Discovery of a Cyclic Choline Analog That Inhibits Anaerobic Choline Metabolism by Human Gut Bacteria

Article abstract of DOI:10.1021/acsmedchemlett.0c00005

The anaerobic conversion of choline to trimethylamine (TMA) by the human gut microbiota has been linked to multiple human diseases. The potential impact of this microbial metabolic activity on host health has inspired multiple efforts to identify small molecule inhibitors. Here, we use information about the structure and mechanism of the bacterial enzyme choline TMA-lyase (CutC) to develop a cyclic choline analog that inhibits the conversion of choline to TMA in bacterial whole cells and in a complex gut microbial community. In vitro biochemical assays and a crystal structure suggest that this analog is a competitive, mechanism-based inhibitor. This work demonstrates the utility of structure-based design to access inhibitors of radical enzymes from the human gut microbiota.

Full text of DOI:10.1021/acsmedchemlett.0c00005

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Letter
Discovery of a Cyclic Choline Analog That Inhibits Anaerobic
Choline Metabolism by Human Gut Bacteria
Maud Bollenbach, Manuel Ortega, Marina Orman, Catherine L. Drennan, and Emily P. Balskus*
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ABSTRACT: The anaerobic conversion of choline to trimethyl-
amine (TMA) by the human gut microbiota has been linked to
multiple human diseases. The potential impact of this microbial
metabolic activity on host health has inspired multiple efforts to
identify small molecule inhibitors. Here, we use information about
the structure and mechanism of the bacterial enzyme choline TMA-
lyase (CutC) to develop a cyclic choline analog that inhibits the
conversion of choline to TMA in bacterial whole cells and in a
complex gut microbial community. In vitro biochemical assays and a
crystal structure suggest that this analog is a competitive, mechanism-
based inhibitor. This work demonstrates the utility of structure-based
design to access inhibitors of radical enzymes from the human gut
microbiota.
KEYWORDS: Gut microbiota, choline, trimethylamine, small molecule inhibitor, cardiometabolic disease
he human gastrointestinal (GI) tract is colonized by
trillions of microorganisms that have coevolved with the
cardiovascular disease,12−16 chronic kidney disease,17,18 type 2
diabetes,19 and artherosclerosis.20,21 If mutations in the gene
encoding FMO3 result in a deficiency of this enzyme, TMA
builds up in the body and its excessive excretion leads to a
distinct odor. This inherited metabolic disorder is known as
trimethylaminuria or “fish odor syndrome”.22,23 The correla-
tion between TMA production by the gut microbiota and
diseases, as well as its prevalence in the human gut microbiota,
makes this metabolic activity a prominent target for
manipulation and further study.
We have discovered and biochemically characterized the
enzymes responsible for the key C−N bond cleavage reaction
that converts choline to TMA: the glycyl radical enzyme
(GRE) choline TMA-lyase (CutC) and its corresponding
radical S-adenosylmethionine (SAM) activating protein
(CutD).6,24−26 GREs are a family of oxygen-sensitive enzymes
that use protein-based radical intermediates to catalyze
chemically challenging reactions. Well characterized family
members include pyruvate formate-lyase, class III (anaerobic)
ribonucleotide reductase, benzylsuccinate synthase, and 4-
hydroxyphenylacetate decarboxylase.27 The glycyl radical is
installed by a dedicated partner enzyme that is a member of the
T
host. These organisms play an important role in maintaining
host health by performing critical biological functions,
including metabolizing inaccessible dietary components,
synthesizing essential vitamins and nutrients, providing
protection from invading pathogens, and priming the immune
system.1,2 Gut microbial metabolic activities have also been
linked to human disease. Indeed, metabolomics experiments
have showed strong correlations between levels of human
serum metabolites made or modified by gut microbes and
various disease states.1,3
Anaerobic gut microbial choline metabolism is a disease-
associated activity that has received much attention as a
potential target for therapeutic intervention. While choline is
an essential nutrient for the host, contributing to cell
membrane function, methyl transfer events, and neuro-
transmission,4 gut anaerobes use choline as a source of carbon
and energy, cleaving its C−N bond to generate acetaldehyde,
which is further processed by alcohol dehydrogenase CutO
and aldehyde oxidoreductase CutF to give ethanol and acetyl
coenzyme A,5,6 and trimethylamine (TMA), a uniquely
microbial metabolite (Figure 1A).7,8 While gut microbes
generate TMA from multiple sources, including the nutrients
carnitine and glycine betaine, choline is considered to be the
major precursor.7 In the human body, TMA is further oxidized
to trimethylamine-N-oxide (TMAO) by the liver enzyme
flavin-dependent monooxygenase 3 (FMO3).9 Elevated levels
of TMAO in human plasma have been linked to multiple
diseases such as nonalcoholic fatty liver disease (NAFLD),10,11
Special Issue: Medicinal Chemistry: From Targets to
Therapies
Received: January 3, 2020
Accepted: March 25, 2020
Published: March 25, 2020
https://dx.doi.org/10.1021/acsmedchemlett.0c00005
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Figure 2. Previously reported small molecule inhibitors of anaerobic
bacterial choline metabolism.
