Novel riboswitch-binding flavin analog that protects mice against Clostridium difficile infection without inhibiting cecal flora

Article abstract of DOI:10.1128/AAC.01282-15

Novel mechanisms of action and new chemical scaffolds are needed to rejuvenate antibacterial drug discovery, and riboswitch regulators of bacterial gene expression are a promising class of targets for the discovery of new leads. Herein, we report the characterization of 5-(3-(4-fluorophenyl)butyl)-7,8-dimethylpyrido[3,4-b]quinoxaline-1,3(2H,5H)-dione (5FDQD) - an analog of riboflavin that was designed to bind riboswitches that naturally recognize the essential coenzyme flavin mononucleotide (FMN) and regulate FMN and riboflavin homeostasis. In vitro, 5FDQD and FMN bind to and trigger the function of an FMN riboswitch with equipotent activity. MIC and time-kill studies demonstrated that 5FDQD has potent and rapidly bactericidal activity against Clostridium difficile. In C57BL/6 mice, 5FDQD completely prevented the onset of lethal antibiotic-induced C. difficile infection (CDI). Against a panel of bacteria representative of healthy bowel flora, the antibacterial selectivity of 5FDQD was superior to currently marketed CDI therapeutics, with very little activity against representative strains from the Bacteroides, Lactobacillus, Bifidobacterium, Actinomyces, and Prevotella genera. Accordingly, a single oral dose of 5FDQD caused less alteration of culturable cecal flora in mice than the comparators. Collectively, these data suggest that 5FDQD or closely related analogs could potentially provide a high rate of CDI cure with a low likelihood of infection recurrence. Future studies will seek to assess the role of FMN riboswitch binding to the mechanism of 5FDQD antibacterial action. In aggregate, our results indicate that riboswitch-binding antibacterial compounds can be discovered and optimized to exhibit activity profiles that merit preclinical and clinical development as potential antibacterial therapeutic agents.

Full text of DOI:10.1128/AAC.01282-15

AAC Accepted Manuscript Posted Online 13 July 2015
Antimicrob. Agents Chemother. doi:10.1128/AAC.01282-15
Copyright © 2015, American Society for Microbiology. All Rights Reserved.
For Submission to: Antimicrobial Agents and Chemotherapy
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A Novel Riboswitch-Binding Flavin Analog that Protects Mice Against
Clostridium difficile Infection without Inhibiting Cecal Flora
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Kenneth F. Blount,a,b,†# Cynthia Megyola,a Mark Plummer,c David Osterman,b,* Tim O’Connell,b,‡
Paul Aristoff,d Cheryl Quinn,e R. Alan Chrusciel,f Toni J. Poel,f Heinrich J. Schostarez,f Catherine A
Stewart,f Daniel P Walker, f Peter G. M. Wuts,f Ronald R. Breakera,g,h
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Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven,
CT 06520, USAa; BioRelix Inc., New Haven, CT 06511, New Haven, CT 06520, USAb; Yale
Center for Molecular Discovery, Yale University, New Haven, CT 06520, USAc; Aristoff
Consulting LLC, Fort Collins, CO 80528, USAd; QnA Pharma Consulting, LLC, Minneapolis, MN
55401e; Kalexsyn, Inc, Kalamazoo, MI 49008, USAf; Department of Molecular Biophysics and
Biochemistry, Yale University, New Haven, CT 06520, USAg; Howard Hughes Medical
Institute, Yale University, New Haven, CT 06520, USAh
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†
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Current Address: Yale Cancer Center, Yale University, New Haven, CT 06520; Alexion
‡
Pharmaceuticals, Cheshire, CT 06410; Department of Microbiology and Immunology, New
York Medical College, Valhalla, NY 10595
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#Address correspondence to Kenneth F. Blount, ken.blount@yale.edu or Ronald R. Breaker,
ronald.breaker@yale.edu
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Running title: Riboswitch-binding C. difficile antibacterial

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Novel mechanisms of action and new chemical scaffolds are needed to rejuvenate
antibacterial drug discovery, and riboswitch regulators of bacterial gene expression are a
promising class of targets for the discovery of new leads. Herein, we report the
characterization of 5-(3-(4-fluorophenyl)butyl)-7,8-dimethylpyrido[3,4-b]quinoxaline-
1,3(2H,5H)-dione (5FDQD)—an analog of riboflavin that was designed to bind riboswitches
that naturally recognize the essential coenzyme flavin mononucleotide (FMN) and regulate
FMN and riboflavin homeostasis. In vitro, 5FDQD and FMN bind to and trigger the function of
an FMN riboswitch with equipotent activity. MIC and time-kill studies demonstrated that
5FDQD has potent and rapidly bactericidal activity against Clostridium difficile. In C57BL/6
mice 5FDQD completely prevented the onset of lethal antibiotic-induced C. difficile infection
(CDI). Against a panel of bacteria representative of healthy bowel flora, the antibacterial
selectivity of 5FDQD was superior to currently marketed CDI therapeutics, with very little
activity against representative strains from the Bacteroides, Lactobacillus, Bifidobacterium,
Actinomyces, and Prevotella genera. Accordingly, a single oral dose of 5FDQD caused less
alteration of culturable cecal flora in mice than the comparators. Collectively, these data
suggest that 5FDQD or closely related analogs could potentially provide a high rate of CDI
cure with a low likelihood of infection recurrence. Future studies will seek to assess the role
of FMN riboswitch binding to the mechanism of 5FDQD antibacterial action. In aggregate, our
results indicate that riboswitch-binding antibacterial compounds can be discovered and
optimized to exhibit activity profiles that merit preclinical and clinical development as
potential antibacterial therapeutic agents.
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Despite nearly 85 years of successful antibacterial chemotherapy, bacterial infections still
pose a serious threat to human health. Continually emerging drug-resistant bacteria make
existing agents less effective, and a paucity of new agents and decreased development effort
from the pharmaceutical industry provide little hope of replenishing the arsenal (1, 2).
