Gram-Negative Antibiotic Active through Inhibition of an Essential Riboswitch

Article abstract of DOI:10.1021/jacs.0c04427

Multidrug-resistant Gram-negative (GN) infections for which there are few available treatment options are increasingly common. The development of new antibiotics for these pathogens is challenging because of the inability of most small molecules to accumulate inside GN bacteria. Using recently developed predictive guidelines for compound accumulation in Escherichia coli, we have converted the antibiotic Ribocil C, which targets the flavin mononucleotide (FMN) riboswitch, from a compound lacking whole-cell activity against wild-type GN pathogens into a compound that accumulates to a high level in E. coli, is effective against Gram-negative clinical isolates, and has efficacy in mouse models of GN infections. This compound allows for the first assessment of the translational potential of FMN riboswitch binders against wild-type Gram-negative bacteria.

Full text of DOI:10.1021/jacs.0c04427

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Article
A Gram-Negative Antibiotic Active Through
Inhibition of an Essential Riboswitch
Stephen E. Motika, Rebecca J. Ulrich, Emily J. Geddes,
Hyang Yeon Lee, Gee W Lau, and Paul J Hergenrother
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.0c04427 • Publication Date (Web): 20 May 2020
Downloaded from pubs.acs.org on May 20, 2020
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A Gram-Negative Antibiotic Active Through Inhibition of an
Essential Riboswitch
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§
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#
Stephen E. Motika, Rebecca J. Ulrich, Emily J. Geddes, Hyang Yeon Lee, Gee W. Lau, and Paul J.
Hergenrother *
§
§Roger Adams Laboratory, Department of Chemistry, University of Illinois, Urbana, Illinois 61801, United States
§Carl R. Woese Institute for Genomic Biology, University of Illinois, Urbana, Illinois 61801, United States
#Department of Pathobiology, University of Illinois, Urbana, Illinois 61802, United States
KEYWORDS: antibacterial, compound accumulation
ABSTRACT: Multi-drug resistant (MDR) Gram-negative (GN) infections for which there are few available treatment options are
increasingly common. The development of new antibiotics for these pathogens is challenging due to the inability of most small
molecules to accumulate inside GN bacteria. Using recently developed predictive guidelines for compound accumulation in
Escherichia coli we have converted the antibiotic Ribocil C, which targets the FMN riboswitch, from a compound lacking whole-cell
activity against wild-type GN pathogens into a compound that accumulates to a high level in E. coli, is effective against Gram-
negative clinical isolates, and has efficacy in mouse models of GN infection. This compound allows for the first assessment of the
translational potential of FMN riboswitch binders against wild-type Gram-negative bacteria.
INTRODUCTION
has efficacy in mouse models of infection using permeability-
deficient strains of E. coli. Interestingly and contrastingly,
exogenous riboflavin does squelch the antibacterial activity of
Ribocil C in permeability defect E. coli grown in culture.
17
Deaths from drug-resistant bacterial infections in the
European Union rose three-fold from 2007 to 2015, with over
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7,22
1
2
7,000 deaths occurring in 2015. Notably, six specific drug-
The only means to truly assess the suitability of the FMN
riboswitch as a target for GN pathogens is to identify
appropriate compounds and evaluate them in vivo;
unfortunately Ribocil C and its derivatives have no notable
resistant bacterial pathogens accounted for this rise, four of
which are Gram-negative (GN), with cephalosporin-resistant
E. coli contributing the greatest to both incidence and deaths
(285,758 infections and 8,750 deaths in 2015). Therefore, new
therapeutics are needed, especially those that engage untapped
1
7, 22
activity against wild-type GN bacteria.
2
-11
cellular targets.
Although there are multiple novel
We suspected that the inactivity of Ribocil C against
wild-type GN pathogens was due to low intracellular
accumulation, as is the case for many antibiotics that are only
active against GP organisms. Thus, if strategic modifications
to Ribocil C could be made to facilitate enhanced bacterial
accumulation without disrupting target engagement, this
approach could provide the first wild-type GN-active
antibiotic that targets the FMN riboswitch. Here we detail the
application of recently described permeation rules to arrive at
Ribocil C variants that accumulate in E. coli. The lead
compound identified through this strategy has activity against
multiple wild-type GN organisms, including dozens of multi-
drug resistant (MDR) GN clinical isolates, and reduces
bacterial burden and extends survival in mice infected with
antibiotic-resistant E. coli.
antibacterial targets and many promising small-molecule
antibiotic leads for Gram-positive (GP) infections, whole-cell
activity in GN pathogens has proven difficult to achieve.