with a different target. Subsequently, Hazen et al. reported
greatly improved rationally designed CutC inhibitors, includ-
ing iodomethylcholine (IMC)28 and various N,N-dimethyl-
prop-2-yn derivatives29−32 (Figure 2). Bhandari and co-
workers recently described a pyridine derivative, trigonelline,
that reduces TMA production from choline by inhibiting
growth of choline metabolizing bacterial strains, like
Citrobacter freundii, and also directly inhibits FMO3 (Figure
2).33 More recently, Boehringer-Ingelheim used a matrix-
assisted laser desorption/ionization time-of-flight (MALDI-
TOF)-based high-throughput screen with whole cells of
Clostridium hathewayi to identify new inhibitors of choline
metabolism.34 This study revealed the ability of an inhibitor of
choline transport to prevent TMA production (Figure 2).
Concurrently, we reported an additional CutC inhibitor,
betaine aldehyde (BA) (in vitro IC50 = 26 μM), which was
identified by screening a focused library of choline analogs for
activity toward the cut gene cluster-containing human gut
isolate Escherichia coli MS 200-1.35 A cocrystal structure of BA
bound to unactivated CutC showed the formation of a
thiohemiacetal with Cys489 of CutC, suggesting this
compound could to be a covalent, reversible inhibitor. We
also found that BA inhibited TMA formation from choline in
whole cell assays with multiple choline-degrading human gut
bacteria (EC50 = 0.4−2.5 mM range).
Building on this proof of concept work, we sought to use
structural information to design more potent CutC inhibitors.
Examining the conformation of choline within the CutC active
site, we noticed that the trimethylammonium group is oriented
gauche to the hydroxyl, with an average dihedral angle of 61°.
The aromatic ring of Phe395 may stabilize this conformation
of choline by forming a face-on cation−π-like interaction with
C2 (C2 to ring center distance: 3.8 Å), which would be
expected to have a partial positive charge due to the adjacent
quaternary ammonium.24 Overlaying choline- and BA-bound
CutC crystal structures showed that BA binds within the active
site in a similar conformation to choline, maintaining contacts
with key active site residues.
Figure 1. Anaerobic choline metabolism is a disease-linked gut
microbial activity and a promising target for inhibition. (A)
Generation of TMA from choline by gut microbes and its subsequent
processing in the human body. (B) Crystal structure of CutC (PDB =
5FAU) and (C) proposed mechanism for choline cleavage.
radical SAM enzyme superfamily. A crystal structure of
unactivated CutC with choline bound in the active site (Figure
1B), together with site-directed mutagenesis, supports a
mechanism in which the glycyl radical of CutC abstracts a
hydrogen atom from an adjacent cysteine (Cys489),
generating a thiyl radical intermediate that initiates the
reaction with choline by abstracting the pro-S hydrogen
atom from C1. Deprotonation of the C1-OH by Glu491,
followed by C−N bond cleavage via a “spin-center shift”,
would then eliminate TMA. Hydrogen atom abstraction from
Cys489 by the resulting acetaldehyde-centered radical would
regenerate the thiyl radical and produce acetaldehyde. Proton
transfer would then prepare the CutC active site for the next
round of catalysis (Figure 1C).