Complications and mortality due to bacterial infections continue to increase, already reaching
epidemic proportions in some areas of the world. If humans are to regain the upper hand in
fighting bacterial infections, then innovation, investment, and new antibacterial agents will be
needed. Although some of the barriers to the discovery and approval of new compounds are
economic or policy related, there is also a desperate need for new targets and new mechanisms
of action. A renewed effort to expand our knowledge of bacterial physiology and to translate
discoveries into the clinic will be needed to address these challenges and to reinvigorate
antibiotic development pipelines.
Recent advances in our understanding of how bacteria maintain physiological homeostasis
revealed a promising class of potential antibiotics targets called riboswitches—non-coding
mRNAs that form a structured receptor (or aptamer) which can directly bind to a specific small-
molecule ligand or ion and thereby regulate gene expression (3-5). Ligand binding to a
riboswitch aptamer stabilizes a conformationally distinct architecture in the mRNA that
modulates the expression of the adjacent coding region(s) (4-8). To date, more than 35
riboswitch classes have been discovered and characterized (7). Three of these riboswitch
classes have been revealed as important cellular targets of antibacterial small molecules whose
mechanism of action had not been previously defined (9-13). More recently, several
publications have demonstrated that novel small molecules can be rationally identified and
optimized that bind to selected riboswitch aptamers with affinity comparable to the cognate
ligand (14-21).
In some cases, synthetic or natural riboswitch ligand analogs have demonstrated potent
antibacterial activity (12-15, 20, 22). For example, the phosphorylated form of roseoflavin (RoF,
Fig. 1A), a naturally produced analog of riboflavin that inhibits the growth of Gram positive
bacteria (23, 24), directly binds to riboswitches that recognize the essential coenzyme flavin
mononucleotide, FMN (12, 13, 22). Accordingly, the presence of RoF in growth media represses
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the expression of FMN riboswitch-regulated riboflavin biosynthesis and transport genes and
inhibits bacterial growth (12, 22). The fact that RoF antibacterial action is partly due to FMN
riboswitch binding underscores the potential for developing riboswitch-targeting antibiotics.
Concurrent with these initial validation studies with RoF, we established a separate research
and development program that identified and progressively improved a class of novel flavin
analogs that bind to FMN riboswitches in vitro with potency equal to FMN (P.D.G. Coish et al.,
20 Jan 2011, PCT/US2010/001876; P.D.G. Coish et al., P.D.G. Coish et al., 13 Oct 2011,
PCT/US2011/000617; 12 Aug 2012, PCT/US2012/024507). A subset of these compounds were
found to exhibit potent and highly selective antibacterial activity against Clostridium difficile.
These findings suggest that a novel class of antibiotics could be developed that target FMN
riboswitches to provide a useful alternative to currently-approved therapies for C. difficile
intestinal infections (CDI).
In a 2013 threat assessment report, the Centers for Disease Control and Prevention
estimated that CDI causes 14,000 deaths and $1B in excess medical costs each year in the
United States (25). The current standard-of-care treatments for CDI, metronidazole or
vancomycin, are usually effective, but the infection recurs in approximately 25% of patients,
with subsequent recurrences even more likely (26-28). The broad spectrum antibacterial
activities of metronidazole and vancomycin are thought to contribute to recurrence by
preventing the regrowth of healthy intestinal flora (29-33). A newer treatment, fidaxomicin, has
a narrower spectrum of activity than vancomycin or metronidazole (34-36) and causes less
alteration to the intestinal flora of CDI patients (32, 33). Consistent with this narrower spectrum
of activity, multiple clinical trials have demonstrated a lower rate of CDI recurrence after
treatment with fidaxomicin than after vancomycin or metronidazole treatment (37, 38). As a
result, CDI therapies currently in development primarily include narrow spectrum antibiotics or
intestinal flora replacements (39-41).
Herein we report the synthesis and characterization of 5-(3-(4-fluorophenyl)butyl)-7,8-
dimethylpyrido[3,4-b]quinoxaline-1,3(2H,5H)-dione (5FDQD, Fig. 1A), an analog of FMN that
was identified via medicinal chemistry optimization of FMN riboswitch-binding ligands. 5FDQD
is rapidly bactericidal against C. difficile in vitro and can cure mice of laboratory-induced CDI
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nearly as effectively as fidaxomicin. Among a collection of bacteria representative of natural
intestinal microflora, 5FDQD has a narrower spectrum than fidaxomicin and vancomycin.
Moreover, administration of a single dose of 5FDQD to healthy mice was less disruptive of
culturable cecal flora than were fidaxomicin or vancomycin.
MATERIALS AND METHODS
Chemicals, oligonucleotides, and bacterial strains. 5FDQD was resynthesized using a
method similar to that published in patent literature (P.D.G. Coish et al., 20 Jan 2011,
PCT/US2010/001876; P.D.G. Coish et al., P.D.G. Coish et al., 13 Oct 2011, PCT/US2011/000617;
12 Aug 2012, PCT/US2012/024507). The detailed synthetic procedures and spectral
characterization are described in the Supplementary Material. Except as specifically noted, all
chemicals were purchased from Sigma-Aldrich (St. Louis, MO). Except as specifically noted, all
growth media were purchased from BD Diagnostic Systems (Sparks, MD) and prepared in
accordance with the manufacturer’s instructions, and blood supplements were purchased from
Cleveland Scientific (Bath, OH).
In-line probing assays. The 165 ribD RNA used for in-line probing assays was prepared by in
vitro transcription using a template generated from genomic DNA from Bacillus subtilis strain
168 (American Type Culture Collection, Manassas, VA). RNA transcripts were
dephosphorylated, 5′ 32P-labeled, and subsequently subjected to in-line probing using protocols
similar to those described previously (42). The KD for each ligand was derived by quantifying the
amount of RNA cleaved at each nucleotide position over a range of ligand concentrations. For
each region where modification was observed (A, B, and C in Fig. 2b), the fraction of RNA
cleaved at each ligand concentration was calculated by assuming that the maximal extent of
cleavage is observed in the absence of ligand and the minimal cleavage is observed in the
presence of the highest ligand concentration. The apparent KD was determined by using
GraphPad Prism 6 software to fit the plot of the fraction cleaved, x versus the ligand
concentration, [L], to the following equation: x = KD ([L] + KD)-1.
