Platform to Discover Protease-Activated Antibiotics and Application to Siderophore-Antibiotic Conjugates

Article abstract of DOI:10.1021/jacs.0c06987

Here we present a platform for discovery of protease-activated prodrugs and apply it to antibiotics that target Gram-negative bacteria. Because cleavable linkers for prodrugs had not been developed for bacterial proteases, we used substrate phage to discover substrates for proteases found in the bacterial periplasm. Rather than focusing on a single protease, we used a periplasmic extract of E. coli to find sequences with the greatest susceptibility to the endogenous mixture of periplasmic proteases. Using a fluorescence assay, candidate sequences were evaluated to identify substrates that release native amine-containing payloads. We next designed conjugates consisting of (1) an N-terminal siderophore to facilitate uptake, (2) a protease-cleavable linker, and (3) an amine-containing antibiotic. Using this strategy, we converted daptomycin - which by itself is active only against Gram-positive bacteria - into an antibiotic capable of targeting Gram-negative Acinetobacter species. We similarly demonstrated siderophore-facilitated delivery of oxazolidinone and macrolide antibiotics into a number of Gram-negative species. These results illustrate this platform's utility for development of protease-activated prodrugs, including Trojan horse antibiotics.

Full text of DOI:10.1021/jacs.0c06987

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Platform to Discover Protease-Activated Antibiotics and Application
to Siderophore−Antibiotic Conjugates
Jonathan H. Boyce, Bobo Dang,* Beatrice Ary, Quinn Edmondson, Charles S. Craik,
William F. DeGrado,* and Ian B. Seiple*
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ABSTRACT: Here we present a platform for discovery of
protease-activated prodrugs and apply it to antibiotics that target
Gram-negative bacteria. Because cleavable linkers for prodrugs had
not been developed for bacterial proteases, we used substrate
phage to discover substrates for proteases found in the bacterial
periplasm. Rather than focusing on a single protease, we used a
periplasmic extract of E. coli to find sequences with the greatest
susceptibility to the endogenous mixture of periplasmic proteases.
Using a fluorescence assay, candidate sequences were evaluated to
identify substrates that release native amine-containing payloads.
We next designed conjugates consisting of (1) an N-terminal
siderophore to facilitate uptake, (2) a protease-cleavable linker, and (3) an amine-containing antibiotic. Using this strategy, we
converted daptomycinwhich by itself is active only against Gram-positive bacteriainto an antibiotic capable of targeting Gram-
negative Acinetobacter species. We similarly demonstrated siderophore-facilitated delivery of oxazolidinone and macrolide antibiotics
into a number of Gram-negative species. These results illustrate this platform’s utility for development of protease-activated
prodrugs, including Trojan horse antibiotics.
bacteria.13 Owing to the promiscuity of their transport systems,
INTRODUCTION
The well-recognized term, “ESKAPEE” (previously ESKAPE),1
■
bacteria also use siderophores in warfare against other
microbes.12,14 For example, streptomycetes produce albomy-
encompasses the names of seven species of clinically relevant
cins, which are natural siderophore−antibiotic conjugates
pathogens (Enterococcus faecium, Staphylococcus aureus, Kleb-
(SACs) and highly effective antibiotics against Gram-negative
siella pneumoniae, Acinetobacter baumannii, Pseudomonas
Enterobacteriaceae.15 Albomycins are recognized by side-
aeruginosa, Enterobacter spp., and Escherichia coli) that are
rophore uptake machinery, transported into the cytoplasm,
associated with resistance to commonly prescribed antibiotics
and activated by peptidase N, which cleaves the N-terminal
and are largely responsible for the world’s nosocomial
serine-amide bond and releases the serine-bound t-RNA
infections.2 Five of these pathogens are Gram-negative species,
synthetase inhibitor to bind to its target.16−19 Inspired by
whose outer membrane and associated resistance−nodula-
Nature’s strategy, we develop an unbiased platform for the
tion−cell division (RND) efflux pumps render them resistant
discovery of linkers that are cleaved by periplasmic proteases,20
to many classes of antibiotics.3 Indeed, the outer membrane
which demonstrates that this platform can produce SACs with
shields the bacteria from molecules that are unable to pass
both broad and narrow spectra of activity. We target proteases
through porins,4 providing an effective barrier to many
in the periplasm, a compartment that contains >20 known
molecules that would otherwise be effective antibiotics against
proteases, because all SACs pass through the periplasm.
these pathogens.5 Gram-negative therapies can be delivered by
There are two categories of SACs, depending on the type of
siderophore-mediated antibiotic delivery using Nature’s
linker they possess: non-cleavable and cleavable. There has
Trojan-horse approach.6−8 Siderophores are small-molecule
been significant progress in the development of non-cleavable
chelators that are produced by bacteria to sequester Fe(III),9
which is an essential nutrient required for bacterial growth and
virulence.10 In the case of Gram-negative pathogens, outer
Received: June 29, 2020
membrane proteins (e.g., TonB-dependent transporters)11
bind ferric siderophores and provide opportunities for their
active transport into the periplasm,12 where they may undergo
further translocation into the cytoplasm by alternate transport
mechanisms depending on the siderophore and the species of
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Figure 1. (A) Selected cleavable linkers that have previously been used for SACs. (B) Concept for SACs that contain a linker that can be cleaved by
periplasmic proteases. (C) Workflow for the development of protease-cleavable SACs.
