Small-Molecule Inhibition of the C. difficile FAS-II Enzyme, FabK, Results in Selective Activity

Article abstract of DOI:10.1021/acschembio.9b00293

Clostridioides difficile infection (CDI) is a leading cause of significant morbidity, mortality, and healthcare-related costs in the United States. After standard therapy, recurrence rates remain high, and multiple recurrences are not uncommon. Causes inc

Full text of DOI:10.1021/acschembio.9b00293

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Article
Small-molecule inhibition of the C. difficile FAS-
II enzyme, FabK, results in selective activity
Jesse A. Jones, Allan M. Prior, Ravi K. R. Marreddy, Rebecca D.
Wahrmund, Julian G. Hurdle, Dianqing Sun, and Kirk E. Hevener
ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.9b00293 • Publication Date (Web): 11 Jun 2019
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Small-molecule inhibition of the C. difficile FAS-II enzyme, FabK,
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results in selective activity
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Jesse A. Jones,a* Allan M. Prior,b* Ravi K.R. Marreddy,c Rebecca D. Wahrmund,a Julian
G. Hurdle,c Dianqing Sun,b# Kirk E. Hevener,a#
aDepartment of Pharmaceutical Sciences, College of Pharmacy, University of Tennessee
Health Science Center, Memphis, Tennessee, 38163, United States
bDepartment of Pharmaceutical Sciences, The Daniel K. Inouye College of Pharmacy,
University of Hawaii at Hilo, Hawaii, 96720, United States
cCenter for Infectious and Inflammatory Diseases, Institute of Biosciences and
Technology, Texas A&M Health Science Center, Houston, Texas, 77030, United States
Running Head: Selective Inhibition of C. difficile FabK
#Address correspondence to Kirk E. Hevener, khevener@uthsc.edu and Dianqing Sun,
dianqing@hawaii.edu.
*J.A.J. and A.M.P. contributed equally to this work.
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Abstract
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Clostridioides difficile infection (CDI) is a leading cause of significant morbidity,
mortality, and healthcare-related costs in the United States. After standard therapy,
recurrence rates remain high and multiple recurrences are not uncommon. Causes include
treatments employing broad-spectrum agents that disrupt the normal host microbiota, as
well as treatment-resistant spore formation by C. difficile. Thus, novel druggable anti-C.
difficile targets that promote narrow-spectrum eradication and inhibition of sporulation
are desired. As a critical rate-limiting step within the FAS-II bacterial fatty acid synthesis
pathway, which supplies precursory component phospholipids found in bacterial
cytoplasmic and spore-mediated membranes, enoyl-acyl carrier protein (ACP) reductase
II (FabK) represents such a target. FabK is essential in C. difficile (CdFabK) and is
structurally and mechanistically distinct from other isozymes found in gut microbiota
species, making CdFabK an attractive narrow-spectrum target. We report here the kinetic
evaluation of CdFabK, the biochemical activity of a series of phenylimidazole analogues,
and microbiological data suggesting these compounds’ selective antibacterial activity
against C. difficile versus several other prominent gut organisms. The compounds display
promising selective, low micromolar CdFabK inhibitory activity without significantly
affecting the growth of other gut organisms, and the series prototype (1b) is shown to be
competitive for the CdFabK cofactor and uncompetitive for the substrate. A series
analogue (1g) shows maintained inhibitory activity while also possessing increased
solubility. These findings represent the basis for future drug discovery efforts by
characterizing the CdFabK enzyme while demonstrating its druggability and potential
role as a narrow-spectrum anti-difficile target.