In vitro RNA transcription termination assays. Single-round RNA transcription termination
assays were conducted following protocols adapted from a previously described method (43).
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The DNA template covered the region −382 to +13 (relative to the start of translation) of the B.
subtilis ribD gene and was generated by PCR from genomic DNA from the 168 strain of B.
subtilis. To initiate transcription and form halted complexes, each sample was incubated at 37°C
for 10 min and contained 1 pmole DNA template, 0.17 mM ApA dinucleotide (TriLink
Biotechnologies, San Diego, CA), 2.5 μM each of ATP, GTP, and UTP, plus 2 μCi 5′-[α-32P]-UTP,
and 0.4 U E. coli RNA polymerase holoenzyme (Epicenter, Madison, WI) in 10 μl of 80 mM Tris-
HCl (pH 8.0 at 23°C), 20 mM NaCl, 5 mM MgCl2, 0.1 mM EDTA and 0.01 mg/mL bovine serum
albumin (BSA, New England Biolabs, Ipswich, MA). Halted complexes were restarted by the
simultaneous addition of a mixture of all four NTPs that yield a final concentration of 50 μM,
0.2 mg/mL heparin to prevent re-initiation, and varying concentrations of ligand as indicated to
yield a final volume of 12.5 μl in a buffer containing 150 mM Tris-HCl (pH 8.0 at 23°C), 20 mM
NaCl, 5 mM MgCl2, 0.1 mM EDTA and 0.01 mg/mL BSA. Reactions were incubated for an
additional 20 min at 37°C, and the products were separated by denaturing 10% polyacrylamide
gel electrophoresis (PAGE) followed by quantitation of the fraction terminated at each ligand
concentration by using a phosphorimager (Storm 860, GE Healthcare Life Sciences, Pittsburgh,
PA).
In vitro antibacterial activity. MIC analyses were conducted by Micromyx, LLC (Kalamazoo,
MI) in accordance with standard microdilution methods for aerobic (44) and anaerobic bacteria
(45). All procedures involving anaerobic bacteria were conducted within a Bactron 300 or
Bactron II anaerobic chamber (Sheldon Manufacturing, Cornelius, OR) using pre-reduced media.
MICs for anaerobic bacteria were determined in supplemented Brucella broth, comprising BBL
Brucella broth, 5 μg/mL hemin, 10 μg/mL vitamin K, and 5% lysed horse blood. MICs for aerobic
bacteria were determined in Mueller-Hinton II broth, and a supplement of 3% lysed horse blood
was included for assays with Streptococcus pneumoniae. Stock solutions of fidaxomicin (Ontario
Chemicals, Guelf, ON) and 5FDQD in dimethylsulfoxide (DMSO) or vancomycin in water were
used to prepare dilution plates such that the final concentration of DMSO in the assay did not
exceed 5%. Time-kill assays for bactericidal activity were conducted at 35 °C in supplemented
Brucella broth. A suspension of C. difficile strain VPI 10463 (ATCC 43255) was prepared from
day-old colonies on supplemented Brucella agar (BBL Brucella agar, 5 μg/mL hemin, 10 μg/mL
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vitamin K, and 5% lysed sheep blood). 10-fold dilutions of the suspension spanning 102 to 105
were prepared and allowed to grow overnight in broth at 35°C. On the following morning, a
culture with a measurable OD600 less than 0.4 was diluted by 40 fold into 12 mL of fresh broth
that had been pre-warmed to 35°C. 2 mL aliquots of this culture were immediately mixed with
100 μL of DMSO or a solution of 5FDQD, fidaxomicin, or vancomycin sufficient to give the final
concentrations indicated in Fig. 2. Serial dilutions of the starter culture into pre-reduced
phosphate-buffered saline (PBS) were plated onto brain heart infusion agar supplemented with
0.5% yeast extract and 0.1% L-cysteine (BHIS) to measure the density at time zero and to
confirm that the starting inoculum was greater than 5 x 105 CFU/mL (46). Serial dilutions of
each test culture were prepared in PBS and plated onto BHIS agar at the indicated time points,
and colonies were counted after 24 h and used to calculate the culture densities in CFU/mL. The
lower limit of detection using this method was 200 CFU/mL.
Preparation of C. difficile spores for infecting mice. C. difficile spores for the mouse
infection model were prepared at Micromyx (Kalamazoo, MI). A frozen culture of C. difficile VPI
10463 was inoculated into 4 L of pre-reduced sporulation medium comprising 80 g/L
Trypticase™ Peptone, 5 g/L Proteose Peptone no. 3, 1 g/L ammonium sulfate, and 1.5 g/L Tris
adjusted to a final pH of 7.4. The culture was incubated in an anaerobic chamber at 35°C for 5
days, centrifuged, resuspended in 50% ethanol, and incubated at room temperature to
eliminate any remaining vegetative bacterial cells. The spores were then washed twice in sterile
PBS and enumerated by dilution plating onto supplemented Brucella agar with 0.1% sodium
taurocholate. Following quantitation, the spores were diluted to a concentration of 2 x 106
spores/mL and stored at -80°C.