SACs,21,22b,23 with the first siderophore−β-lactam conjugate
Fetroja (cefiderocol) recently approved by the FDA.24
However, their use is often limited to periplasmic-targeting
antibiotics (e.g., daptomycin, vancomycin, and β-lactams). The
few examples of cytoplasmic-targeting, non-cleavable SACs
may be less effective than the parent antibiotic for two
reasons:25,26b−d,27−34 (1) the conjugate may not pass through
the inner membrane to reach the cytoplasm, or (2) the bulky
siderophore component may interfere with binding to the
target.12,25b,35 Therefore, cleavable linkers are traditionally
thought to be required for SAC compatibility with
cytoplasmic-targeting antibiotics.21 The majority of Gram-
positive antibiotics are cytoplasmic-targeting and may require a
cleavable linker to be converted into SACs for Gram-negative
pathogens.12,21,36 We show that protease-cleavable linkers
improve the activity of SACs containing periplasmic- and
cytoplasmic-targeting antibiotics.
ciprofloxacin or norfloxacin,40b,41 which are already active
against Gram-negative pathogens. These SACs provide a
method to study siderophore-mediated antibiotic delivery
because release of the antibiotic from the conjugate is often
required to observe significant growth inhibition.21,25,26,35,37,38
In line with this trend, we investigate a macrolide that is
already active in Gram-negative bacteria as one strategy to
interrogate SAC cleavage.
Protease-cleavable SACs are classified as prodrugs.42,43
Protease-activated prodrugs that are cleaved by mammalian
proteases include antibody−drug conjugates,44−50 antibody−
antibiotic conjugates,51,52 peptide−drug conjugates,53 macro-
molecular prodrugs,42,54 and protease-activatable photosensi-
tizers,55,56 with the cathepsin B-sensitive valyl-citrulline (Val-
Cit) linker being the most successful and widely known.47,49
Although most protease-activated prodrugs target cancer,
antibody−antibiotic conjugates are undergoing clinical trials
for intracellular bacterial infections associated with difficult-to-
treat persisters.51,52 However, antibody−antibiotic conjugates
have been limited to the treatment of intracellular S. aureus,
using a linker that was optimized for cleavage by mammalian
proteases. Protease-activated prodrugs that are activated by
bacterial proteases would clearly be of value for treatment of a
variety of Gram-negative bacterial infections.
Several technologies have been developed to screen libraries
of protease substrates,57 including indexed arrays of fluoro-
genic substrates,58,59 positional scanning of synthetic combi-
natorial libraries,60,61 substrate phage,62,63 multiplex substrate
profiling by mass spectrometry,57,64−66 and others.58,67−71
Given the large size of achievable libraries, substrate phage
display62 provides an unbiased selection tool to discover
cleavable linkers for SACs. Conventional phage display has
been used in vitro and in vivo to design targeted-peptide
conjugates.72 For example, Wilfred van der Donk used phage
display to select for ribosomally synthesized and post-
translationally modified peptides (RiPPs) that bind to lipid
II.72g However, “substrate phage display” has not been applied
Over the past 30 years, only a few cleavable linker strategies
have been developed for SACs and a number of challenges
remain.22,25−27,35,21,37−39 Despite optimization for hydrolytic
stability,35 ester linkers for SACs (e.g., A, Figure 1A) are
susceptible to premature cleavage prior to bacterial-cell
entry.25,26c,d,27,35,39 SACs with disulfide (e.g., B) and
trimethyl-lock linkers based on reduction- (e.g., C),
phosphatase (e.g., D), and esterase-triggered cleavage mech-
anisms were less active than the parent antibiotic.26,38 Recent
work by Nolan demonstrated that cytoplasmic siderophore
degradation by the siderophore-hydrolase IroD confers high
activity to a conjugate with a non-cleavable linker.37 Miller and
co-workers developed a dual drug conjugate with the cleavable
β-lactam linker E.22a However, more alternatives are needed as
the physiological instability of β-lactams can lead to hyper-
sensitivity and allergic reactions in patients.40a Protease-
cleavable linkers have the potential to overcome the limitations
of cleavable SACs reported to date.