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Keywords: Clostridioides difficile, enoyl-ACP reductase II, FabK, phenylimidazole,
enzyme inhibitor, antibacterial, narrow-spectrum
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Clostridioides difficile (formerly Clostridium difficile) is a Gram-positive, spore-
forming, anaerobic pathogen that is the leading cause of health care-associated diarrhea
and gastroenteritis-associated deaths in the U.S.1, 2 Clinical consequences of C. difficile
infection (CDI) range from asymptomatic or mild diarrhea to recalcitrant diarrhea and
fatal pseudomembranous colitis.3 CDI is caused by the eradication of key gut flora, such
as Bifidobacterium sp. and Bacteroides sp., which normally suppress C. difficile
overgrowth.4 This typically occurs via the use of broad-spectrum antibiotics (e.g.,
clindamycin, fluoroquinolones, and beta-lactams).4-6 The first-line CDI treatment,
vancomycin, exhibits activity against beneficial gut flora and results in their eradication,
contributing to high rates of recurrence.7, 8 Further contributing to relapse is the
pathogen’s ability to produce treatment-resistant spores that survive in the intestinal
tract.9, 10 The emergence of epidemic strains, namely BI/NAP1/027 (North American
pulsed-field gel electrophoresis type 1, ribotype 027), correlated with an increase in
incidence and severity of CDI.7 While the alternative first-line drug fidaxomicin is
narrow-spectrum and reduces recurrence, it has failed to show evidence of doing so in
NAP1-mediated infections.11, 12 Fecal microbiota transplantation (FMT) shows promise,
but long-term safety data is still lacking, including non-infectious microbiota-related
metabolic diseases like obesity.13, 14 While new agents are currently in clinical trials,
recent failures highlight the pressing need for novel anti-CDI agents to maintain the
discovery pipeline.6, 15 These issues emphasize that C. difficile remains a high-priority
candidate for the development of novel narrow-spectrum antibacterial agents.
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During cyclic fatty acid elongation within the bacterial type II fatty-acid synthesis
(FAS II) pathway, enoyl-acyl carrier protein (ACP) reductase enzymes catalyze the
critical, rate-limiting terminal reduction step. The FAS-II system provides fatty acid
precursory components vital to bacterial organisms for various biological processes like
membrane phospholipid production and sporulation.16 Strong scientific precedent
substantiates these enzymes as antimicrobial drug targets, exemplified by isoniazid, a
marketed inhibitor of tubercular InhA (enoyl-ACP reductase); triclosan, a well-known
antibacterial FabI inhibitor; and the anti-staphylococcal FabI inhibitor afabicin (Debio-
1450) currently in clinical trials.17-20 While the existence of four distinct enoyl reductase
isozymes (FabI, FabK, FabL, and FabV) in bacteria precludes the development of broad-
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spectrum antibacterials targeting them, it affords an ideal opportunity for the rational
design of narrow-spectrum agents.21-23 Bioinformatics analyses of sequenced C. difficile
genomes indicate it expresses the FabK isozyme as its sole enoyl-ACP reductase.24, 25
FabK, a member of the triosephosphate isomerase (TIM) barrel family of proteins, is a
less abundant isozyme among the four enoyl-ACP reductases, and lacks sequence
similarity to FabI, FabL, and FabV, which are members of the short-chain alcohol
dehydrogenase/reductase (SDR) superfamily (Figure 1).22, 26 Like the other isozymes,
FabK requires either NADH or NADPH as a cofactor for activity, but unlike its SDR
counterparts, FabK is a flavoenzyme that utilizes flavin mononucleotide (FMN) as an
active site prosthetic group. FabK is also unique in that it uses a bi-bi double
displacement (Ping-Pong) enzymatic mechanism for reducing its enoyl substrate.22, 26-33
The SDR enzymes, however, possess a classical Rossman fold for binding their
NAD(P)H cofactor and rely on an ordered sequential enzyme mechanism for reducing
their enoyl substrates.34, 35 While there has been recent debate about the essentiality of the
FAS-II pathway in Gram-positive bacteria,36-38 several groups have shown that
possession of the FapR regulatory system over FabT, as seen in S. aureus, prevents full
bypass of FAS-II inhibition by exogenous fatty acid uptake.39, 40 Indeed, we recently
showed that C. difficile, which also possesses FapR, behaves in a similar manner as
exogenous fatty acids were unable to rescue the pathogen from FAS-II inhibition.25 C.
difficile also has unique branched fatty acid requirements, further limiting the likelihood
of escape from FAS-II inhibition.41, 42 Our recent work also demonstrated the ability of
FAS-II inhibition to decrease spore formation by nearly 90%, which in principle could
reduce potential for spore survival, associated endogenous recurrence, and spore
dissemination.25
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Figure 1. Comparison of FabK and FabI Structures with Bound Cofactors. A. P.