Mouse model of CDI. CDI efficacy studies were conducted by Aragen Biosciences (Morgan
Hill, CA) and were reviewed and approved by Aragen’s Institutional Animal Care and Use
Committee (IACUC). Female C57BL/6 mice weighing 18-21 g were ordered from Harlan
Laboratories (Indianapolis, IN), housed five per cage in a dedicated room, and allowed to
acclimatize for at least three days. Animals were fed Teklad Global Rodent Diet 2018-R (Harlan
Laboratories, Indianapolis, IN) throughout the study. For the seven days following
acclimatization (days -10 through -4), mice were provided an antibiotic cocktail in their
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autoclaved drinking water that included kanamycin (0.5 mg/mL), gentamicin (0.044 mg/mL),
colistin (1062.5 U/mL), metronidazole (0.269 mg/mL), ciprofloxacin (0.156 mg/mL), ampicillin
(0.1 mg/mL) and vancomycin (0.056 mg/mL). Mice were then returned to normal autoclaved
drinking water for three days (days -3 through -1), followed by a single oral dose of 10 mg/kg
clindamycin in a volume of 0.5 mL. Immediately following clindamycin dosing, animals were
randomized and distributed into new cages of five animals such that the average weight across
the groups of five animals was similar. On the following day (day 0), an inoculum of 2,000
spores of C. difficile VPI 10463 (ATCC 43255) in 0.5 mL of sterile PBS was orally administered to
each mouse. Within one hour of inoculation, infected mice were administered the treatments
indicated in Fig. 3 by oral gavage twice a day (with approximately 10 hours elapsing between
AM and PM treatments) for five days, with ten animals per treatment group. All test agents
were dosed as a suspension in 0.5% aqueous methylcellulose. Body weights were measured
three times a week prior to C. difficile infection and daily following infection. Animals were
examined in the morning and evening for eight consecutive days following spore inoculation for
clinical signs of morbidity, evidence of diarrhea, and mortality. At the end of the study,
surviving animals were humanely euthanized.
Pharmacokinetic analysis. The Yale University IACUC reviewed and approved all animal
experimental protocols for pharmacokinetic analysis (protocol number 20114-11652). Female
C57BL/6 mice weighing 18-21 g were ordered from Harlan Laboratories, housed five per cage,
and allowed to acclimatize for fourteen days. Animals were fed Teklad Global Rodent Diet
2018-R throughout the study. After acclimatization, 24 mice each received a single oral dose of
0.2 mL of a 1 mg/mL suspension of 5FDQD in aqueous 0.5% methylcellulose for a total dose of
10 mg/kg. At 1, 2, 4, 6, 8, and 24 h after dosing, three mice per time point were anesthetized
with isoflurane and exsanguinated via cardiac puncture with a pre-heparinized syringe, and the
collected blood was transferred to lithium heparinized tubes and held on ice until further
processing. After exsanguination the mice were humanely euthanized by cervical dislocation,
the ceca were immediately removed, and the cecal contents were collected into pre-weighed
tubes and frozen at -80°C until extraction. The previously collected blood samples were
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centrifuged at 4°C for 10 min at 6,000 RPM, and the plasma supernatant was collected into a
separate tube and frozen at -80°C until extraction.
Sample extraction and bioanalyses were conducted by Northeast Bioanalytical (Hamden,
CT). Cecal content samples were suspended in 2.0 mL of extraction/precipitation solution that
contained 50% acetonitrile, 50% methanol, and 0.1% formic acid. The resulting suspensions
were placed on a shaker table for 20-30 min, followed by centrifugation. Thawed plasma
samples were combined with three volumes of extraction/precipitation solution, mixed, and
allowed to warm to room temperature. A 100 µl aliquot of the cleared cecal extraction
supernatant or plasma solution was transferred into a labeled autosampler vial and combined
with 100 µl of an internal standard compound and 200 µl of extraction/precipitation solution.
Samples were then capped, vortexed, and analyzed by liquid chromatography-tandem mass
spectrometry (LC-MS/MS).
Test samples were injected onto a Phenomenex Synergi Polar-RP column (150 x 4.6 mm)
with a corresponding guard column. The column temperature was maintained at 50°C. The
analytes were separated using a variable composition of eluent A (acetonitrile) and eluent B
(HPLC grade water with 0.1% formic acid). The initial composition of 40% eluent A and 60%
eluent B was maintained at a flow rate of 1,000 μL/min for 1 min, at which point the
composition of eluent A was increased to 95% over 0.5 min and held at 95% for 4 min at a flow
rate of 1,300 μL/min. The composition of eluent A was then decreased to 40% at a flow rate of
1,100 μL/min and held at 40% for 0.5 min, at which point the flow rate was reduced to 1,000
μL/min and re-equilibrated before injecting another sample. Analyte detection was performed
by a Sciex API-4000 tandem mass spectrometer fitted with a Turbo Ion Spray source operating
in positive ion mode. Samples were quantitated against a calibration standard curve
constructed from a stock solution of a known concentration of 5FDQD and using a linear
regression of theoretical concentration of calibrators versus the response ratio (where the
response ratio = analyte peak area/labeled internal standard peak area). The regression used a
1/x2 weighting factor in the following equation y = mx + b. An internal standard that is a
structural analog of 5FDQD was included in all samples. The lower and upper limits of
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quantitation for 5FDQD in plasma and cecal contents were 12 ng/mL and 5,000 ng/mL,
respectively.