With few exceptions,22a,27,39 cleavable SACs have incorpo-
rated a DNA-gyrase-inhibiting fluoroquinolone antibiotic,
B
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to prodrug development,63 and its use for profiling complex
biological mixtures is limited.73 Here, we extend substrate
phage display to identify cleavable peptide linkers for SACs
that are activated by periplasmic proteases.
In designing protease-activated prodrugs, there are advan-
tages to targeting multiple proteases over an individual
protease.74−76 For example, in vivo deletion mutants of
cathepsin B retain the ability to release prodrugs from the
combined activity of several proteases.77,78 Indeed, targeting
multiple proteases might minimize resistance when designing
antibiotic prodrugs. Thus, we screened broadly for peptides
that are cleaved by the proteases present in an unfractionated
periplasmic extract.
C-terminus to mimic the GGS spacers in the phage library. A
tryptophan was added to the N-terminus to facilitate detection
by HPLC. Each peptide was found to be cleaved to varying
extents following treatment with periplasmic extract for 18 h at
37 °C. The cleavage sites and extent of proteolysis were
evaluated by LC/MS (Table S5), which revealed that the
sequences WSWC-KWASG (dash indicates site of cleavage)
and WSPKYM-RFG may be optimal linkers for cleavable
SACs.82 In addition to their promising cleavage profiles
described in Table S5, the presence of these sequences in the
original library contributed to their selection as potential linker
candidates. In protease substrate nomenclature, the Cys and
Met residues at the N-terminal side of the scissile bond for
WSWC-KWASG and WSPKYM-RFG are designated as the P1
positions, while the Lys and Arg residues at the C-terminal side
are designated as the P1′ positions. Although the residues on
the P′ side are sometimes important for efficient cleavage,83
this is not always the case.84
RESULTS
■
Substrate Phage Display Leads to WSPKYM-RFG and
WSWC-KWASG as Substrates for Periplasmic Cleavage.
To discover efficient peptide substrates using the method of
substrate phage,62 we built a random hexapeptide library
genetically fused to the pIII gene of M13 bacteriophage. A
phagemid vector allows monovalent display of the correspond-
ing protein on the tip of the phage. A GGS spacer was
incorporated at each end of the randomized peptide to
enhance flexibility. An AviTag sequence was also incorporated
at the N-terminus for biotinylation of the displayed peptides.
The biotin is used to immobilize the phage library on a
streptavidin-coated surface, and a protease can then cleave at
favorable peptide sequences. Proteolysis releases the phage,
which are then amplified and sequenced to determine the
favorable substrates for the protease of interest.
WSPKYM Conjugates Are Efficiently Cleaved without
a P′ Peptide. With the candidate sequences WSWC-KWASG
and WSPKYM-RFG in-hand, we asked whether the residues
on the C-terminal P′ sequence were required for proteolysis
(Figure 2A). To probe this question, we used a solid-phase
The process of “biopanning” entails the following steps: (1)
enzymatic biotinylation of the AviTag sequence,79,80 (2)
immobilization of the biotinylated library on streptavidin 96-
well plates, (3) cleavage of the immobilized library by
incubation with the periplasmic extract of E. coli K12
MG1655 at 37 °C, (4) amplification of the eluted phage
using E. coli TG-1 cells, and (5) isolation and purification of
phage for the next round of selection. The periplasmic extract
used in panning was obtained by osmotic shock of E. coli K12
MG1655.81 We carried out four rounds of selection (see
Supporting Information, section VB, page S79), with the
stringency being increased with each succeeding round by
reducing extract concentration and decreasing the incubation
time. The phagemids from the input library and final round of
biopanning were isolated, barcoded, and submitted for next-
generation sequencing.82 Sequences for further character-
ization were ranked based on the extent of enrichment relative
to the original library (Table 1).
Figure 2. (A) Peptides with turn-on fluorophore, acc, as an antibiotic
surrogate. (B) Evaluation of acc cleavage from peptides 1 and 2 in a
periplasmic extract of E. coli K12 MG1655 (400 μg/mL) at 37 °C.
method to synthesize fluorescent substrates. A 7-amino-4-
carbamoylmethylcoumarin (acc)85,86 was coupled directly to
the P1 Cys and Met residues as an antibiotic surrogate (see
Supporting Information, section IIIF, pp S67−S72). We were
indeed pleased to find that the treatment of peptide 1 (25 μM)
with periplasmic extract led to rapid release of the fluorescent
amino coumarin. HPLC analysis also showed the substrate was
fully consumed at the end of the reaction (Figure S9). As
expected, the rate of cleavage was dependent on the substrate
concentration, showing partial saturation at concentrations
over 12.5 μM (Figures S4 and S5). However, we did not
attempt to fit a value of KM, given the heterogeneous nature of
the proteolytic composition of the extract. On the other hand,
the amount of acc released from peptide 2 (WSWC-coumarin)
was comparably insignificant under these conditions (Figure
2B). Thus, WSPKYM was determined to be more suitable than
WSWC for the development of cleavable SACs.
