12003−12013

2003

Article

Figure 1. Structures of the antibiotics discussed in the study. Thiomarinol A is a hybrid of a dithiolopyrrolone (DTP, red) and marinolic acid, a

pseudomonic acid derivative. Pseudomonic acids A, B, and C are components of the drug mupirocin. Pseudomonyl C holothinamide (PAC-holo)

is a synthetic analogue of thiomarinol A. Thiomarinol, holothin, holomycin, and thiolutin are all DTP natural products.

unknown. Everninomicin P is a recently discovered hybrid of

the octasaccharide antibiotic everninomicin F and a glycosy-

lated macrolide, rosamicin, linked through a unique nitrone

Staphylococcus aureus (MRSA), a World Health Organization

high-priority pathogen that is responsible for approximately

30% of deaths from antibiotic-resistant infections in the United

States. Isolated from Pseudomonas fluorescens NCIMB 10586

as a mixture of pseudomonic acids A, B, and C (PAA, PAB,

and PAC, respectively; Figure 1), mupirocin and PAA are often

referenced interchangeably since PAA is the major species

moiety (Figure S1). While it is unclear if everninomicin P is

naturally produced or generated under laboratory culture

conditions, the hybrid is eﬀective against Escherichia coli

mutants resistant to either macrolides or octasaccharides by

targeting the orthogonal octasccharide- or macrolide-binding

site on the 70S ribosome, emphasizing the ability of hybrid

antibiotics to overcome resistance.

The thiomarinols were ﬁrst identiﬁed from Pseudoalteromo-

22,23

(>90% w/w).

All pseudomonic acids contain a monic acid

moiety linked to an octanoic acid. PAA and PAC diﬀer in

structure at C-10 by either an epoxide or an alkene,

respectively, and are equally eﬀective against various S. aureus

nas sp. SANK 73390 (thiomarinol A−G, Figure S1 ) in

strains when tested individually. PAB contains an additional

2−14

993.

The major species, thiomarinol A, consists of a

hydroxyl at C-8 (R ) and is 16−32-fold less eﬀective than PAA

or PAC (minimal inhibitory concentrations (MICs) of 4 μM

vs 0.25 μM against S. aureus Newman). PAC is most similar

in structure to marinolic acid A, which also contains the C-10

dithiolopyrrolone (DTP), holothin, and a polyketide, marinolic

acid A (Figure 1). DTPs, including holothin, holomycin, and

thiolutin, are a class of broad-spectrum antimicrobial natural

products that contain a characteristic bicyclic ene-disulﬁde

moiety (Figure 1).

intracellular metal chelators upon cytoplasmic reduction.

alkene and lacks the hydroxyl at C-8.

15−17

DTPs exert a unique mode of action as

Mupirocin binds and inhibits the essential enzyme isoleucyl-

tRNA (tRNA) synthetase (IleRS), including prokaryotic

paralogues from both Gram-positive MRSA and Gram-

IleRS is a class I aminoacyl-

tRNA synthetase (aaRS) that activates L-Ile as an aminoacyl-

adenylate and couples the activated L-Ile with its cognate tRNA

Bacterial aaRSs exhibit signiﬁcant

structural diﬀerences compared to their human counterparts,

which enable selective antibiotic targeting; for example,

mupirocin is 8000-fold more selective for prokaryotic IleRS

Thus, prokaryotic aaRSs are

explored as antibiotic targets in drug discovery eﬀorts. In

fact, nine diﬀerent bacterial aaRSs are targeted by over a dozen

Reduced DTPs can sequester both labile and enzyme-bound

metal ions, particularly zinc, which leads to dysregulation of

metal homeostasis and inhibition of metalloenzymes respon-

sible for essential bacterial processes, such as respiration and

25−28

negative E. coli in vitro.

29,30

glycolysis. Thiolutin, the N-methyl analogue of holomycin,

for protein synthesis.

inhibits several Zn-dependent proteases including Rpn11 in

the 26S proteasome, a promising anticancer target. Inhibition

of these proteases was also observed for a panel of DTPs

including thiomarinol and its synthetic analogue pseudomonyl

C holothinamide (PAC-holo, Figure 1). Thus, intracellular

metal chelation represents a central mechanism for the

antimicrobial and anticancer activities of DTPs.

32,33

over mammalian IleRS.

29,34

natural products,

including the IleRS inhibitors SB-203207

The second component of thiomarinol A, marinolic acid A,

is a close analogue of the pseudomonic acids that compose the

antibiotic mupirocin (Figure 1). Mupirocin has been widely

used to treat topical infections caused by methicillin-resistant

and furanomycin (Figure S2); however, these compounds

suﬀer from low speciﬁcity and high toxicity. Mupirocin remains

the only aaRS inhibitor used to treat human infections, albeit

for topical use only due to low bioavailability. Crystal

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Journal of the American Chemical Society

Article

Table 1. Minimal Inhibitory Concentrations (MICs) of Selected Antibiotics against Mupirocin-Susceptible and -Resistant

MRSA Strains

minimal inhibitory concentration (μM)