Cecal flora analysis. The Yale University IACUC reviewed and approved all animal
experimental protocols for cecal flora analysis (protocol number 20114-11652). A total of 28
female C57BL/6 mice weighing 18-21 g were ordered from Harlan Laboratories, housed four per
cage, and allowed to acclimatize for seven days. Animals were fed Teklad Global Rodent Diet
2018-R throughout the study. After acclimatization, mice were randomized into groups of four
mice per cage for each treatment group. All four mice in a given treatment group received an
oral dose of 0.2 mL of aqueous 0.5% methylcellulose (vehicle control) or 0.2 mL of an aqueous
0.5% methylcellulose suspension of 5FDQD, fidaxomicin, or vancomycin in aqueous 0.5%
methylcellulose such that the total doses administered were 10 or 50 mg/kg 5FDQD, 3 or 50
mg/kg fidaxomicin, or 50 mg/kg vancomycin. 24 h after dosing, mice were humanely
euthanized by cervical dislocation, the ceca were immediately removed, and the cecal contents
were collected into preweighed tubes, weighed, and transferred immediately into an anaerobic
chamber such that cecal contents were never exposed to ambient atmosphere for greater than
10 min. Within the anaerobic chamber, the cecal contents were resuspended in 1 mL of pre-
reduced 0.05% yeast extract per 100 mg of contents and homogenized by vortex mixing. A
series of 10-fold serial dilutions ranging from 101 to 107 were prepared in 0.05% yeast extract,
and 50 μL aliquots of each dilutions were plated in parallel onto agar media selective for each
bacterial taxa, including Bacteroides bile esculin agar (47), Bifidobacterium-selective agar (48),
Lactobacillus-selective agar (49), Enterococcosel, MacConkey agar, and mannitol salt agar. To
enumerate Clostridium spores, an aliquot of the 101 and 102 dilutions for each animal was
mixed with an equal volume of ethanol and incubated for 1 h to kill vegetative cells. 50 μL of
each dilution was then plated onto egg yolk agar (Anaerobe Systems, Morgan Hill, CA). After
growth at 35°C for the number of days appropriate to each medium and bacterial type, colonies
were counted, categorized according to visible morphology and were used to calculate the
density of each taxa in the starting cecal contents in units of log CFU/mL. The reported densities
represent an average of all countable dilution plates from four mice per treatment group. Each
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colony type was presumptively identified to the genus or species level based on visible
appearance, Gram stain, and microscopic appearance. Where necessary, catalase, indole, and
oxidase tests and antimicrobial disc susceptibility testing were used to confirm the presumptive
identifications (47). Clostridium spiroforme was presumptively identified by colony morphology
on egg yolk agar, Gram stain, ability to form spores, a unique helical shape viewable under a
microscope, and antimicrobial susceptibility pattern including rifampicin resistance (50, 51). The
relative density of vegetative C. spiroforme was enumerated in parallel by dilution plating of
selected cecal content samples onto brucella agar that contained 5% defibrinated sheep blood
and 50 μg/mL rifampicin.
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Results
The FMN analog 5FDQD is bound by FMN aptamers and modulates riboswitch activity. An
in-line probing assay (42) was used to assess the binding of 5FDQD to the ribD FMN riboswitch
aptamer from B. subtilis (Fig. 1B) and to determine an apparent dissociation constant (KD) for
the binding interaction. This assay exploits the fact that the internucleotide linkages in
unstructured regions of an RNA will usually undergo phosphoester cleavage more rapidly than
those in structured regions (52). The heightened flexibility of unstructured regions usually
permits more frequent access to a conformation necessary for spontaneous cleavage by an
internal RNA phosphoester transfer reaction. As the secondary and tertiary structures of the
RNA change upon binding to a ligand, the reactivity of some of the internucleotide linkages will
change, resulting in a different pattern of degradation products. The differences in RNA
degradation products and the location of structural changes can be detected and quantified via
denaturing PAGE.
Using the in-line probing assay, 5FDQD and FMN induce nearly identical structural changes
in the B. subtilis ribD FMN aptamer (Fig. 1C, regions 1 through 6). The only observable
difference was that 5FDQD binding increases spontaneous cleavage of the internucleotide
linkage between G33 and G34, whereas FMN decreases reactivity at this same position (Fig. 1C,
arrowhead). The three-dimensional x-ray structure model of a Fusobacterium nucleatum FMN
riboswitch aptamer complexed with FMN predicts an interaction between G34 and a hydroxyl
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group of the ribityl side-chain of FMN (53). This same interaction cannot be formed in a
complex with 5FDQD because the analog lacks hydroxyl groups, which likely explains this single
difference in reactivity as revealed by in-line probing. Therefore, the natural FMN ligand and
the FMN analog 5FDQD are most likely recognized by the same binding pocket and induce
nearly identical structural changes in the aptamer.
RNA-ligand binding curves were established by evaluating the relative change in cleavage at
regions 1 through 6 as a function of compound concentration (Fig. 1D). The concentrations of
ligands needed to half-maximally modulate RNA aptamer structure, representing KD values,
were 7.5 nM and 6.4 nM for 5FDQD and FMN, respectively. Thus, 5FDQD and FMN bind to the
B. subtilis ribD FMN aptamer in vitro with equivalent potency. Moreover, the binding curves
suggest that both compounds bind a single saturable site with 1-to-1 stoichiometry.
An in vitro transcription termination assay was performed to determine whether 5FDQD can
induce FMN riboswitch function (Fig. 1E,F). Using a double-stranded DNA template that
included the B. subtilis ribD endogenous promoter, FMN riboswitch region, and the first 13
nucleotides of the open reading frame, the concentration at which FMN or 5FDQD induce half-
maximal transcription termination (T50) was determined. The T50 values for 5FDQD and FMN
were 0.5 μM and 0.6 μM, respectively, indicating that 5FDQD can induce riboswitch function in
vitro, again with the same potency as FMN.
5FDQD exhibits robust and selective antibacterial activity. Against a panel of 21 strains of
C. difficile, including four hypervirulent North American pulsed-field gel electrophoresis type 1
(NAP1) ribotype 027 strains (54, 55) and 14 isolates from hospital patients, the MIC50 and MIC90
of 5FDQD were determined to be 1 μg/mL by broth microdilution methods (Table 1), compared
to MIC50 and MIC90 values of 0.06 and 0.12 μg/mL for fidaxomicin. Although less potent than
fidaxomicin, 5FDQD shows equivalent potency to published values for both vancomycin and
metronidazole (56). Notably, there was less variability among the MIC values for 5FDQD than
for fidaxomicin among the isolates tested.