gingivalis FabK structure (PDB 4IQL) shows the C-terminal domain in orange and the
conserved TIM barrel domain in blue. The FMN prosthetic group is shown as ball & stick
structure with yellow carbons. B. The S. aureus FabI structure represents the SDR
protein family structure shared by FabI, FabL, and FabV. The conserved Rossman fold is
shown as orange ribbons and the NADPH cofactor is shown as ball & stick structure with
yellow carbons.43
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CDI pharmacotherapy has shown that maximizing therapeutic success includes
preventing collateral damage to the GI microbiome. Important gut species such as Gram-
negative Bacteroides sp.44 express FabI with or without FabK, while Bifidobacterium sp.
lack FabK entirely.25 Importantly, published FabK inhibitors are inactive against FabI
and FabI-bearing strains.29, 32 Similarly, FabK has demonstrated resistance to FabI
inhibitors like triclosan, which are consequently inactive against strains carrying FabK.22,
27, 45, 46 Lastly, the ability of bacterial strains carrying two enoyl reductases to survive the
deletion of one isozyme suggests that both enzymes would need to be dually inactivated
to inhibit the growth of such organisms.21 This data justifies the investigation of FabK as
a narrow-spectrum target for selective anti-difficile therapy.
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FabK from S. pneumoniae (SpFabK) was shown by Takahata et al. to be
specifically inhibited by a compound (AG-205) they found during a high throughput
screening campaign, which led to the development of the more potent phenylimidazole-
derived class of inhibitors via iterative medicinal chemistry modifications (e.g., replacing
the amide linker with urea functionality, exploring various substitutions at the solvent
exposed head region, un-fusing the benzimidazole ring systems, and evaluating various
substitutions at the tail region).29-33 Despite substantial gains in potency, further
development was ceased, possibly due to the unfortunate fact that Streptococci are able to
bypass FAS-II inhibition as they carry the FabT regulatory system.36 Because C. difficile
harbors the FapR regulatory system, it presents an opportunity to renew the development
of phenylimidazole FabK inhibitors.25 In an effort to characterize the enzyme target, we
therefore performed essential kinetic evaluations of CdFabK, including substrate and
cofactor Km determinations, inhibition modality, and substrate inhibition effects. We also
report that phenylimidazoles display potent on-target biochemical activity against
CdFabK (Table 1) and are amenable to modifications that improve physiochemical
properties (Scheme 1; Materials and Methods) while maintaining biochemical and anti-
C. difficile activity, and demonstrate a promising, narrow-spectrum antibacterial profile
against C. difficile over key members of the gut flora (Table 2).
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Scheme 1. Synthesis of phenylimidazole derivatives 1a-h. aReagents and conditions:
(a) i. NaOMe, MeOH, 25 ºC, 8 h; ii. NH4Cl, 25 ºC, 3 days, 91%; (b) substituted 2-
bromoacetophenone, K2CO3, THF/H2O (10:1), reflux, 18 h; (c) HCl, H2O, reflux, 3 h, 27-
69% (over 2 steps); (d) K2CO3, DMF, 80 ºC, 1.5 h, 67%; (e) CDI (2 equiv.), DCM, 25 ºC,
18 h, 74%; (f) Et3N, CHCl3, 25 ºC, 2 h, 30%-89%; (g) BBr3, DCM, 0-25 ºC, 45 min,
92%.
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Table 1. Inhibitory activity of Enoyl-ACP isozymes by phenylimidazole analogues.