This inactivity is due to the impermeability of GN bacteria to
most drug-like molecules, conferred through an outer
membrane of densely packed lipopolysaccharides (LPS) and
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13-16
promiscuous efflux pumps.
A creative cell-based screen conducted at Merck & Co
identified Ribocil C (Figure 1A) as a synthetic compound with
potent activity against GP bacteria (S. aureus) and against E.
coli strains with permeability defects. Ribocil C targets the
flavin mononucleotide (FMN) riboswitch, ultimately
inducing bacterial death through riboflavin starvation.
Importantly, there are no human homologues of the FMN
riboswitch and indeed no riboswitches are known in
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RESULTS
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mammalian systems,
suggesting the potential for bacterial
cell selectivity for compounds that engage this target. While
Ribocil C kills S. aureus in culture, GP organisms can
22
scavenge riboflavin from their environment thus tempering
enthusiasm for Ribocil C specifically, and the FMN
riboswitch target in general, for S. aureus. In contrast, E. coli
strains with deletions within genes in the riboflavin
biosynthesis pathway appear unable to scavenge exogenous
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riboflavin during mouse models of infection, and Ribocil C
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coli ∆rfaC) or efflux pump deficiency (E. coli ∆tolC) (Figure
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1A), suggesting that if the compound were tailored to
accumulate in wild-type E. coli, it would be a potent
antibiotic.
To convert Ribocil C into a GN-active antibiotic, the
eNTRy rules were used to guide the design of a small
compound collection. Ribocil C already meets two eNTRy
rules criteria (globularity and rotatable bonds), therefore the
installation of an unhindered primary amine was the chief
objective. For placement of the amine, the x-ray crystal
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structure of a similar compound, Ribocil B, in complex with
the FMN riboswitch aptamer of Fusobacterium nucleatum
was analyzed and priority was given to solvent exposed sites
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(red in Figure 1B), such as in proximity to the northern
pyrimidine. As such, two general classes of Ribocil C-amine
variants were envisioned, possessing amines at this priority
location (Figure 1C). Class A variants would be designed with
amine tethers containing varying levels of saturation, so as to
assess the effects of geometric/rotational freedom on target
engagement, whole cell accumulation, and GN antimicrobial
activity. Class B analogues were also considered as these
possess low rotatable bonds, but slightly larger globularity
scores.
Synthesis and antimicrobial evaluation of Ribocil C
variants predicted to accumulate in GN pathogens. The
construction and preliminary assessment of target compounds
proceeded through the process depicted in Figure 2A. Specific
compounds with varying carbon and carbon-heteroatom
linkers were synthesized, with the objective of positioning an
Figure 1. Ribocil C as a starting point to access a GN-
active antimicrobial that engages the essential FMN
riboswitch. A) Accumulation in E. coli MG1655 and
Minimum Inhibitory Concentration (MIC) values against
strains. MICs were performed in M9-MOPS media per
CLSI guidelines. B) Location prioritized for amine
installation, based on the co-crystal of Ribocil B bound to
the FMN riboswitch aptamer of F. nucleatum (PDB 5C45).
C) Target compounds designed to have enhanced
accumulation in GN bacteria.
Design of GN-active versions of Ribocil C. We recently
reported the elucidation of physicochemical traits that bias
23
compounds for accumulation in E. coli. These “eNTRy
24
rules” detail that a high accumulating molecule should
possess an ionizable nitrogen (with a primary amine being
most favorable), low three-dimensionality, and a low number
of rotatable bonds. Using a recently developed web-
application for calculating eNTRy parameters (www.entry-
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way.org) it was observed that Ribocil C meets two of the
three eNTRy criteria, with low three-dimensionality
(globularity=0.058) and five rotatable bonds, but lacking a
primary amine. The synthesis of Ribocil C (See Supporting
Information) and subsequent evaluation (Figure 1A) revealed
that indeed this compound accumulates very poorly in E. coli
12
(Figure
1A,
accumulation=139
nmol/10
CFUs,
accumulation relative to positive and negative controls shown
in Supporting Figure 1) and demonstrates no detectable
activity at its solubility limit in standard microbial
susceptibility assays against reference strains of wild-type GN
bacteria (Figure 1A). However, Ribocil C has marked activity
against E. coli strains possessing either a truncated LPS (E.