The bactericidal activity of 5FDQD, fidaxomicin, and vancomycin were compared via a time-
kill assay with C. difficile VPI 10463 (ATCC 43255, Fig. 2). At a concentration 4- or 8-fold higher
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than the MIC, 5FDQD reduced the viable density of C. difficile by 99.9% to 1.7 x 103 and 8.0 x
103 CFU/mL, respectively, within 4 h. Within 6 h, incubations containing these concentrations of
5FDQD reduced viable cells below the 200 CFU/mL limit of quantitation. Similar bactericidal
activity was observed for 5FDQD against C. difficile strain 630 (data not shown). In contrast, at a
concentration 4-fold above their MIC values fidaxomicin and vancomycin only reduced the
viable density by 6.3- and 64-fold, respectively, which is consistent with their previously
reported activity (57). Based on these data, 5FDQD appears more rapidly bactericidal than
these well-established CDI therapeutic compounds.
To assess the selectivity of 5FDQD antibacterial activity, we measured MIC values against a
diverse panel of anaerobic and aerobic bacteria that included many common intestinal flora
species (Table 1). Neither 5FDQD nor fidaxomicin showed appreciable activity against B. fragilis
group species, whereas vancomycin and metronidazole are known to inhibit the B. fragilis
group at pharmacologically relevant concentrations (35, 39, 58). Likewise, little to no activity
was observed for 5FDQD and fidaxomicin against other anaerobic Gram negative rods, which
are usually susceptible to metronidazole but not vancomycin. Interestingly, 5FDQD showed no
appreciable activity against the Bifidobacterium, Lactobacillus, or Actinomyces species tested,
whereas fidaxomicin inhibited these strains as potently as it inhibits C. difficile. The activity of
fidaxomicin, vancomycin, and metronidazole against these species has been well documented
elsewhere (35, 58). Based on these data, the selectivity of 5FDQD with respect to anaerobic
intestinal flora appears to be at least as good as, or superior to, currently marketed CDI
therapeutic compounds. 5FDQD also exhibited very little activity against representative aerobic
pathogens, with the exception of activity against some strains of Enterococcus faecalis.
In vivo efficacy. An antibiotic-induced mouse CDI model was used to evaluate the in vivo
efficacy of 5FDQD, fidaxomicin, and vancomycin. In accordance with published protocols (59),
CDI was established in C57BL/6 mice by administering an antibiotic cocktail via their drinking
water to disrupt intestinal flora and an oral dose of C. difficile VPI 10463 spores. If the infected
mice are not subsequently treated with an effective antibiotic, they exhibit severe diarrhea,
lethargy, weight loss, failure to groom, and 40-60% mortality within 48 h (59, 60).
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Using this model, the efficacy of 5FDQD was compared to that of fidaxomicin and
vancomycin, with all test agents suspended in aqueous 0.5% methylcellulose and administered
orally twice each day for five days, except vancomycin, which was administered once a day for
five days. Mice that received only the 0.5% methylcellulose vehicle orally developed signs of
severe CDI within three days of inoculation, and 40% died by day 6 (vehicle, Fig. 3A). The
average weight of surviving vehicle-treated mice relative to their weight at the time of
inoculation decreased by up to 17% during the study (Fig. 3B).
By contrast, treatment with 10 mg/kg 5FDQD prevented any mortality or weight loss, with
the average weight increasing by 6% by day 8. Furthermore, although symptoms of mild
diarrhea developed in one mouse, the symptoms resolved by day 3 of dosing. The activity of 10
mg/kg 5FDQD is comparable to the activity of both 3 mg/kg fidaxomicin and 50 mg/kg
vancomycin. Symptoms of mild diarrhea were observed in two of the fidaxomicin-treated mice
and one vancomycin-treated mouse, but the symptoms resolved by day 4. These data indicate
that 5FDQD prevented the onset of severe CDI in mice as effectively as fidaxomicin and
vancomycin.
Pharmocokinetics of 5FDQD are advantageous for treating intestinal tract infections. To
assess the pharmacokinetic distribution of 5FDQD at an efficacious dose, mice were
administered a single 10 mg/kg oral dose of 5FDQD, and the concentration of the compound in
the cecal contents and plasma was measured at specific time points after dosing. Levels of
5FDQD in the cecal contents were 192, 204, 174, 86, 82, and 0.3 μg/g at 1, 2, 4, 6, 8, and 24 h
after dosing, respectively. This rapid transit into the lower intestinal tract followed by a
sustained concentration over several hours is comparable to the distribution observed after a
single dose of oral fidaxomcin (61). Levels of 5FDQD in the plasma were 23 ng/mL at 1 h and
below the limit of quantitation (12 ng/mL) at the remaining times. These very low plasma levels
would constitute a potential safety advantage for a drug intended to treat an intestinal tract
infection such as caused by C. difficile.
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Cecal flora is only modestly affected by 5FDQD. Since the disruption of endogenous
intestinal flora is closely linked with both initial C. difficile infection and with recurrence rates
(40, 62), we evaluated the extent to which 5FDQD, fidaxomicin, or vancomycin altered the
culturable cecal flora in mice. A single oral dose of each compound or of the vehicle alone was
administered to uninfected, healthy C57BL/6 mice. After 24 h, the mice were euthanized, the
visible morphology of the ceca were recorded, and the densities of representative bacterial
taxa within the cecal contents were determined by plating onto agar media selective for B.
fragilis, bifidobacteria, lactobacilli, enterococci, enterobacteriaceae, or staphylococci. In
addition, the density of Clostridium spores in the cecal contents was determined by incubating
an aliquot of the cecal contents with ethanol for 1 h followed by dilution plating onto egg yolk
agar (63). The identity of each countable colony type on each media type subsequently was
confirmed to the genera or species level (see MATERIALS AND METHODS).
Ceca from mice treated with the aqueous 0.5% methylcellulose vehicle were visually
indistinguishable from ceca isolated from untreated mice. The cecal contents from vehicle-
treated mice had a high density of culturable B. fragilis, bifidobacteria, and lactobacilli (2.1 x
107, 9.3 x 108, and 2.0 x 107 CFU/g, respectively) and a lower density of Clostridium spores,
enterococci, enterobacteriaceae, and staphylococci (6.8 x 104, 7 x 104, 4.6 x 103, and 3.0 x 104
CFU/g, respectively) (Fig. 4). This distribution was very similar to that measured for cecal
contents from untreated mice (data not shown) and is consistent with previous
characterizations of mouse cecal flora (64-66).