MRSA strain

protein variant

mupirocin

thiomarinol A

holomycin

COL

MR COL

IleRS

0.25

100

0.002

0.08

0.04

0.50

0.02

0.01

2.5

5.0

IleRS_V₅₈₈_F_/_V₆₃₁_F

IleRS_F₅₈₇_V_/_V₆₃₁_F

IleRS_V₅₈₈_F

MupA

100

>8000

3.13

6.25

^H^LMR BAA-1556

thiomarinol-resistant COL

IleRS_G₅₉₃_D

IleRS_P₆₀₆_S

structures of mupirocin complexed to either MRSA or Thermus

thermophilus IleRS revealed that mupirocin occupies the

binding site of the isoleucyl-adenylate reaction intermedi-

tography coupled with high-resolution mass spectrometry

(Figures S5 −S7). Due to the instability of holothin, we

synthesized holomycin, the stable prototypical DTP with an

exocyclic N-acetyl group. We also generated a synthetic

analogue of thiomarinol: pseudomonyl C holothinamide

(PAC-holo), a hybrid of holothin and pseudomonic acid C

(PAC), the pseudomonic acid whose structure is most similar

5,36

ate.

Mupirocin is a slow, tight-binding inhibitor that forms

an initial enzyme−inhibitor complex with IleRS that converts

into a stable complex with much higher binding aﬃnity. The

inhibitory mechanism of mupirocin formed the basis for

rational design eﬀorts that yielded femtomolar inhibitors of

IleRS by linking L-Ile and the monate core of mupirocin via a

to marinolic acid A (Figure 1, Figures S8 −S11). Compared

with thiomarinol, PAC-holo contains an extra methylene in the

sulfamate to mimic the isoleucyl-adenylate intermediate.

linker region and lacks a hydroxyl group at C-4 (R group).

Rising clinical resistance to mupirocin is observed in MRSA

Both thiomarinol and PAC-holo display potent inhibitory

activities against hospital-associated methicillin-resistant S.

aureus (MRSA) COL with MICs of 0.002 μM, over 125-fold

more potent than mupirocin or PAC (MICs of 0.25 and 0.52

μM, respectively) and 1250 times more potent than holomycin

(MIC of 2.5 μM, Table 1). We also quantiﬁed the

cytotoxicities of thiomarinol and holomycin against the

HEK293T human cell line (EC₅₀of 3.0 ± 0.3 μM, Figure

S12 ). The cytotoxic concentration is 3 orders of magnitude

higher than the MIC for thiomarinol against MRSA.

strains, including both low-level ( MR) and high-level

mupirocin resistance ( MR), with minimal inhibitory

concentrations (MICs) in the range 16−512 μM and greater

37,38 LL

than 1024 μM, respectively.

MR most commonly arises

from spontaneous mutations in the mupirocin binding site of

7,38

the native IleRS,

whereas

MR results from the

acquisition of a plasmid containing a mupirocin-resistant,

eukaryotic-like IleRS variant, MupA or MupB (Figure

9−41

S3 ).

Although less frequently observed, the

MRSA isolates are of critical concern medicinally because

We performed a chemical genomics screen comparing the

eﬀects of PAC-holo, mupirocin, and holomycin on essential

cellular processes (Figure 2, Figure S13 , and Data Sets 1 and

they render mupirocin treatments ineﬀective.

The hybrid antibiotic thiomarinol A has been reported to be

,48

signiﬁcantly more potent than mupirocin against MRSA in

2 ). Since antibiotics generally target essential processes,

2−14

vitro.

Thiomarinol A also exhibits a broader antimicrobial

used a CRISPR interference (CRISPRi) knockdown library

spectrum that includes multiple Gram-negative bacteria, such

that targets 94% (242/257) of essential genes in Bacillus subtilis

as E. coli and ESKAPE pathogens Pseudomonas aeruginosa and

168, a model bacterium for Gram-positive bacteria. Of the

Enterobacter spp., against which mupirocin is ineﬀective. In

257 essential B. subtilis genes, 91% have orthologues within S.

addition, preliminary studies indicate that thiomarinol can

aureus and 77% are also essential in S. aureus (Figure S14 );

inhibit MRSA expressing the MR IleRS variant, MupA.

thus, hits from the B. subtilis chemical genomics screen can be

further explored in MRSA. The B. subtilis library contains

strains with chromosomally integrated CRISPRi machinery

that reduces target gene expression by ∼3-fold; the reduced

expression does not substantially perturb bacterial growth, yet

it leads to partial depletion of essential gene products to allow

The potent and broad-spectrum activity of thiomarinol against

antibiotic-resistant pathogens showcases the therapeutic

potential of this hybrid antibiotic. Despite the discovery of

the thiomarinols almost three decades ago, the mode of action

had not been elucidated. We aimed to investigate the mode of

action of thiomarinol and discern the contribution of each

constituent to the eﬃcacy of the hybrid antibiotic.

identiﬁcation of chemical−gene interactions. Using PAC-

holo, mupirocin, holomycin, thiolutin, and gliotoxin, a fungal

toxin that also contains a redox-active disulﬁde, we screened

the B. subtilis CRISPRi library along with 31 other growth

perturbing chemicals that have published chemical−gene

RESULTS

■

To identify the mode of action of the hybrid antibiotic, we set

out to investigate thiomarinol activity in comparison to

mupirocin and holomycin, respective analogues of the

marinolic acid and holothin constituents of thiomarinol. We

extracted and puriﬁed thiomarinol A (Figure 1, hereafter

thiomarinol) from Pseudoalteromonas luteoviolacea 2ta16 with

interactions for a total of 36 unique chemical conditions at

variable concentrations.