Figure 2. eNTRy rule guided design of amine analogues of
Ribocil C that accumulate within and kill GN pathogens.
A) Workflow utilized to develop new Ribocil C variants
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that possess activity against wild-type GN pathogens. B)
generation leads. All three of these compounds were found to
have notably elevated accumulation in E. coli relative to
Ribocil C while maintaining antibacterial activity against
membrane compromised GN strains (Figure 2B).
Additionally, compounds (-)3, (-)4, and (-)5 also have marked
antibacterial activity in Enterobacteriaceae strains with intact
outer membranes and functional efflux pumps, including
strains of E. coli, E. cloacae, and Klebsiella pneumoniae
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Lead compounds identified with the ability to accumulate
in wild-type E. coli and inhibit growth. Antimicrobial
assessment of lead derivatives against various GN
pathogens. MC E. coli: Membrane Compromised E. coli.
Accumulation in E. coli MG1655, units are nmol/10
CFUs. MICs were performed in M9-MOPS media per
CLSI guidelines.
1
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(
Figure 2B). These compounds were inactive against the other
amine in a non-intrusive region of the FMN riboswitch
aptamer. To construct the target molecules, a two fragment
cou- pling was adopted, harnessing a late stage reductive
amination between aldehydes (represented by 1) and cyclic
amine 2. Using this focused workflow, next-generation
Ribocil C derivatives were rapidly constructed and then
evaluated for antimicrobial activity (MIC assessments in
wild-type E. coli and outer-membrane compromised E. coli),
with any active compounds being subjected to enantiomer
resolution and further biological evaluation (Figure 2A);
multiple novel Ribocil C derivatives with amine extensions
were constructed and assessed for antimicrobial activity (the
full list is shown in Supporting Figure 2). Using this protocol,
enantiopure compounds (-)3, (-)4, and (-)5 emerged as next-
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GN ESKAPE pathogens, Acinetobacter baumannii and
Pseudomonas aeruginosa, unsurprising based on the low
homology of the FMN riboswitch target of these organisms
with that of Enterobacteriaceae, although these compounds do
accumulate to some extent in A. baumannii and P. aeruginosa
(Supp Table 1). As compound (-)3 was the most active
antibiotic (Figure 2B) and also displayed little toxicity against
normal human cells in culture (Supp Table 2), it was
prioritized and named as Ribocil C-PA; (+)3 is devoid of
antibiotic activity (Supporting Figure 2).
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Systematic construction of Ribocil variants to probe
influence of functional groups on activity. With antibiotics
active against wild-type GN bacteria in hand, the next
Figure 3. Synthetic modifications of compound 3 to probe the effect of functional groups on antimicrobial activity.
Accumulation was performed in E. coli MG1655 and is represented in nmol/1012 CFUs. The accumulation of compound 6
could not be determined due to limitations of aqueous solubility. The MIC values for all compounds are reported for the
racemates. The accumulation values for all compounds are reported for the racemates, with the exception of compound 3,
which is reported for (-)3. MIC values were determined for E. coli BW25113 (WT E. coli), E. coli JW3596 (E. coli ∆rfaC), and
E. coli JW5503 (E. coli ∆tolC). MICs were performed in M9-MOPS media per CLSI guidelines.
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Page 4 of 8
objective was to construct compounds that could illuminate
K. pneumoniae (n=54). MICs were performed in M9-
MOPS media per CLSI guidelines. See Supporting Table
3 for list of resistance genes in clinical isolate panels. A full
listing of this MIC data is in Supporting Tables 4-5.
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the influence of various functional groups on antibiotic
activity and whole-cell accumulation. These synthetic efforts
were focused on modifications to derivative 3. Towards this
end and as shown in Figure 3, Boc-protected 6 was converted
to free amine 3, which was then transformed to dimethyl
amine (compound 7) and acetamide (compound 8) variants.