Treatment with a single 10 mg/kg or 50 mg/kg oral dose of 5FDQD did not affect the visible
morphology of the ceca and had very little effect on the densities of the cultured cecal flora
groups compared to the vehicle-treated mice. The only difference observed was a 5-fold
increase in the density of enterobacteriaceae. Thus, even at a dose 5-fold above the efficacious
dose, 5FDQD did not dramatically alter the cecal morphology or flora. Speculation on why
5FDQD is highly selective is presented in the Discussion section (see below).
Ceca isolated from mice treated orally with 3 mg/kg fidaxomicin appeared normal, but
those isolated from mice treated orally with 50 mg/kg fidaxomicin were visibly larger than
those from vehicle- or 5FDQD-treated mice, and the mass of their cecal contents was increased
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by 40%. Treatment with a single 3 mg/kg oral dose of fidaxomicin also resulted in a 10-fold
reduction of B. fragilis, a 10-fold increase of enterobacteriaceae, and a 3-fold increase in
Clostridium spores relative to vehicle-treated mice. These changes are consistent with
alterations to bowel flora observed in human patients treated with fidaxomicin (32, 33).
Treatment with 50 mg/kg fidaxomicin altered the flora to a greater extent, most notably a
>1,000-fold increase in enterobacteriaceae (Fig. 4). Although the density of Clostridium spores
was not dramatically altered by either dose of fidaxomicin, the diversity of clostridial species
was decreased as judged by colony visual appearance and microscopic examination. One colony
type presumptively identified as C. spiroforme (51) was increased 10-fold by fidaxomicin
treatment to become the predominant culturable Clostridium species. Overall, fidaxomicin
exhibited a moderate, dose-responsive effect on both cecal morphology and culturable cecal
flora populations.
Ceca from mice treated orally with 50 mg/kg vancomycin mice were significantly enlarged,
had visible gas bubbles, and contained 80% more contents by weight than ceca from vehicle- or
5FDQD-treated mice. Vancomycin also profoundly altered the culturable cecal flora (Fig. 4). B.
fragilis, Clostridium spores, and staphylococci were decreased by 10,000-, 200-, and 10-fold,
respectively, and lactobacilli, enterococci, and enterobacteriaceae were increased by 100-, 32,
and >30,000-fold, respectively. These changes are consistent with the large alterations of bowel
flora that vancomycin causes in human patients (30, 32). Overall, vancomycin treatment
dramatically alters both cecal morphology and culturable cecal flora.
Discussion
Given the urgent need for new antibiotics targets and lead compounds, we have established
a research program aimed at identifying antibacterial compounds that target riboswitches.
From a starting point of FMN and RoF, an intensive medicinal chemistry optimization effort
yielded a series of flavin analogs that are potent FMN riboswitch ligands in vitro and that inhibit
the growth of certain Gram positive bacteria (P.D.G. Coish et al., 20 Jan 2011,
PCT/US2010/001876; P.D.G. Coish et al., P.D.G. Coish et al., 13 Oct 2011, PCT/US2011/000617;
12 Aug 2012, PCT/US2012/024507). Herein, we report the in vitro and in vivo characterization
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of one of these compounds 5FDQD—a flavin analog that differs from FMN in that it has an N1-
deaza flavin core and in that the ribityl phosphate moiety is replaced by an aryl-alkyl moiety.
The observation that in vitro 5FDQD binds to and triggers the function of an FMN riboswitch
with the same potency as FMN is remarkable given the network of interactions that the
aptamer appears to form with the ribose-phosphate moiety of FMN in crystallographic
structure models (53, 67). Our in-line probing assay data indicate that 5FDQD binds to the
riboswitch in the same region as FMN and causes nearly the same structural rearrangements,
suggesting that the aptamer pocket is capable of forming equally favorable interactions with
the aryl-alkyl side chain of 5FDQD. The transcription termination data confirm that these
interactions are sufficient for 5FDQD to trigger riboswitch function in vitro with the same
potency as FMN.
Replacement of the ribityl-phosphate moiety of FMN with a hydrophobic moiety to yield
compounds that retain riboswitch binding function constitutes a key medicinal chemistry
advance. This modification obviates the need to deliver a phosphorylated drug to the target cell
or to deliver a pro-drug that the target cell needs to phosphorylate in order to achieve full
potency. These differences greatly expands the diversity of possible functional analogs. By
contrast, the weak riboswitch-binding potency of the natural compound RoF in vitro suggest
that it needs to be phosphorylated to RoF-P inside cells to be active (12), and this necessity
likely diminishes the range of compound variations that could serve as effective antibiotics.
Thus, 5FDQD and related analogs have two key advantages that are common characteristics of
some classes of drugs (68). First, molecules in this series are uncharged and have reduced
polarity compared to FMN or RoF, which might favor their uptake by target cells. Second, the
compounds bind their RNA targets with high affinity without the need for modification by
cellular enzymes. Moreover, our results suggest that 5FDQD is a representative of a much
larger class of compounds that could be created based on this scaffold and that additional
compounds could be generated and tested to further optimize the function of FMN riboswitch-
binding antibiotic compounds.
In its current form, 5FDQD is a potent C. difficile antibacterial agent with an MIC90 of 1
μg/mL among strains tested herein, including four hypervirulent NAP-1 isolates. The C. difficile
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MIC90 values for 5FDQD are equivalent to those reported for metronidazole, vancomycin, and
surotomycin, a phase III investigational agent for CDI therapy (56, 69). Although 5FDQD is less
potent against C. difficile than fidaxomicin, the available fecal concentration of 5FDQD after a
single oral dose is well above the MIC and at a concentration that rapidly kills C. difficile in vitro.