Targeting microbial choline metabolism to reduce TMA
generation (leading to lowered TMAO levels) has been
proposed as a potential therapeutic approach for the treatment
of cardiometabolic diseases. As CutC is the enzyme
responsible for TMA generation, it may be an ideal therapeutic
target. Hazen and co-workers reported the first CutC inhibitor,
3,3-dimethyl-1-butanol (DMB), replacing the nitrogen atom of
choline by a carbon (Figure 2).21 They demonstrated that
orally administered DMB reduced plasma TMAO levels in
mice. They also observed that DMB treatment reduced foam
cell formation induced by a high choline diet in an
atherosclerosis mouse model. However, in our hands DMB
poorly inhibits CutC activity in vitro and in bacterial cell
culture, suggesting these effects may arise from interactions
Together, these structures suggested the possibility of
building this conformation into an inhibitor scaffold by
forming a ring between C1 and an N-methyl group, leading
either to N-methyl-3-pyrrolidinol 1 or N-methyl-3-piperidinol
2 (Figure 3). These modifications could potentially improve
inhibitor potency by constraining its conformation and thereby
reducing the entropic penalty of binding.36,37 These com-
pounds (synthesis detailed in the SI) were tested for inhibition
B
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indicating that installing an electrophile would not improve
activity. This loss of potency could also arise from the absence
of a hydrogen atom on C3 if its abstraction is critical for
activity.
We next introduced an alkene into the piperidine ring
(compound 6), hypothesizing that this modification could
affect potency by activating the C3 hydrogen atom for
abstraction and/or altering the piperidine ring conformation.
This modification improved inhibitory activity 100-fold both in
whole cells and in vitro. The addition of a methyl group to this
derivative at position 5 (compound 7) decreased activity. An
acetylated analog (compound 8) was 100-fold less active than
6 in whole cells.
Figure 3. Strategy for rigidifying choline analogs leading to N-methyl-
3-pyrrolidinol 1 or N-methyl-3-piperidinol 2.
Table 1. Inhibitory Activity of Compounds 1−8
We then turned our attention toward the N-methyl group
(Table 2), replacing it with both a carbon atom (compound 9)
and an oxygen atom (compound 10). The methylene-
containing derivative 9 was 25- and 230-fold less active than
6 in vitro and in whole cells, respectively. While 10 possessed
activity similar to that of 6 in the whole cell assay, it was 1,000-
fold less active toward CutC in vitro, suggesting that it may
interact with an additional, unknown target. These results
highlight the importance of the amine for activity toward CutC
and suggest this part of the molecule may be mimicking the
trimethylammonium group of choline.
Next, we varied the substituent on the piperidine nitrogen
atom. Introduction of bulkier substituents like n-propyl
(compound 11), cyclobutyl (compound 12), and benzyl
(compound 13) all reduced activity. We also tested the
dimethylated quaternary ammonium analog 14, and we found
this compound possessed similar activity to 6 in whole cells but
was slightly less potent against CutC in vitro. Finally, we
synthesized and tested both enantiomers of compound 6.
While (S)-6 possessed similar activity to the racemic mixture,
(R)-6 was at least 3-fold less active both in whole cells and in
the CutC assay. Overall, this optimization effort revealed (S)-6
to be the most promising inhibitor, with a high ligand
efficiency (0.98) and fit quality score (1.38).38
IC50 against E. coli MS 200−1
(μM)
In vitro IC50 against CutC
(μM)
Compound
1
2
3
4
5
6
7
8
6,300 1,800
950 340
1,600 300
760 150
840 100
2,000 300
7,000 2,000
2,600 1,100
>10,000
27,000 4,000
10
4
2.9 0.7
920 80
1,800 600
420 100
a
N/A
a
N/A = not available.
Table 2. Inhibitory Activity of Compounds 9−14
To gain further structural insights into the interactions
between (S)-6 and CutC, we obtained a cocrystal structure of
an unactivated enzyme−inhibitor complex. Following incuba-
tion of wild-type CutC with 10 mM (S)-6, CutC was
crystallized using similar conditions to those reported
previously.24 The resulting structure determined to 2.3-Å
resolution revealed nonproteinogenic electron density within
the CutC active site. Based on the observed electron density
we modeled compound (S)-6 (Figure 4A), which fits well in
the composite omit map density (Figure 4B). Overall, inhibitor
(S)-6 bound in a similar fashion as choline, exploiting some of
the same key binding interactions observed within the CutC-
choline complex (Figure 4A,C). Of interest, the alcohol moiety
of (S)-6 is within hydrogen bond distance (2.7 Å) of Glu491,
placing the C3 hydrogen within 3.8 Å of Cys489. Similar to the
CH−O bond interactions observed in the CutC−choline
bound structure that are involved in securing the trimethy-
lammonium moiety of choline in place, comparable CH−O
interactions are observed between the N-methyl group, C2,
and C6, which are adjacent to the tertiary amine of (S)-6, and
residues Asp216, Tyr506, and Tyr208, respectively. Disruption
of these interactions could provide a rationale for the decrease
in activity observed for compounds 9−14 where the N-methyl
functionality has been substituted. Perhaps unexpectedly, the
two carbons that we anticipated would mimic the choline
backbone (C2 and C3) are not located in the same region of
IC50 against E. coli MS In vitro IC50 against
Compound
X
200-1 (μM)
CutC (μM)
6
9
10
11
12
13
14
(S)-6
(R)-6
N-Me
10 0.4
260 110
80 22
200 60
190 60
370 70
7.6 2.4
9.8 1.9
2.9 0.7
710 270
2,900 1,300
140 20
200 90
100 10
7.0 1.0
2.4 0.3
CH2
O
N-nPr
N-cyclobutyl
N-Bn
N-Me2
N-Me
N-Me
31
5
22
3
of both anaerobic choline metabolism by E. coli MS 200-1 and
CutC activity in vitro (Tables 1 and 2).