Late stage reduction of 6, followed by protecting group
removal, provided saturated analogue 9. Finally, amine
analogues with alternative linker lengths were accessed via
reductive amination between 10a/10b/10c and 2 to provide,
after deprotection, amines 11a-c (Figure 3B).
Assessing the activity of Ribocil C-PA against a large
panel of MDR clinical isolates of E. coli and K.
pneumoniae. Extensive panels of MDR clinical isolates of E.
coli (n=42) and K. pneumoniae (n=54) were assessed for their
susceptibility to (-)3, also called Ribocil C-PA. Within these
collections of clinical isolates there are strains with resistance
to a wide variety of antibiotics, including carbapenems,
tetracyclines, aminoglycosides, sulfonamides, trimethoprim,
and colistin (see Supporting Table 3 for full list of resistance
profiles). Ribocil C-PA markedly out performs Ribocil C in
these susceptibility studies (Figure 4).
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Antibacterial activity of novel Ribocil variants.
Compounds were assessed for their ability to accumulate in E.
coli, in addition to their antibacterial activity against both
wild-type E. coli and permeabilized strains. The activity of
compounds against these latter strains provides some
indication of their ability to engage the FMN riboswitch
target, without the complication of cell permeability. As
shown by the data in Figure 3, compound 3 had the best
antibacterial activity against wild-type E. coli, with all
modifications (6-9, acylation, dimethylation, or saturation of
the linker) resulting in inactive variants against wild-type E.
coli; however, all four of these compounds retained some
activity against the membrane-compromised strains,
suggesting a retention of some degree of target engagement.
Linker length appeared important as compounds with shorter
(e.g. 11a) or longer (e.g. 11c) linkers lost either target
engagement or accumulation.
ns
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(
100
Figure 5. Flavin reduction in E. coli BW25113 at sub-
inhibitory concentration (2 µg/mL) of Ribocil C-PA. The
inactive enantiomer, (+)3, was also assessed at 2 µg/mL
and demonstrates no inhibition of flavin production.
Flavin monitoring was performed after cultures were
treated with either vehicle or compound for 20 hours. Data
are shown as meansꢀ±ꢀs.d. and statistical significance was
determined by one-way ANOVA with Tukey’s multiple
comparisons. NS, not significant (P>ꢀ0.05). *P<ꢀ0.015).
80
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20
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(-)3, Ribocil C-PA
Ribocil C
Mode-of-action and resistance frequency of Ribocil
C-PA. To assess the mode-of-action of Ribocil C-PA, two key
experiments were conducted. The first was to evaluate E. coli
production of flavins in the presence of Ribocil C-PA. As
detailed in previous reports, non-native FMN riboswitch
ligands inhibit the downstream transcription and translation of
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[Compound] (µg/mL)
[Compound] (g/mL)
B. 54 MDR K. pneumoniae clinical isolates
1
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key enzymes involved in riboflavin biosynthesis. Riboflavin
serves as a precursor to FMN and flavin adenosine
dinucleotide (FAD), therefore depletion of these intracellular
flavins would be expected for an antibiotic that acts through
targeting this riboswitch. Using an LC-MS/MS assay to
monitor flavin levels in E. coli cultures treated with Ribocil
C-PA (at 0.5x the MIC), reduction in flavins was observed
(Figure 5), indicating that Ribocil C-PA selectively and
competitively engages the FMN riboswitch. A control
experiment showed the inactive enantiomer, compound (+)3,
had no effect on flavin production in E. coli (Figure 5).
100
(-)3, Ribocil C-PA
80
Ribocil C
60
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As a second experiment, spontaneous E. coli mutants
resistant to Ribocil C-PA were generated, and the FMN
riboswitch was sequenced; this experiment was conducted in
E. coli BW25113, K. pneumoniae ATCC 27736, and E. coli
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[Compound] (µg/mL)
[Compound] (g/mL)
[Compound] (µg/mL)
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Figure 4. Assessment of Ribocil C-PA and Ribocil C
against MDR clinical isolates of A) E. coli (n=42) and B)
∆
tolC (JW5503). Significant target-based resistance was
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observed in all of these strains (Supporting Figure 3, at 8X frequencies of resistance against E. coli ∆tolC (Supporting
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MIC E. coli FOR= 2.8 x10 ; at 8X MIC K. pneumoniae FOR=
Figure 4, at 8X MIC Ribocil C FOR=3.2 x10 ; Ribocil C-PA
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2.4 x10 ). Ribocil C and Ribocil C-PA yield similar
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Figure 6: Mutations observed after sequencing the FMN riboswitch in Ribocil C-PA resistant clones of E. coli BW25113
generated at 8X the MIC (32 µg/mL) of Ribocil C-PA.