To quantify chemical−gene interactions, the colony sizes of

a knockdown strain across all conditions were analyzed by use

of a variance bound t-test to generate a chemical−gene score

44,45

slight modiﬁcations to previous methods.

The thiomarinol

(Data Set 1). For a global analysis of each chemical−gene

interaction, we used the median chemical−gene score from a

range of concentrations for the same compound (Figure 2 and

Data Set 2). Consistent with IleRS as the direct target of

gene cluster in P. luteoviolacea 2ta16 shares 81% sequence

identity with the original producer, Pseudoalteromonas sp.

SANK 73390 (Figure S4). The structure and purity of

thiomarinol were characterized by NMR and liquid chroma-

25−28

mupirocin,

we found that ileS was the most sensitized

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Figure 2. Hybrid antibiotic PAC-holo targets IleRS in B. subtilis and exerts distinctive eﬀect from mupirocin. The knockdown of ileS is most

sensitized in the library to treatment with (A) mupirocin or (B) PAC-holo. Genes in the top and bottom 2.5% outliers of the chemical−gene score

(

y-axis) are labeled with their gene name (ileS in red, genes involved in peptidoglycan biosynthesis in blue, and genes involved in cell shape

regulation in yellow). Gene order (x-axis) corresponds to the order the genes appear in the genome of B. subtilis.

knockdown under mupirocin treatment (i.e., most negative

chemical−gene score) (Figure 2A). We also identiﬁed ileS as

the most sensitized knockdown under PAC-holo treatment,

indicating that the hybrid antibiotic retains activity against

IleRS (Figure 2B). However, other outlier strains diﬀered

between mupirocin and PAC-holo treatments (Figure 2),

suggesting that the hybrid PAC-holo has distinctive eﬀects on

essential processes compared to its constituent, mupirocin. In

contrast, the ileS knockdown was not sensitized to treatment

with holomycin, thiolutin, or gliotoxin (Figure S13), hinting

that these disulﬁde natural products do not target IleRS in

cells.

We also conducted gene ontology (GO) analysis to identify

cellular pathways enriched in the outlier strains of knockdowns

for each antibiotic. B. subtilis knockdowns hypersensitive to

PAC-holo are enriched in genes involved in peptidoglycan

synthesis and cell shape (Figure 2B, Data Set 3). While the ileS

knockdown is the predominant hypersensitive outlier for

mupirocin, resistant outliers also include valanyl-tRNA

synthetase (valS), aspartyl-tRNA synthetase (aspS), and

subunits of phenylalanyl-tRNA synthetase (pheT and pheS);

knockdowns of these genes might mitigate the eﬀect of

mupirocin by slowing translation. Lastly, the knockdowns

hypersensitive to holomycin, thiolutin, and gliotoxin are

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Figure 3. Chemical−gene interactions of PAC-holo correlate with mupirocin. A heat map of the Pearson correlation coeﬃcients (r) between

chemical treatments of the CRISPRi library in B. subtilis is shown. Higher correlation (r) of chemical−gene scores between antibiotics indicates

more similar chemical−gene interactions. The range of r values is depicted with a color scale from yellow (high) to indigo (low). Self-correlations,

which always have an r of 1, are shown in white, while an r of 0 denotes no correlation. Dendrograms at the top and left side of the ﬁgure show the

relationship between compounds, with shorter branches indicating more similarity in chemical−gene interactions. Compounds investigated in this

study are in bold.

enriched in metabolic genes, including those involved in

sulfamonomethoxine and trimethoprim (r = 0.53), and the

menaquinone biosynthesis (menA) and cytochrome biogenesis

cell wall biosynthesis inhibitors D-cycloserine and fosfomycin

(

resC).

(r = 0.41). Holomycin, thiolutin, and gliotoxin cluster

together and exhibit the strongest correlations of all conditions

in the analysis (e.g., r = 0.75 for holomycin and thiolutin),

suggesting a similar mode of action for these compounds.

PAC-holo clusters with mupirocin but not with holomycin or

thiolutin (Figure 3), indicating that the action of PAC-holo is

more similar to mupirocin than to either holomycin or

thiolutin under our screening conditions; however, the

correlation between PAC-holo and mupirocin is relatively

modest (r = 0.37), suggesting that their modes of action do not

overlap completely.

To identify broad patterns in chemical−gene interactions,

we correlated chemical−gene scores across conditions and

clustered the resulting matrix to group compounds that likely

perturb similar essential processes (Figure 3, Data Set 3 ).