Both 1 and 2 inhibited TMA formation from choline in vitro
and in bacterial cell culture. We decided to prioritize
compound 2 for further optimization as it was more active
in whole cells. We found that moving the hydroxyl group to
the 4-position (compound 3) led to a 3- and 2-fold loss in vitro
and in whole cell activities, respectively. Expanding the size of
the heterocyclic ring (compound 4) did not improve activity
relative to 2. Oxidation of the alcohol to a ketone (5) led to a
compound that was 20-fold less active toward whole cells,
C
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Scheme 1. Potential Mechanism of CutC Inhibition by (S)-6
Table 3. Inhibitory Activity of Compounds 6 and 15
Figure 4. Binding of (S)-6 to CutC reveals key interactions important
for recognition. (A) Crystal structure of unactivated CutC soaked
with (S)-6 reveals interactions between bound inhibitor and CutC
active site residues. Black dashes represent H-bond interactions,
purple dashes represent CHO interactions, gray dashes represent a
potential π−π interaction, and orange dashes represent the path of
radical transfer. Chemical structure of (S)-6 with numbered atoms is
shown next to the structure. (B) Simulated annealing composite omit
map (2Fo − Fc, blue mesh) contoured at 1.5σ showing the location of
the inhibitor. (C) Previously described crystal structure of unactivated
CutC bound to choline (PDB 5FAU). Interactions between CutC
and choline are color coded as described above with the exception of
a cation−π interaction which is shown in gray dashes. (D)
Comparison of choline-bound (protein residues are colored in light
green) and (S)-6-bound (colored in green) CutC active sites. Choline
and (S)-6 were omitted from the structure for clarity.
IC50 against E. coli MS 200-1
(μM)
IC50 against CutC
a
Compound
R
(μM)
6
15
H
D
10
30
3
6
a
Compounds 6 and 15 were assayed at the same time.
and assayed it both in whole cells and in vitro (Table 3).
Racemic 15 displays a 3- and 2-fold loss of activity in whole
cells and in vitro against CutC in comparison to 6. This
preliminary result provides support for our mechanistic
hypothesis, as the loss in activity could potentially arise from
a reduced rate of deuterium abstraction due to a kinetic
isotope effect. An alternative mode of inhibition that we cannot
yet rule out would be the oxidation of 6 to an enone followed
by conjugate addition of C489 at C5.
The cut genes are widely distributed among human gut
bacteria and likely encode a major pathway for choline
utilization in the GI tract. We therefore tested the potency of
our best lead compound (S)-6 and our reference inhibitor BA
for inhibition of TMA production from choline by additional
Gram-negative (Proteus mirabilis, Escherichia coli MS69-1,
Klebsiella sp. MS 92-3) and Gram-positive (Clostridium
sporogenes) choline-metabolizing human gut isolates. Com-
pound (S)-6 was effective against all strains analyzed, with IC50
values ranging from 5 to 97 μM, which is at least 20-fold more
active than betaine aldehyde (Table 4). Finally, (S)-6 inhibited
the conversion of choline to TMA by a human fecal suspension
the active site; instead, (S)-6 binds in a conformation
amenable for π−π interaction between C4−C5 of (S)-6 and
F395.39 This interaction may contribute to the boost in
potency observed upon introducing this functional group. The
similar binding orientation of (S)-6 compared to choline is also
reflected in the observation that active site residues do not
rearrange upon binding the different molecules (Figure 4D).
Overall, the structures are very similar, with low RMSD values
(0.28 Å over 792 residues out of 846).