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FOR=2.1 x 10 ). After generating and sequencing 20
to mice at 100 mg/kg (IP), twice-a-day for three days, and this
dosing regimen was thus used in the infection models. Ribocil
C-PA was evaluated head-to-head versus Ribocil C in two E.
coli infection models, an acute pneumonia infection (bacterial
burden model) and septicemia (bacterial survival model). In
these experiments, a nearly 2-log reduction in bacterial burden
was achieved for Ribocil C-PA in mice infected with two
different pathogenic strains of E. coli (Figure 7A), while 80%
(12/15) of mice burdened by septic infection were
successfully rescued (Figure 7B); Ribocil C showed
different E. coli BW25113 clones resistant to Ribocil C-PA,
6 different mutations were observed, all of which mapped
1
back to either the FMN riboswitch aptamer or expression
platform (Figure 6). Ribocil C-PA resistant bacteria were
assessed for changes in fitness by comparing their growth
rates to those of wild-type E. coli. As shown in Supporting
Figure 5, growth rates for resistant mutants and wild-type E.
coli were nearly identical, suggesting limited changes in cell
culture fitness and viability between these distinct genotypes.
Efficacy in mouse models of GN infection. Both
Ribocil C and Ribocil C-PA were tolerated when administered
E. coli AR0493
E. coli ELZ4081
A.
B.
*
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****
ns
Ribocil C-PA
Ribocil C
ns
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c
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c
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e
ibo
l
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ibo
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Figure 7. A) Bacterial burden model of acute pneumonia in E. coli. Acute pneumonia infections initiated in seven-week-old
9
-1
9
-1
male CD-1 mice with E. coli AR0493 (1.7x10 CFU mouse intranasally) or E. coli ELZ4081 (3.1x10 CFU mouse
intranasally). Mice were treated with vehicle (n=8) or compound (Ribocil C or Ribocil C-PA, 100 mg/kg IP, n=8 for each
group) 8, 16, 30 and 48h post-infection, and the bacterial burden was evaluated 48h post-infection. MIC=8 µg/mL and 4
µg/mL for Ribocil C-PA against E. coli AR0493 and E. coli ELZ4081 respectively. B) Kaplan-Meier survival curve for the
7
mouse efficacy model of E. coli sepsis. Seven-week-old male CD-1 mice were infected with E. coli AR0493 (7.91x10 CFU
-1
mouse ; 15 mice per group) via intravenous injection. Mice were treated with compound (Ribocil C or Ribocil C-PA, 100
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mg/kg IP, n=15 for each group) twice daily post-infection for 5 days. In A, data are shown as meansꢀ±ꢀs.d. and statistical
significance was determined by one-way ANOVA with Tukey’s multiple comparisons. NS, not significant (P>ꢀ0.05),
****P<ꢀ0.0001. In B, statistical significance was determined by two-tailed log-rank (Mantel–Cox) test, ***P<0.001.
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no statistically significant reductions in bacterial burden in
vivo against either E. coli strain tested, and was ineffective at
rescuing mice in a model of E. coli septicemia (Figure 7).
are potent against membrane-compromised E. coli, but only
primary amine 3 accumulates in and has antibiotic activity
against wild-type GN strains. As reinforced with this
conversion of Ribocil C to Ribocil C-PA, the existence of
structural data (ideally an x-ray structure with the parent
antibiotic bound to its target) and/or significant structure-
activity relationship data facilitates the design of new
compounds that meet the eNTRy rule criteria and maintain
target engagement.
DISCUSSION
The bacterial-specific and essential nature of the FMN
riboswitch suggests it as a promising antibiotic target.