Structurally related antibiotics cluster in this analysis, including

the quinolones ciproﬂoxacin and nalidixic acid that inhibit

DNA gyrase (r = 0.62) and the β-lactams aztreonam and

cefoxitin that inhibit penicillin-binding proteins in cell wall

biosynthesis (r = 0.62). Antibiotics of diﬀerent structural

classes that target diﬀerent enzymes in the same pathway also

cluster, such as the fatty acid synthesis inhibitors cerulenin and

triclosan (r = 0.27), the folate synthesis inhibitors

With these ﬁndings, we set out to examine IleRS as a target

for PAC-holo and the natural product thiomarinol in

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Figure 4. Thiomarinol inhibits MRSA IleRS. Dose−response curves for inhibition of 6.5 nM IleRS by (A) thiomarinol A and (B) mupirocin with

app

values of 19 ± 4 and 12 ± 2 nM, respectively. The relative adenylation activity of IleRS for L-Ile is plotted over antibiotic concentrations.

Curves depict the average of four replicates for thiomarinol A and three for mupirocin. Data were ﬁt by using the Morrison equation for tight-

binding, competitive inhibitors. The inhibition constants, K ’s, were estimated to be 370 ± 70 pM for thiomarinol A and 240 ± 20 pM for

mupirocin.

comparison to mupirocin using biochemical and biophysical

methods. To evaluate the potency of thiomarinol against IleRS,

we expressed recombinant IleRS from MRSA COL in E. coli

titration (Figure S18), and these data aligned well with the

reported value of −15 kcal/mol. Due to the low aqueous

solubility of thiomarinol, the reverse titration of IleRS into

thiomarinol was performed and a single exothermic binding

event was observed (Figure 5). We observed widened

diﬀerential power peaks during the initial additions of IleRS

into thiomarinol under reducing conditions (Figure S19); thus,

we opted to perform the titration of IleRS into thiomarinol

under nonreducing conditions to obtain a reliable ΔH°′

measurement for the binding interaction. Overall, thiomar-

inol−IleRS binding was less enthalpically favorable than

mupirocin binding with a ΔH°′ of −12.4 ± 0.4 kcal/mol

compared to −14.6 ± 0.4 kcal/mol (Figure 5, Figures S18 and

S19, Table S1). The binding isotherms of both mupirocin and

thiomarinol are step functions, indicating both antibiotics are

extremely tight binders of IleRS with dissociation constants

Figure S15 ) and measured the adenylation activity for L-Ile

under steady-state conditions using an ATP-[ P]-

pyrophosphate (PP ) exchange assay. We conducted an

active site titration of IleRS that yielded 0.65 ± 0.03 active

site per enzyme (Figure S16). Subsequently, we obtained an

−

adjusted turnover number (k ) of 12.0 ± 0.5 s and a

cat

Michaelis−Menten constant (K ) of 40 ± 5 μM (Figure S16),

which are comparable to values previously reported (k = 18

cat

−

2 s and K = 10 ± 2 μM). With active IleRS in hand, we

measured inhibition of IleRS catalysis by mupirocin,

thiomarinol, and holomycin under reducing conditions. Due

to the slow, tight-binding mechanism of mupirocin for IleRS,

we adopted the steady-state conditions by Pope et al. by

preincubating the compounds with IleRS before initiating

(K ’s) outside the reliable range for ITC determination.

ATP-[ P]PP exchange. The amino acid adenylation activity of

To quantify the binding aﬃnities of mupirocin and

thiomarinol with IleRS, we used circular dichroism (CD)

thermal melts as a complementary method. A change in the

IleRS was measured in the presence of increasing concen-

trations of each antibiotic at a saturating L-Ile concentration of

mM (Figure 4). Using the Morrison equation for tight-

melting temperature (T ) of IleRS in the presence of an

binding inhibitors, we determined the apparent inhibitory

antibiotic correlates to the stability of that protein−antibiotic

complex. A CD scan of IleRS from 270 to 190 nm reveals a

signal with local minima at ∼220 and 210 nm, representative of

app

constants (K_i) for mupirocin (12 ± 2 nM) and thiomarinol

(

19 ± 4 nM) against 6.5 nM IleRS. The K_i^a^p^pvalues of both

antibiotics approach the IleRS conentration which is character-

istic of tight-binding inhibitors. Holomycin did not inhibit 50

nM IleRS at concentrations up to 100 μM (Figure S17). Using

the K_Mvalues for L-Ile and [L-Ile], we calculated the

an α-helical secondary structure as described for IleRS; this

signal remains constant in the presence of diﬀerent antibiotics,

indicating that no detectable secondary structural changes

occur in IleRS upon antibiotic binding (Figure S20). Protein

ellipticity at 210 nm was measured as a function of temperature

to assess IleRS stability in the presence of various antibiotics

approximate inhibitory constants (K ) of mupirocin (240 ± 20

pM) and thiomarinol (370 ± 70 pM) for the competitive

inhibition mode. Our results indicate that thiomarinol is a

tight-binding inhibitor of IleRS with a potency similar to that

of mupirocin.