Six highly enriched sequences (SKNQSLGG, SGSDSSVG,
SNHADVHG, SKSEMLSG, SWCKWASG, and SPKYMRFG)
were synthesized with a Ser at the N-terminus and a Gly at the
Table 1. Highly Enriched Sequences Found through
Substrate Phage Display
sequence
reads
initial reads
enrichment factor
KNQSLG
GSDSSV
NHADVH
KSEMLS
WCKWAS
PKYMRF
10652
9239
8138
7742
15307
13192
0.5
0.5
0.5
0.5
1
21304
18478
16276
15484
15307
13192
1
C
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Table 2. Modular Synthetic Platform for SAC Synthesis
a
For detailed synthetic methods, see Supporting Information.
To determine the type of protease responsible for acc release
in a periplasmic extract of E. coli, we evaluated a variety of
protease inhibitors (Figure S6), which revealed that the
enzymes of interest include a metalloprotease or a calcium-
dependent protease. We also used alanine positional scanning
to evaluate the sequence dependence for efficient cleavage.
The starting peptide Ac-WSPKYM-acc (1) was the most
efficient substrate (Figure S7). Individual substitutions of Ala
at Tyr (P2), Trp (P6), and Lys (P3) resulted in large decreases
in rate, while substitutions at other positions resulted in an
approximately 2-fold decrease in rate. These findings indicate
that substrate phage was effective in discovering an optimized
substrate for cleavage.
We also examined the ability of human and mouse serum to
cleave 1. We were pleased to find that under conditions where
1 is rapidly cleaved by periplasmic extract, human serum
released acc with a half-life of approximately 3−4 h (Figure
S8). However, the compound is cleaved more rapidly by
mouse serum, indicating that some optimization would be
necessary for applications using mouse models.
Design and Synthesis of SACs That Incorporate
Solithromycin, Daptomycin, and Eperezolid-NH2. To
explore the versatility of SACs, we selected three structurally
and mechanistically diverse antibiotics that act on targets in
either the periplasm or the cytoplasm. Each antibiotic has an
amine, which can be unmasked upon proteolysis of the
WSPKYM linker. The lipopeptide daptomycin (4) interacts
with the cytoplasmic membrane in Gram-positive bacteria,
leading to increased membrane permeability and membrane
depolarization.87a−c However, it is ineffective against Gram-
negative bacteria and challenging to functionalize without loss
of potency.87d Nevertheless, the Miller group has shown that
daptomycin can gain activity in Gram-negative species if
conjugated to a siderophore with a non-cleavable linker.22b,23
Here, we examine the use of a protease-cleavable linker for this
system.
We also chose two ribosomal protein synthesis inhibitors,
amino-oxazolidinone 5 and solithromycin 6, as examples of
antibiotics that must gain access to the cytoplasm to be
active.22a Since both oxazolidinones and macrolides bind deep
within the large ribosomal subunit in fairly occluded binding
D
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sites, siderophore conjugates without cleavable linkers have
been met with limited success, potentially due to interference
of the linkers with binding.12,21,36,88,89 Our strategy would
avoid this complication by enabling release of the parent
antibiotics.
For attachment to the N-terminal side of the linker, we
sought a siderophore that was synthetically accessible and
compatible with a variety of bacterial siderophore uptake
systems. The bis-catecholate, azotochelin-like90 siderophore
siderophore conjugate became increasingly active at higher
concentrations of dipyridyl (Table S3A−C). This phenomen-
on can be explained by the enhanced expression of outer-
membrane transport proteins for siderophore uptake in iron-
deficient media.91e The absence of dipyridyl from the growth
medium dramatically attenuated siderophore−conjugate activ-
ities without influencing the MIC of the free antibiotic.82
These results correlate well with expected growth-inhibitory
activity of SACs.
̈
(Heinisch−Mollmann−Miller (HMM) siderophore, Table
We included 15 bacterial strains in our assay (14 Gram-
negative and one Gram-positive)94−98 and have highlighted
selected activities below (for full-activity tables and strain
details, see Table S1B). Two genetically modified strains of E.
coli were included: a ΔsurA strain that is deficient in outer-
membrane proteins and has increased permeability,95a and a
ΔbamBΔtolC mutant, which has a deficient BamACDE outer-
membrane-assembly complex and lacks the TolC-transport
protein.95a,b This strain is widely used because it is defective in
small-molecule efflux.