However, GP bacteria are able to scavenge riboflavin from the
environment, likely making compounds that engage this target
unsuitable for GP infections. With conflicting evidence about
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The development of Ribocil C-PA indicates that the
inhibition of the FMN riboswitch in the Enterobacteriaceae
its suitability for GN infections,
we set out to answer this
question through the creation and assessment of an antibiotic
active versus wild-type GN organisms through inhibition of
the FMN riboswitch. The discovery herein of Ribocil C-PA,
active in cell culture and in vivo against wild-type GN bacteria
through inhibition of the FMN riboswitch, demonstrates that
indeed this riboswitch could be an outstanding antibacterial
target for wild-type GN pathogens. In addition, Ribocil C-PA
exhibits 16-fold more potent activity against efflux deficient
E. coli compared to wild-type organisms (Figure 2B),
suggesting a next-generation derivative that evades efflux
could have even better activity against wild-type GN
pathogens.
(and likely in other GN bacteria) is an effective antibacterial
strategy. While the elevated frequency of resistance that is
apparently associated with this target is not ideal, this issue
has been surmounted for other antibiotics, for example
3
2
through a large front-loading dose (e.g. fusidic acid ) or
through combination regimens such as commonly employed
6
for rifampicin, trimethoprim, and sulfamethoxazole and as is
1
1
common when treating Mycobacterium tuberculosis. The
validation of novel antibiotic targets will be critical in the
continued battle against drug-resistant bacteria.
ASSOCIATED CONTENT
Supporting Information
An intriguing opportunity afforded when targeting this
bacterial riboswitch is the possibility for narrow-spectrum
(
pathogen specific) antibiotics. Unlike many macromolecular
Supporting Figures and Tables, materials and methods, figures
detailing syntheses of compounds, experimental procedures and
characterization of chemical products including spectra. This
material is available free of charge via the internet at
http://pubs.acs.org.
targets, the FMN riboswitch does not have high conservation
across GN organisms, with marked sequence differences
between the Enterobacteriaceae, A. baumannii, and P.
aeruginosa versions of this target (sequence identity of
aptamer relative to E. coli): K. pneumoniae (90%); E. cloacae
(
87%); P. aeruginosa (72%); A. baumannii (61%)). Ribocil
AUTHOR INFORMATION
Corresponding Author
C-PA demonstrates activity against constituents of the
Enterobacteriaceae, including in large panels of clinical
isolates, but not A. baumannii and P. aeruginosa. Given the
sequence divergence of the FMN riboswitch, complementary
compounds that have specificity for A. baumannii or P.
aeruginosa can be envisioned. Of course as infections from
Enterobacteriaceae are of particular concern – drug-resistant
*
E-mail hergenro@illinois.edu
Funding Sources
We are grateful to the NIH (AI136773) and the University of
Illinois for supporting this work. S.E.M was partially supported
by F32AI143207 and R.J.U. is an NSF predoctoral fellow.
1
E.coli but also carbapenem-resistant K. pneumoniae, which
has a 30-50% mortality rate
2
7-28
– the GN-active Ribocil C
analogues described herein hold considerable promise.
With its successful conversion into a GN active
antibacterial, Ribocil C joins a growing list of compounds
where GN activity and/or high accumulation has been imbued
ACKNOWLEDGMENT
We thank L. Li (Metabolomics Center, Roy J. Carver
Biotechnology Center, UIUC) for LC-MS/MS analysis. We thank
Gyumin Lee for assistance with the mouse efficacy studies.
23, 25, 29-31
via derivative synthesis guided by the eNTRy rules.
In this context it is interesting to note that compounds (-)3, (-
)4, and (-)5, all of which accumulate in E. coli, exist just
outside the five rotatable bonds recommended by the eNTRy
rules. As shown in Supporting Figure 6, energy minimization
of these structures provides evidence of a preferable
conformation that may reduce rotational freedom in this
compound class. Analysis of activity data of derivatives
ABBREVIATIONS
CLSI, Clinical and Laboratory Standards Institute; FAD, flavin
adenosine dinucleotide; FMN, flavin mononucleotide; FOR,
Frequency of Resistance; GN, Gram-negative; GP, Gram-
positive; LPS, lipopolysaccharides; MDR, Multi-drug resistant;
MIC, Minimum Inhibitory Concentration; MTD, Maximal
tolerated dose
(Figure 3) once again suggests that primary amines are most
proficient at facilitating compound accumulation when
compared to other ionizable nitrogens (such as the dimethyl
amine), consistent with previous observations from
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