(Figure 5). The T of IleRS in buﬀer (49.0 ± 0.4 °C) agrees

well with the literature value of 52 °C and was not aﬀected

by the addition of holomycin (49.0 ± 0.4 °C), implying that

app

As K_ivalues can be an underestimate of the potency of

the DTP moiety alone does not bind IleRS. The T of IleRS

tight-binding inhibitors, we also quantiﬁed the binding

interaction between thiomarinol and IleRS in comparison to

mupirocin and holomycin. With the use of isothermal titration

calorimetry (ITC), mupirocin was titrated into IleRS at room

temperature (25 °C) and a 1:1 binding event was observed

with an exothermic heat signature of −14.6 ± 0.4 kcal/mol

signiﬁcantly increases in the presence of mupirocin, PAC-holo,

or thiomarinol (Figure 5, Table S2 ), supporting tight binding

of all three antibiotics to IleRS as an enzyme target.

We evaluated the stability of antibiotic−IleRS complex by

comparing the diﬀerences in the T of IleRS in the presence

and absence of antibiotics. The T ’s of IleRS in the presence of

(Figure S18 ). The reverse titration of IleRS into mupirocin

mupirocin and PAC-holo show no statistically signiﬁcant

yielded enthalpies (ΔH°′) similar to those of the forward

diﬀerence, 69.3 ± 0.2 and 68.3 ± 0.6 °C respectively (Figure 5,

2008

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Figure 5. Thiomarinol binds MRSA COL IleRS with high aﬃnity. (A) Representative ITC data of 250 μM IleRS titrated into 30 μM thiomarinol A

ThioA) at 25 °C. The titration curve depicts an exothermic 1:1 binding event. (B) Representative thermal shift assay of 2.5 μM IleRS in the

(

presence of 5 μM of each antibiotic. The CD signal, ellipticity (mdeg), of IleRS at 210 nm was measured as a function of temperature to determine

the melting temperatures (T ’s). (C) Table of IleRS T ’s in the presence of each antibiotic obtained from thermal shift assay, enthalpies of binding

at 25 °C, pH 7.4 (ΔH°′) obtained from ITC, and calculated dissociation constants (K_d_,₂₅_°_C).

Table S2). However, the T of IleRS binding to thiomarinol

ATCC BAA-1556 MRSA strain that contains a mupirocin-

was 79.9 ± 0.8 °C, 31 °C higher than that of IleRS alone and

resistant isoleucyl-tRNA synthetase, MupA, and conﬁrmed it

0 °C higher than that of IleRS in the presence of either PAC-

to exhibit high-level mupirocin resistance ( MR, MIC of

holo or mupirocin; the substantially higher T_msuggests

thiomarinol binds IleRS considerably tighter than all other

antibiotics tested (Figure 5, Table S2 ). Additionally, IleRS

maintained a similar T_min the presence of thiomarinol or

holomycin under reducing and nonreducing conditions,

implying the redox state of the DTP does not a ﬀ ect the

overall binding of thiomarinol or holomycin (Figure S21).

Combining the enthalpy of binding obtained from ITC and the

T_mof IleRS with and without ligands, we calculated the K_d

values of mupirocin and thiomarinol binding to IleRS using

derivations of the van’t Hoﬀ equation described by Brandts et

>8000 μM). The MICs of thiomarinol and holomycin were

measured against mupirocin-sensitive MRSA COL, MR

mutants of MRSA COL, and the MR strain BAA-1556.

Thiomarinol retains activity against both MR MRSA and

MR MRSA, although its MIC increases from 0.002 μM to

0.08 ( MR) and 0.5 μM ( MR) as the mupirocin resistance

of MRSA increases (Table 1). In contrast, the MICs of

holomycin remain constant at 2.5−5.0 μM against MRSA

COL and both MR and MR MRSA strains, supporting

that holomycin does not target IleRS.

Given that both DTP-containing molecules remain eﬀective

al. These calculations estimate that mupirocin binds IleRS at

8 ± 7 pM, while thiomarinol binds at 0.011 ± 0.006 pM,

against MupA-containing

MR MRSA, against which

mupirocin is inactive, we proposed thiomarinol may enact

additional modes of action against MRSA that are similar to

other DTPs. Thus, we measured the ability of thiomarinol to

chelate zinc ions. UV-vis analysis showed formation of a new

thiomarinol−zinc species at 373 nm (pH 7.4) under reducing

conditions (Figure S22), indicating that thiomarinol chelates

zinc in vitro. To assess the eﬀect of thiomarinol on metal

availability in bacterial cells, we obtained the MRSA Newman

mutant ΔadcAΔcntA that is defective in zinc import and

measured cell growth with and without zinc at subinhibitory

concentrations of thiomarinol and holomycin (Figure S23). As

reported by Grim et al., the ΔadcAΔcntA mutant grows slower

under zinc-deﬁcient conditions than wild type (WT) MRSA (p

approximately 1600-fold more tightly. Thermodynamic calcu-

lations reveal that binding of both mupirocin and thiomarinol

to IleRS is enthalpically driven; however, entropy contributes

signiﬁcantly to thiomarinol interactions with IleRS while the

entropy component of mupirocin−IleRS complexation is too

small to calculate (Table S1). Thus, thiomarinol forms an

exceptionally tight, inhibitory complex with IleRS that is driven

by both enthalpy and entropy and is estimated to bind 3 orders

of magnitude tighter than mupirocin.