2)22,91a,b was selected due to its ease of synthesis and its
ability to carry large cargo (e.g., daptomycin) into A.
baumannii, E. coli, and P. aeruginosa.22,23 Furthermore, β-
lactam conjugates with non-cleavable linkers that used tetra-
acetate derivatives of siderophore 10a were active against
MDR strains capable of efflux.91c We used a modified version
of Miller’s protocol to access siderophore 10, which has acid-
labile ketal protecting groups that can be removed concom-
itantly with tert-butyl and tert- butoxycarbonyl (Boc)
protecting groups on the amino acid side chains.82
Solithromycin is active in many Gram-negative and Gram-
positive species, and the strategy of using a Gram-negative
antibiotic is frequently used to evaluate the efficiency of linker
cleavage for cleavable SACs (see Introduction).21,25,26,35,37,38
The L-linker solithromycin conjugate 9 was comparably active
to solithromycin in several pathogenic Gram-negative strains,
and the D-linker conjugate 16 was inactive (Table S1A,B). In E.
coli, however, conjugates 9 and 16 had similar activity,
suggesting that the D-linker conjugate should not be used to
evaluate linker cleavage in this species. We found that this may
be due to the ability of the entire conjugate to inhibit the 70S
E. coli ribosome (Figure S1A). However, the differences in
activity between the D- and the L-solithromycin conjugates in
pathogenic strains suggested that the linker may enable the
release of Gram-positive antibiotics (Table S1A and Figure
S1A). With this promising result in-hand, we then proceeded
to investigate Gram-positive-only antibiotics (vide infra).
Oxazolidinone Conjugate 8 Was Active against E. coli
ΔbamBΔtolC (Table 3). The oxazolidinone class of anti-
biotics are active against Gram-positive bacteria, but members
of this class lack activity against Gram-negative bacteria, due to
the presence of endogenous efflux pumps (Figure S1B).
Nevertheless, mutants of E. coli such as ΔbamBΔtolC are
susceptible to oxazolidinones because these bacterial strains
have disruptions in their efflux systems. This strain is
susceptible to eperezolid (Table S1B), but the corresponding
amine variant, eperezolid-NH2 (5), was inactive (MIC > 171
μM). This is likely a result of the inability of 5 to diffuse
through the outer and inner membranes as indicated by the
data in Table 3 (vide infra). We therefore asked whether
conjugate 8 could deliver 5 into a ΔbamBΔtolC mutant of E.
coli. We were pleased to discover an MIC of 1 μM for 8 in this
mutant; the corresponding derivative 14 with an all-D linker
showed strongly decreased activity with an MIC of 19 μM,
which is consistent with low activities of previously reported
oxazolidinone conjugates with non-cleavable linkers.39 In
contrast to eperezolid-NH2 (5), eperezolid conjugate 8
displayed only 10% inhibition in a cell-free translation assay
at a concentration of 38 μM (Figure S1A), indicating that the
intact conjugate does not inhibit the ribosome. These findings
suggest that potent inhibition of bacterial growth requires
enzymatic cleavage of the linker. Supporting this suggestion,
34% cleavage of 8 to the parent antibiotic eperezolid-NH2
occurred after 11 h of incubation with bacterial periplasmic
We developed a modular synthetic route that enables the
facile incorporation of a variety of linkers, antibiotics, and
siderophores (Table 2). Gram-scale linker assembly and
subsequent siderophore attachment were accomplished on
solid-phase to provide the partially protected intermediate 3 in
50% overall yield, and the antibiotic was then coupled to the
C-terminus in solution. Following acidolytic deprotection, the
final SACs (7−9) were obtained in 12−53% yield over two to
four steps.
Several aspects of our route merit further discussion. The
majority of the synthesis proceeds on solid phase, simplifying
purification and facilitating parallel synthesis of analogues.
Antibiotics are directly attached in the penultimate step,
enabling rapid access to the final antibiotic conjugates from
intermediate 3.82 The synthesis requires only one HPLC
purification, and the final products are purified by trituration.
Daptomycin and solithromycin are commercially available and
the oxazolidinones were synthesized following the protocols of
Miller22a and Rafai Far.91d
We also synthesized several controls to probe the
mechanism of action of 7, 8, and 9 using modifications of
our existing protocol (11−18, Table 2). These included
conjugates with D-amino acid linkers (e.g., 13, 14, and 16),
which are not readily cleaved by proteases, enabling us to
determine if proteolytic linker cleavage is responsible for the
observed activity (see Supporting Information, section VIII,
page S139).92 To confirm on-target activity of the antibiotic,
we evaluated conjugates that lack an antibiotic or contain an
inactive enantiomer of the antibiotic (e.g., 11, 12, and 15). To
compare the effectiveness of conjugates containing a peptide
linker that did not release acc in periplasmic extract (Figure
2B), we synthesized WSWC conjugate 17. We also synthesized
a siderophore-free conjugate (18) to determine the depend-
ence of activity on the siderophore.