Together, our inhibitor optimization studies and structural
data allow us to propose that cyclic compound 6 may be a
mechanism-based inhibitor (Scheme 1). The similarity of its
binding mode to that of choline, coupled with the proximity of
the C3 hydrogen atom of (S)-6 to Cys489, suggests that this
compound could undergo C−H abstraction to form an
inhibitor-centered ketyl radical. Notably, such a radical
would be stabilized by the adjacent alkene, which could derail
catalysis and lead to inhibition. This proposal is consistent with
the dramatic improvement in activity observed upon
introducing the alkene into the cyclic scaffold. It is also
consistent with improved potency for the (S) versus (R)
enantiomer, as the (S) enantiomer should position the C3
hydrogen optimally for abstraction.24 To test this hypothesis,
we synthesized an analog of 6 that is deuterated at C3 (15)
Table 4. Activity of BA and (S)-6 toward Diverse Human
Gut Bacteria and Human Fecal Suspension
Isolate
IC50 (μM) for BA
IC50 (μM) for (S)-6
E. coli MS 69-1
P. mirabilis
Klebsiella sp.
C. sporogenes
Fecal suspension
465
2,488
212
1,000
1,273
22
92
4
12
60
D
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ex vivo with an EC50 of 60 μM, confirming its efficacy in a
complex community more reminiscent of the gut microbiota
(Table 4). Although (S)-6 possesses a somewhat modest
biochemical IC50, the small shift between our in vitro and ex
vivo IC50 values demonstrates its potential utility as a tool for in
vivo studies.
In summary, we have discovered a cyclic choline analog that
inhibits anaerobic choline metabolism by both Gram-negative
and Gram-positive human gut isolates as well as a more
complex gut community. Structural information and prelimi-
nary mechanistic studies suggest 6 may be a mechanism-based
inhibitor that is processed by CutC to generate a stabilized
radical intermediate. This finding adds another molecular
scaffold to the growing arsenal of small molecules that inhibit
gut microbial choline metabolism.
General Medical Sciences from the National Institutes of
Health (P30GM124165). The Pilatus 6M detector on the 24-
ID-C beamline is funded by a NIH-ORIP HEI grant (S10
RR029205). This research used resources of the Advanced
Photon Source, a U.S. Department of Energy (DOE) Office of
Science User Facility operated for the DOE Office of Science
by Argonne National Laboratory under Contract No. DE-
AC02-06CH11357. We acknowledge the laboratory of
Federico Rey (University of Wisconsin Madison) for supplying
the fecal sample.
ABBREVIATIONS
■
BA, betaine aldehyde; CutC, glycyl radical enzyme choline
TMA-lyase; DMB, 3,3-dimethyl-1-butanol; FMO3, flavin-
dependent monooxygenase 3; GI, human gastrointestinal;
GRE, glycyl radical enzyme; NAFLD, nonalcoholic fatty liver
disease; SAM, S-adenosylmethionine; TMA, trimethylamine;
TMAO, trimethylamine-N-oxide.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
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REFERENCES
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AUTHOR INFORMATION
Corresponding Author
■
Emily P. Balskus − Department of Chemistry and Chemical
Biology, Harvard University, Cambridge, Massachusetts 02138,
United States; orcid.org/0000-0001-5985-5714;
Email: balskus@chemistry.harvard.edu
Authors
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Maud Bollenbach − Department of Chemistry and Chemical
Biology, Harvard University, Cambridge, Massachusetts 02138,
United States
Manuel Ortega − Department of Chemistry, Massachusetts
Institute of Technology, Cambridge, Massachusetts 02139,
United States
Marina Orman − Department of Chemistry and Chemical
Biology, Harvard University, Cambridge, Massachusetts 02138,
United States; orcid.org/0000-0002-1925-4612
Catherine L. Drennan − Department of Chemistry, Department
of Biology, Howard Hughes Medical Institute, and Center for
Environmental Health, Sciences, Massachusetts Institute of
Technology, Cambridge, Massachusetts 02139, United States;
orcid.org/0000-0001-5486-2755
Complete contact information is available at:
https://pubs.acs.org/10.1021/acsmedchemlett.0c00005
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We acknowledge support from the Packard Fellowship for
Science and Engineering and a Blavatnik Biomedical
Accelerator Award (E.P.B.). C.L.D. is a Howard Hughes
Medical Investigator. This work was supported by the National
Institutes of Health grant R35 GM126982 (to C.L.D). M.O. is
a Merck Fellow of the Life Sciences Research Foundation and
a Postdoctoral Enrichment Program Grant recipient from the
Burroughs Wellcome Fund. This work is based upon research
conducted at the Northeastern Collaborative Access Team
beamlines, which are funded by the National Institute of
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