We measured the eﬀectiveness of thiomarinol against both

mupirocin-sensitive MRSA and mupirocin-resistant MRSA in

culture. Mupirocin-sensitive MRSA COL (MIC of 0.5 μM)

was subjected to increasing concentrations of mupirocin to

< 0.01). In the absence of zinc, treatment of ΔadcAΔcntA

with either thiomarinol or holomycin leads to a signiﬁcant

growth defect in comparison to the antibiotic-free control (p <

0.001). The addition of zinc restores the growth of the

ΔadcAΔcntA strain in the presence of antibiotics to WT levels,

suggesting that both thiomarinol and holomycin aﬀect zinc

availability in S. aureus. These results support the additional

generate mutants that exhibit low-level mupirocin resistance

(

MR, MIC of 100 μM). Sequencing of ileS from these strains

reveals spontaneous point mutations near the active site of

IleRS: V588F/V631F, F587V/V631F, and V588F, among

which V588F/V631F mutations are the most commonly

observed MR genotype in clinical isolates. We obtained the

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Journal of the American Chemical Society

Article

mode of action of the hybrid antibiotic in reducing metal

availability in a Gram-positive pathogen.

We characterized inhibition of IleRS by thiomarinol using

biochemical and biophysical methods. Thiomarinol potently

inhibits IleRS with a K_iof 19 ± 4 nM under steady-state

app

Having shown that thiomarinol maintains the modes of

action of its constituents, we examined the interactions

between DTP and pseudomonic acids A or C (mupirocin or

PAC) in combination for potential synergistic eﬀects.

Interestingly, checkerboard assays against MRSA COL reveals

an additive relationship for the combination of mupirocin and

holomycin as well as that of PAC and holomycin (Figure S24).

A concentration of 0.25 μM PAC or mupirocin in combination

with 0.3 μM holomycin is required to fully inhibit growth of

MRSA COL; thus, both combinations are approximately 100-

fold less potent than the hybrids PAC-holo and thiomarinol

conditions, similar to that of 12 ± 2 nM for mupirocin.

Combining the data obtained from ITC and CD thermal melt

experiments allowed for the calculation of an estimated K_d_,₂₅_°_C

of 18 ± 7 pM for mupirocin binding to IleRS and an

exceptionally low K_d_,₂₅_°_Cof 11 ± 6 fM for thiomarinol binding

to IleRS, a 1600-fold higher aﬃnity than mupirocin. The much

app

lower K than K

value could be due to the nature of

bisubstrate inhibition as well as the sensitivity limit of the

inhibition assay. Mupirocin is a bisubstrate inhibitor of IleRS

and exhibits a competitive inhibitory mechanism with both

(

MICs of 0.002 μM). These data reveal that the covalent

substrates, L-Ile and ATP. We expect that thiomarinol

exhibits a mode of inhibition similar to that of mupirocin given

the structural resemblance between the two antibiotics;

therefore, the bisubstrate inhibition complicates the calculation

linkage to the DTP is required to signiﬁcantly potentiate the

antibiotic activity of mupirocin or PAC against MRSA.

We investigated the development of thiomarinol resistance

by culturing MRSA COL in the presence of increasing

concentrations of thiomarinol using the same protocol that was

used to generate MR mutants (Figure S25 ). These strains

are 5−10-fold more resistant to thiomarinol and 12.5−25-fold

more resistant to mupirocin than wild type MRSA COL

of the true inhibition constant of thiomarinol, which is likely

app

much lower than K_iand the estimated K for competition

with a single substrate L-Ile. Additionally, the low picomolar

sensitivity limit of the ATP-[ P]PP exchange inhibition assay

cannot capture femtomolar potencies. Due to the competitive

(Table 1). The increase in resistance is modest compared to

mechanism of inhibition, the K values we obtained using

the 400-fold increase in MIC to mupirocin in the resistant

calorimetry and stability shift assays more likely reﬂect the

actual potencies of mupirocin and thiomarinol, which are in

the low picomolar and femtomolar range.

mutants that were raised in the presence of mupirocin ( MR

MRSA) under the same conditions. We looked for

spontaneous mutations in IleRS in the thiomarinol-resistant

strains and identiﬁed a G593D mutation in two strains and a

P606S mutation in the third strain, which may contribute to

thiomarinol resistance. While a Gly593 to Val mutation was

Notably, the synthetic hybrid PAC-holo complexed with

IleRS displays a stability similar to that of mupirocin as

determined by CD thermal melt experiments while the DTP

holomycin does not inhibit or bind to IleRS. Together, the

data indicate that the DTP makes no signiﬁcant interactions

with IleRS by itself or when linked with PAC. The marinolic

acid A portion of thiomarinol A diﬀers from mupirocin and

PAC by alkyl chain length (n of 7, 8, and 8, respectively) and

37,38

reported in a MR MRSA mutant,

an IleRS mutation at

Pro606 had not been observed. Thiomarinol remains highly

eﬀective against the G593D and P606S IleRS mutants (MICs

of 0.02 and 0.01 μM).