Determination of the Antibacterial Activity of SACs
7−9 and Iron-Dependent Activity. The minimum
inhibitory concentrations (MICs) of conjugates 7−9 were
evaluated according to the standard CLSI antimicrobial
susceptibility testing guidelines in Meuller−Hinton-II (MH-
II) broth with dipyridyl to sequester iron from the media and
promote siderophore-mediated transport (Tables 3 and 4,
Table S1A,B).82,93 Controls that lacked a siderophore did not
show activity dependence on dipyridyl concentration, while the
E
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extract (Table 3). Finally, as expected, conjugate 8 was not
active in wild-type strains with functional endogenous efflux
pumps (Table S1B).
used to treat Gram-positive infections, but it lacks activity
against Gram-negative species. Therefore, we were gratified to
find that the cleavable L-linker daptomycin conjugate 7 showed
species-specific activity against A. nosocomialis, A. baumannii,
and E. coli, with MIC values in the 1 to 10 μM range, while
daptomycin itself was inactive against these species (MIC > 39
μM). The highest activities were observed in Acinetobacter
species (MIC = 1−5 μM). Similarly, Miller and co-workers
revealed that non-cleavable daptomycin conjugates displayed
selective activity against A. baumannii.22b,23 These findings
indicate that this approach has the potential to produce
precision antibiotics with relatively narrow-spectrum activity.
Moreover, as expected, 7 was inactive against S. aureus as it was
not proteolytically activated due to the absence of a periplasm
and periplasmic proteases. The activities of ornithine-function-
alized daptomycin analogs in S. aureus are highly dependent on
the side chain, and the WSPKYM linker reduces the activity of
daptomycin in this species.87d The activity of 7 cannot be
compared to previously reported non-cleavable linkers because
the specific linker contributes to the activity of non-cleavable
daptomycin conjugates (Figure S3).22b,23 Therefore, we limit
our comparisons of the cleavable conjugate 7 to its closely
related non-cleavable diastereomer 13.
Eperezolid-NH2 (5) has only minimal activity (MIC = 43
μM) against both E. coli ΔsurA and S. aureus Newman, which
lack outer membrane proteins, indicating that the cytoplasmic
membrane of both Gram-positive and Gram-negative bacteria
provide a barrier for the diffusion of 5 into the cytoplasm.
There are two possible explanations for the potent activity of 8
given the lack of activity of 5 in E. coli ΔbamBΔtolC: (1)
conjugate 8 may be actively transported to the cytoplasm and
activated by a cytoplasmic protease99 or (2) cleavage in the
periplasm may lead to large differences in the concentrations of
molecules in the periplasm versus the cytoplasm, enhancing
the effective concentration of 5 in the cytoplasm and hence
potency.
Table 3. Antibacterial Activity (MIC in μM) and In Vitro
Cleavage of Eperezolid-NH2 Conjugate 8 and Derivatives
a
Thereof
The D-linker conjugate 13 was 2- to 11-fold less active than
7 against E. coli, A. baumannii, and A. nosocomialis and did not
entirely lose activity as would be expected because daptomycin
can still engage its target in the periplasm with a noncleavable
linker.22,23 Nevertheless, the L-linker conjugate 7 improved the
activity up to 11-fold, suggesting that cleavable linkers may
benefit SACs containing periplasmic-targeting antibiotics.
Finally, derivatives of 7 lacking the daptomycin payload (11
and 12) were essentially inactive. Taken together, the data in
Table 4 indicates that we have successfully repurposed
daptomycin for Gram-negative bacteria, and the enhanced
activity of 7 relative to 13 is consistent with our guiding
hypothesis of stereospecific proteolytic activation. It is also
clear that proteolytic activation likely occurred in the
periplasm, rather than by an extracellular protease in the
medium given that daptomycin lacks activity against Gram-
negative species.
To understand the lower activity observed for E. coli (MIC =
11 μM) relative to Acinetobacter species (MIC = 1−5 μM), we
note that the release of daptomycin from conjugate 7 in
periplasmic extract of E. coli was not observed by HPLC
analysis (see Supporting Information, section VIII, pp S120−
S124), which may be due to unfavorable steric interactions
between the approaching protease(s)101 and the large
daptomycin molecule. Following incubation of 7 in periplasmic
extracts of A. baumannii and A. nosocomialis, however, the
HPLC trace revealed cleavage products with retention times
that overlapped with daptomycin (see Supporting Information,
section VIII, pp S115−S119).
a
For strain descriptions and extract cleavage procedure, see
Supporting Information, Table S1B. ND = not determined.