Accordingly, 5FDQD can completely prevent the onset of severe antibiotic-induced CDI in mice
at an oral dose of 10 mg/kg twice daily for five days. In three repeated side-by-side
comparisons, 10 mg/kg 5FDQD showed equivalent potency to 3 mg/kg fidaxomicin. Given the
high cecal bioavailability after dosing, it is likely that lower doses would also be effective. Future
studies could be designed to determine a minimal effective dose of 5FDQD. However, it might
also be beneficial to determine the maximal tolerated dose and whether higher concentrations
of drug might clear an infection sooner and with fewer doses. Overall, the antibacterial activity
and in vivo efficacy of 5FDQD against C. difficile are comparable to known CDI therapeutics or
development candidates.
At present, the mechanism of 5FDQD antibacterial activity has not been definitively
confirmed. Preliminary attempts to isolate 5FDQD-resistant C. difficile by single-step or serial
passage methods have not yielded isolates with a stably increased MIC. These effortsindicate
that the frequency of resistance development is <1 x 10-9 (data not shown). If confirmed, this
low propensity to develop resistance could suggest either that mutations in the primary target
are not well tolerated or that there are multiple targets. In support of a riboswitch-targeting
component of the mechanism, 5FDQD binds to and triggers the function of an FMN riboswitch
in vitro with potency comparable to that of the active phosphorylated form of RoF, whose
antibacterial activity is at least partly attributable to the repression of FMN riboswitch-
regulated genes (12, 13, 22). Moreover, among a collection of 5FDQD analogs, a correlation
exists between MIC and in vitro riboswitch binding potency (data not shown). However, it is
also conceivable that inhibition of flavoprotein-related processes could be a component of the
5FDQD antibacterial activity as has been suggested for RoF (70, 71). Efforts are currently
underway in the Breaker laboratory to isolate 5FDQD-resistant C. difficile and to definitively
characterize the mode of action for 5FDQD, including elucidating the relative importance of
FMN riboswitch binding.
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It is well established that healthy, diverse intestinal flora protects against C. difficile
colonization and reduces the likelihood of recurrence after an initial CDI episode (72-74).
Although the specific taxa responsible for colonization resistance have not been unequivocally
established, it is currently thought that bacteria of the Bacteroides, Bifidobacterium, and
Prevotella genera as well as the Ruminococcaceae, Lachnospiraceae, and Porphyromonadaceae
families are major contributors (75-80). Like fidaxomicin, 5FDQD is inactive against most of the
aerobes and Gram negative anaerobes tested herein, including the B. fragilis group and
Prevotella species. Favorably, 5FDQD is less active than fidaxomicin against the Bifidobacterium,
Lactobacillus, and Actinomyces strains tested herein. Likewise, the MICs of 5FDQD against
representatives of these genera are higher than the published MICs of fidaxomicin, vancomycin,
metronidazole, and two clinical development candidates for CDI treatment—LFF-571 and
surotomycin (39, 41, 58, 81). Thus, the spectrum of 5FDQD appears to be more narrow
compared to established and developmental CDI therapeutics and should theoretically permit
the regrowth of intestinal flora that protect against recurrence. At this time, the basis for the
remarkable selectivity of 5FDQD is not entirely clear. Earlier analogs of 5FDQD were found to be
modestly inhibitory toward an efflux-deficient ΔnorA strain of S. aureus (82) or chemically-
permeabilized E. coli (83), suggesting that 5FDQD either does not readily enter or is effluxed
from most of the bacterial species tested (data not shown). Future studies will seek to confirm
the narrow spectrum of 5FDQD against a larger collection of representative species.
The narrower spectrum of 5FDQD compared to fidaxomicin and vancomycin was evident
from the effect of each agent on the spectrum of bacteria cultured from mouse cecal flora. At
comparable doses in healthy mice, 5FDQD was significantly less disruptive of cultured cecal
flora than both fidaxomicin and vancomycin. No changes in excess of one order of magnitude
were observed after a single oral dose of up to 50 mg/kg 5FDQD, whereas a single fidaxomicin
dose caused reduced cecal B. fragilis density and Clostridium diversity and increased
enterobacteriaceae density. Interestingly, overgrowth of enterobacteriaceae is positively
correlated with a number of intestinal disorders including C. difficile colonization (74, 84-86).
Although the taxa cultured herein represent a subset of the total flora, the measured changes
are consistent with the known effects of fidaxomicin and vancomycin on bowel flora (32, 33).
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The lower impact of fidaxomicin on human bowel flora compared to vancomycin is widely
believed to be the basis for a lower rate of CDI recurrence. Therefore, it is conceivable that
treatment with a selective agent like 5FDQD would lead to fewer recurrences in humans.
Efforts are now underway to evaluate the likelihood of CDI recurrence after treatment utilizing
a hamster model (87) and a recently developed mouse recurrence model (88).
In summary, our efforts to optimize new FMN riboswitch-targeting antibiotics resulted in
the identification of a novel series of flavin analogs that are potent and highly selective C.
difficile antibacterial compounds. The most potent of these analogs examined to date, 5FDQD,
can prevent lethal CDI in mice without disrupting the regrowth of beneficial flora. Further
exploration of this compound series appears to be a promising route for the development of
new CDI therapeutics. The success of this program also underscores the potential for the
development of riboswitch-targeting compounds for treating other bacterial infections.
Acknowledgements
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The original design and synthesis of 5FDQD was supported by funds from BioRelix, Inc. (New
Haven, CT). Resynthesis of 5FDQD was funded by HHMI research support to RRB and by the
Yale Center for Molecular Discovery. Animal testing was supported by BioRelix, Yale University,
and by HHMI. We thank Malavika Ghosh and Rashmi Munshi at Aragen for conducting in vivo
studies. We thank Dean Shinabarger and Beverly Murray at Micromyx for conducting MIC
studies. We thank Ron Mays and Vipin Agarwal at Northeast Bioanalytical for conducting
pharmacokinetic bioanalyses.
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