an additional hydroxyl at C-4 (R ). These unique structural

DISCUSSION

features may play a role in enhancing interactions between

thiomarinol and IleRS at the active site. Although PAC-holo

exhibits a weaker in vitro interaction with IleRS than the

natural product thiomarinol, PAC-holo is equally eﬀective as

thiomarinol against MRSA in laboratory culture, hinting at the

therapeutic potential of this natural product derivative. In

summary, our results reveal that thiomarinol is one of the

tightest IleRS binders to date, on par with the femtomolar

binders obtained from an extensive medicinal chemistry

■

We identiﬁed IleRS as a target for thiomarinol and its synthetic

analogue, pseudomonyl C holothinamide (PAC-holo). Using a

chemical genomics screen of an essential gene knockdown

library in B. subtilis, we found that the ileS knockdown is the

most sensitized to PAC-holo and mupirocin among the 242

essential genes analyzed; PAC-holo most strongly correlates

with mupirocin among the 36 compounds tested. In agreement

with our chemical genomics ﬁndings, the thiomarinol

biosynthetic gene cluster contains an ileS homologue (tmlM)

that likely provides self-resistance for the producing

campaign. Our discovery highlights the potency and

therapeutic potential of hybrid natural products and the

derivatives thereof.

bacterium. In addition, the chemical genomics screen

indicates that the other DTPsholomycin and thiolutin

and gliotoxin do not target IleRS. These compounds exhibit

diﬀerent chemical−gene interactions from PAC-holo and

mupirocin: the ileS knockdown was insensitive, while knock-

downs in menA (menaquinone biosynthesis) and resC

We demonstrated that thiomarinol remains eﬀective against

clinical MRSA strains that have acquired low-level or high-level

mupirocin resistance ( MR or MR). Although the activity

of thiomarinol is reduced by 40- and 250-fold against MR

and MR MRSA, respectively, the potency of the hybrid is

(

cytochrome biogenesis) were hypersensitive. Further, the

still higher than or comparable to the activity of mupirocin

against sensitive MRSA. Compared to holomycin, which is

disulﬁde-containing natural products, holomycin, thiolutin, and

gliotoxin, exhibit the strongest correlations observed in this

screen, corroborating the results from our previous chemical

genomics screen that involved 3900 nonessential E. coli gene

knockouts and further suggesting these compounds exert a

similar mode of action. This mode of action may involve

inhibition of metabolic processes as suggested by the

enrichment of metabolic genes (e.g., menA and resC) in the

knockdowns hypersensitized to these disulﬁde-containing

antibiotics.

equally active against mupirocin-sensitive, MR, and MR

MRSA, thiomarinol is 3 orders of magnitude more active

against mupirocin-sensitive MRSA and 10-fold more active

against MR MRSA. Thus, the DTP portion of thiomarinol

likely plays a more signiﬁcant role in antimicrobial activity as

the level of mupirocin-resistance increases in MRSA. Like

holomycin and thiolutin, the DTP of thiomarinol may also

disrupt metal homeostasis in the bacterial cell, which may help

thiomarinol overcome high-level mupirocin resistance. In

2010

J. Am. Chem. Soc. 2021, 143, 12003−12013

Journal of the American Chemical Society

North Carolina 27599, United States; orcid.org/0000-

Article

support of the metal chelation mechanism, we demonstrate

that thiomarinol can chelate zinc in vitro and its inhibitory

eﬀect against MRSA involves the reduction of zinc availability.

Additionally, both thiomarinol and PAC-holo inhibit zinc

Materials, methods, and supplemental ﬁgures (PDF)

Chemical genomics data including B. subtilis CRISPRi

library information, chemical treatment conditions,

chemical−gene scores, and GO analysis (XLSX)

enzymes in vitro. These data indicate that the zinc chelation

properties of thiomarinol contribute to antibiotic activity. A

dual mechanism has been described for the hybrid natural

product everninomicin P that overcomes resistance to either of

its constituents: a macrolide and octasaccharide; both

AUTHOR INFORMATION

Corresponding Author

■

Bo Li − Department of Chemistry, University of North

Carolina at Chapel Hill, Chapel Hill, North Carolina 27599,

United States; Department of Microbiology and Immunology,

University of North Carolina at Chapel Hill, Chapel Hill,

components target the 70S ribosome and inhibit translation.

Our results suggest that the mupirocin and DTP constituents

of thiomarinol likely enact two very diﬀerent antimicrobial

mechanismstargeting translation by inhibiting IleRS and

causing metal dysregulation by chelating metals, respectively

to overcome antibiotic resistance.