We also synthesized a number of additional control
molecules to probe the antibacterial mechanism of 8, including
conjugate 17 with the WSWC linker (Table 3); this linker did
not release an acc fluorophore from peptide 2 (Figure 2). Not
surprisingly, this analogue had only weak (MIC = 37 μM)
activity, as did compounds 11, 12, and 15 that lacked an active
antibiotic payload. Similarly, the conjugate 18 without a
siderophore was inactive. However, methyl ester 15 retained a
modicum of activity (9 μM), possibly by a mechanism similar
to many non-helical proline-containing cationic antimicrobial
peptides.100
Miller and co-workers published a highly similar eperezolid
conjugate using the same siderophore and eperezolid-NH2
derivative with a cleavable β-lactam linker.22a This conjugate
exhibited activity against a number of strains of Gram-negative
bacteria, including some with high-level β-lactam resistance.
Against strains with high β-lactamase content for which the β-
lactam linker did not measurably contribute to activity (up to
50 μM), the MIC of the conjugates was 6 μM. We did not
observe activity of conjugate 8 in the same A. baumannii strain
(ATCC BAA-1797), which could be due to inherent
differences in the two linkers.
These results are also of interest with respect to the
mechanism of action of daptomycin. It has been previously
reported that the target for daptomycin (4) may be absent in
Gram-negative species due to the differing membrane
compositions between Gram-positive and Gram-negative
bacteria.102 Given that 7 is active against E. coli, it would
appear that daptomycin is able to act on the cytoplasmic
membranes of these Gram-negative species once they gain
access. Also, our finding that daptomycin itself is equipotent
against S. aureus Newman and in the outer membrane-
Daptomycin Conjugate 7 Exhibits High Activity
against Acinetobacter Species (Table 4). Daptomycin is
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a
Table 4. Antibacterial Activity (MIC in μM) of Daptomycin SAC 7 and Derivatives Thereof
a
For strain descriptions, see Supporting Information. ND = not determined. Daptomycin is a calcium-dependent lipopeptide. MIC assays of
daptomycin conjugates were conducted in the presence of 100 μg/mL CaCl2 in MH-II media.103
compromised E. coli ΔsurA (MIC = 0.6 μM) is consistent with
be enhanced by limited structure−activity relationships (Figure
S8). The peptide WSPKYM also contains more than one
cleavage site (Table S5), which may complicate the drug
activation mechanism. Our conjugates do not show significant
activity against P. aeruginosa (Table S1B and Figure S2), a
pathogen prone to resistance, which could be due to the
following: (1) insufficient linker cleavage may not lead to
growth inhibition because the linker was optimized for E. coli,
(2) the target for daptomycin may be present in minimal
amounts, depending on the strain or species of Gram-negative
bacteria,102 or (3) solithromycin and eperezolid might be efflux
substrates in this organism.
In summary, this work provides a robust methodology for
selection and screening of Trojan-horse prodrugs applied to
the persistent and growing problem of antibacterial resistance.
The resulting conjugates from this platform improve upon
existing cleavable linkers for SACs. Using phage display, one
can rapidly screen vast peptide libraries, and by varying the
selection strategy one can screen for linkers with desired
characteristics. For example, by using periplasmic extracts from
different species of bacteria in succeeding selections, one can
ensure broad activity over the desired range of bacteria.
Alternatively, negative selection could be incorporated to select
against cleavage of serum proteases or beneficial members of
the microbiome. Thus, the potential for fine-tuning the
protocol for future practical applications is substantial.
this conclusion.
CONCLUSION
■
The strategy developed here should be broadly applicable for
discovery of protease-activated peptide prodrugs for a variety
of applications. Here, we focused on delivering antibiotics by
designing protease-cleavable siderophore conjugates. By
targeting E. coli periplasmic proteases, we were able to design
conjugates that act against a broad (or narrow) spectrum of
Gram-negative bacteria, illustrating the potential of this
approach. Our results provide strong support for the overall
mechanism of proteolytic release of the antibiotic from
conjugates 7−9. Although we have not yet identified the
proteases responsible for activity against our substrates, we
purposefully avoided targeting a single protease to decrease the
chances of resistance arising from mutants of a single protein.
Moreover, the use of chemically stable amide linkers provides
an advantage to targeting proteases over esterases and β-
lactamases by avoiding the need for esters and β-lactams,
which are chemically more labile. Importantly, the modular
design and facile synthetic route provides opportunity for rapid
synthesis of SACs with different siderophores, linkers, and
antibiotics. This has led to the discovery of cleavable
conjugates with activity against several clinically relevant
Gram-negative pathogens.
Throughout the course of this work, we made a number of
unexpected discoveries with impacts that extend beyond the
scope of protease-cleavable prodrugs. The L-linker daptomycin
conjugate 7 completely lacks the Gram-positive activity of
daptomycin and has gained Gram-negative activity, effectively
“flipping” the spectrum of activity of this potent antibiotic. We
found that conjugates with D-linkers, which are unlikely to be
cleaved proteolytically, have moderate activity against several
strains of Gram-negative bacteria. Perhaps the most
unexpected results are the activities of the solithromycin
conjugates 9 and 16 in a cell-free translation assay, which
indicate that these large (MW > 2000) conjugates may directly
inhibit the ribosome in E. coli (see Supporting Information,
Figure S1A). These results are extremely surprising in the
context of solithromycin−ribosome structural data104 and may
provide the basis for new macrolide−peptide hybrid anti-
biotics.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/jacs.0c06987.
■
sı
Supplementary Tables S1−S5 and Figures S1−S9;
general information; experimental procedures and
compound charcterization; MIC, periplasmic cleavage
assays, and cell-free translation; substrate phage display;
references cited; select NMR spectra; and HPLC traces
for cleavage in periplasmic extract (PDF)
AUTHOR INFORMATION
Corresponding Authors
■
Bobo Dang − Key Laboratory of Structural Biology of
Zhejiang Province, School of Life Sciences, Westlake
University, Hangzhou, Zhejiang 310024, China; Center for
Infectious Disease Research, Westlake Laboratory of Life
Sciences and Biomedicine, Hangzhou, Zhejiang 310024,
China; Institute of Biology, Westlake Institute for Advanced
Additional studies will be required to optimize the current
linkers for use as practical drugs. The current linker has
reasonable stability in human serum, which could doubtlessly
G
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Article
pathogens in an intensive care unit of Monterrey, Mexico.
Chemotherapy 2012, 58, 475−481.
Study, Hangzhou, Zhejiang 310024, China;
Email: dang.bobo@westlake.edu.cn
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of the ESKAPE pathogens. Expert Rev. Anti-Infect. Ther. 2013, 11,
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William F. DeGrado − Department of Pharmaceutical
Chemistry, University of California, San Francisco, California
94158, United States; Cardiovascular Research Institute,
University of California, San Francisco, California 94158,
United States; orcid.org/0000-0003-4745-263X;
Email: bill.degrado@ucsf.edu
Ian B. Seiple − Department of Pharmaceutical Chemistry,
University of California, San Francisco, California 94158,
United States; Cardiovascular Research Institute, University
of California, San Francisco, California 94158, United
States; orcid.org/0000-0002-8732-1362;
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Jonathan H. Boyce − Department of Pharmaceutical
Chemistry, University of California, San Francisco, California
94158, United States; Cardiovascular Research Institute,
University of California, San Francisco, California 94158,
United States; orcid.org/0000-0003-3697-9292
Beatrice Ary − Department of Pharmaceutical Chemistry,
University of California, San Francisco, California 94158,
United States
Quinn Edmondson − Department of Pharmaceutical
Chemistry, University of California, San Francisco, California
94158, United States
Charles S. Craik − Department of Pharmaceutical Chemistry,
University of California, San Francisco, California 94158,
United States; orcid.org/0000-0001-7704-9185
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Complete contact information is available at:
https://pubs.acs.org/10.1021/jacs.0c06987
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Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
■
We thank Adam Cotton, Peter Rowheder, Dr. Sam Ivry, and
Dr. Matthew Ravalin for helpful discussions. We thank Bruk
Mensa for helpful discussions and for providing P. aeruginosa
ATCC 10145, S. typhi ATCC 700931, S. aureus Newman, E.
coli K12 MG1655, E. coli BW25113 ΔtolC, and E. coli
BW25113 ΔsurA. We thank Neha Prasad for helpful
discussions and for providing K. pneumoniae MGH 78578, E.
cloacae ATCC 13047, E. aerogenes ATCC 13048, P. aeruginosa
PA01, P. aeruginosa PA14, and S. enterica 14028s. We thank
Jenna Pellegrino for providing E. coli BW25113 ΔbamBΔtolC
and for helpful discussions on in vitro translation assays. We
thank Professor Joanne Engel for a generous gift of A.
nosocomialis M2. J.H.B. was supported by the National
Institutes of Health under the Ruth L. Kirschstein National
Research Service Award 5T32HL007731-27 from the National
Heart Lung and Blood Institute (NHLBI). Q.E. was supported
by the National Science Foundation Graduate Research
Fellowship Program under Grant no. 1650113. B.A. was
supported by the National Institutes of Health under Grant no.
T32 Al 0605357. This project was supported by the David and
Lucile Packard Foundation (I.B.S.) and the National Institutes
of Health under Grant no. R35 GM122603 (W.F.D.).
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