002-8019-8891 ; Email: boli@email.unc.edu

Authors

We found that MRSA could develop spontaneous

thiomarinol resistance, but to a much lesser degree than

mupirocin resistance during the same number of serial

passages; the resulting MRSA mutants are only 5−10-fold

more resistant to thiomarinol and remain sensitive to

mupirocin. These data suggest that the hybridity of

thiomarinol helps reduce the rise of resistance. We showed

that the two antimicrobial components of thiomarinol and its

synthetic analogue, PAC-holo, are additive in combination and

must be covalently linked to signiﬁcantly increase eﬃcacy

against mupirocin-sensitive MRSA. Increased cellular accumu-

lation is a common mechanism to potentiate antibiotic eﬃcacy

that is used by the newly FDA-approved bifunctional

Rachel A. Johnson − Department of Chemistry, University of

North Carolina at Chapel Hill, Chapel Hill, North Carolina

7599, United States; orcid.org/0000-0003-1958-2218

Andrew N. Chan − Department of Chemistry, University of

North Carolina at Chapel Hill, Chapel Hill, North Carolina

27599, United States; orcid.org/0000-0002-4257-7436

Ryan D. Ward − Pharmaceutical Sciences Division, School of

Pharmacy, University of Wisconsin-Madison, Madison,

Wisconsin 53705, United States; Laboratory of Genetics,

University of Wisconsin-Madison, Madison, Wisconsin

53706, United States

Caylie A. McGlade − Department of Chemistry, University of

North Carolina at Chapel Hill, Chapel Hill, North Carolina

antibiotic, ceﬁderocol, which evades antibiotic resistance

through increased compound uptake. PAC-holo-hypersensitive

knockdowns are enriched with genes involved in peptidoglycan

synthesis and cell shape regulation; this observation hints at a

cell envelope target or increased cellular accumulation for

PAC-holo that could contribute to the synergy of the hybrid

27599, United States

Breanne M. Hatﬁeld − Department of Chemistry, University

of North Carolina at Chapel Hill, Chapel Hill, North

Carolina 27599, United States

Jason M. Peters − Pharmaceutical Sciences Division, School of

Pharmacy, University of Wisconsin-Madison, Madison,

Wisconsin 53705, United States; Great Lakes Bioenergy

Research Center, Wisconsin Energy Institute, University of

Wisconsin-Madison, Madison, Wisconsin 53726, United

States

9,60

molecule.

The mechanism by which hybridity potentiates

the activity will be the focus of future studies.

CONCLUSION

■

Our work demonstrates that the hybrid antibiotic thiomarinol

is a dual acting antibiotic: a potent inhibitor of IleRS and a

metal chelator that reduces intracellular zinc availability. By

conjugating an analogue of the IleRS-inhibiting antimicrobial

mupirocin with a metal-chelating antibiotic, holothin, thio-

marinol retains the ability to target IleRS but binds to IleRS

signiﬁcantly more tightly than mupirocin at femtomolar

aﬃnities. In comparison to mupirocin, thiomarinol exhibits

higher potency against MRSA in culture and remains active

against mupirocin-resistant MRSA at concentrations below

cytotoxic levels. Additionally, a lower level of resistance

develops in MRSA toward thiomarinol than toward mupirocin.

We also investigated PAC-holo, a highly eﬀective synthetic

hybrid and thiomarinol derivative; we show that for both

thiomarinol and PAC-holo covalent linkage between the

antibiotic constituents is required for the high potency. The

broad antimicrobial spectrum, low level of resistance, and

activity against mupirocin-resistant isolates render thiomarinol

a promising therapeutic lead and a potential model for hybrid

antibiotics to overcome antimicrobial resistance.

Complete contact information is available at:

https://pubs.acs.org/10.1021/jacs.1c02622

Author Contributions

R.A.J. and A.N.C.: These authors contributed equally to this

study

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

The authors thank Walter Wever for making PAC-holo;

Elissabetta Massolo, and Xiaoyan Chen (UNC Chapel Hill)

for synthesizing holomycin; Carol Gross (UCSF), Gary Pielak,

Charlie Carter, Tishan Williams, and Candice Crilly (UNC

Chapel Hill), and Thomas Meek (Texas A&M) for helpful

discussions; Joseph Turner (North Carolina State University)

for assistance with MATLAB coding; Thomas Kehl-Fie

(University of Illinois Urbana−Champaign) for MRSA New-

man strains; Gerry Wright (McMaster University) for access to

instrumentation for antibiotic testing in microplates; Ash

Tripathy (UNC Macromolecular Interactions Facility) for

assistance with ITC and CD thermal melt experiments; and

Rachel M. Johnson for manuscript review. This work is

supported by the National Institutes of Health

ASSOCIATED CONTENT

sı Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/jacs.1c02622.

■

2011

J. Am. Chem. Soc. 2021, 143, 12003−12013

Journal of the American Chemical Society

DP2HD094657 to B.L.), the Packard Fellowship for Science

and Engineering (B.L.), a Career Transition Award from the

NIH National Institute of Allergy and Infectious Diseases

15) Li, B.; Wever, W. J.; Walsh, C. T.; Bowers, A. A.

Article

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K22AI137122 to J.M.P.), and UNC Chapel Hill. R.D.W. was

supported by the Predoctoral Training Program in Genetics

5T32GM007133-46). R.A.J. and B.M.H. acknowledge sup-

(

05−23.

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16) Ettlinger, L.; Gäumann, E.; Hutter, R.; Keller-Schierlein, W.;

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