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Compound
CdFabK IC50 [95% CI];
% Inhibition at 10 M
>100 M
SpFabK IC50 [95% CI]
Triclosan
>100 M
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1a
>10 M; 38.51%
0.730 M [0.434, 1.456]
0.067 M [0.054, 0.084]
0.242 M [0.214, 0.274]
0.198 M [0.178, 0.222]
0.078 M [0.066, 0.093]
0.163 M [0.124, 0.214]
0.085 M [0.072, 0.101]
0.538 M [0.433, 0.675]
5.32 M [4.378, 6.809]
1b
3.31 M; 95% CI [2.68, 4.19]
>10 M; 49.86%
1c
1d
>10 M; 44.80%
1e
2.86 M [2.02, 4.35]
4.63 M [3.12, 8.02]
7.35 M [5.35, 11.65]
>10 µM; 35.58%
1f
1g
1h
AG-205
4.15 M [3.324, 5.597]
CI, Confidence Interval
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Table 2. MIC values in the presence and absence of efflux inhibitor verapamil suggesting inhibitors are specific for FabK
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Organism
Enoyl-ACP
reductase
MIC (M)
MIC (M)
(-) Verapamil
(+) Verapamil
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1b
1e
1f
1g
VAN
1b
1e
1f
1g
VAN
C. difficile R20291
S. pyogenes
FabK
4
8
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1.6
4
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2
0.8
ATCC19615†
FabK
FabI
0.125 <0.0625 <0.0625 0.125 0.2 0.125 <0.0625 <0.0625 0.125
0.4
6.4
6.4
3.2
3.2
C. perfringens HM310
B. ovatus ATCC8485
B. fragilis ATCC25285
B. breve ATCC 11863
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
6.4
6.4
3.2
3.2
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
FabI & FabK >128
FabI
*
>128
>128
*Yeast like fatty acid synthase
† Streptococcus species harbor the FabT regulatory system, allowing for bypass of FASII inhibition in the presence of exogenous fatty
acids.25
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Results
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The CdFabK enzyme possesses intrinsic NADH oxidative activity and double
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substrate inhibition.
The CdFabK enzyme displayed NADH oxidative activity in the absence of enoyl
substrate (Figure 2). Apparent Km (Kmapp) values were determined via 1.5-fold serial
dilutions of the NADH cofactor while holding the enoyl substrate, crotonyl coenzyme A
(Cro-CoA), constant at 150 M; likewise, 1.5-fold serial dilutions of Cro-CoA were
tested while holding the NADH cofactor constant at 150 M; and an alternative enoyl
substrate, octenoyl coenzyme A (Oct-CoA), was tested similarly via 1.5-fold dilutions
while holding NADH constant at 150 M. Initially, Kmapp values were determined via
standard Michaelis-Menten non-linear regression fits. However, upon further analysis,
the data for cofactor and substrates all fit a substrate inhibition model (R2 = 0.9854;
0.7607; and 0.8479, respectively) better than the poorly fit standard Michaelis-Menten
models. As such, respective Kmapp values for NADH, Cro-CoA, and octenoyl coenzyme
A were determined to be 138.1 M, 327.0 M, and 420.8 M, as opposed to 23.0 M,
176.0 M, and 65.5 M observed with the standard Michaelis-Menten model,
respectively.
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Figure 2. Enzyme Kinetics of CdFabK vs. Velocity. (A) Non-linear regression curve
fit of velocity of CdFabK with varying concentrations of NADH (1.5-fold dilutions from
350 M to 9.1 M) at a fixed concentration of Cro-CoA (325 M) demonstrating NADH
substrate inhibition. Standard Michaelis-Menten fit (dashed blue line) and substrate
inhibition line (solid black line with dots) shown for comparison. (B) Non-linear
regression curve fit of velocity of CdFabK with varying concentrations of enoyl substrate
Cro-CoA (1.5-fold dilutions from 4500 M to 78 M and 0 M) at a fixed concentration
of NADH (125 M) demonstrating Cro-CoA substrate inhibition. (C) Non-linear
regression curve fit of velocity of CdFabK with varying concentrations of alternative
enoyl substrate octenoyl-CoA (1.5-fold dilutions from 2500 M to 43 M and 0 M) at a
fixed concentration of NADH (125 M) demonstrating octenoyl-CoA substrate
inhibition.
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Phenylimidazole-derived compounds selectively inhibit the C. difficile FabK enzyme.
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Phenylimidazole compounds have been shown to inhibit SpFabK (PDB 2Z6J) and
lack inhibitory activity against FabI from E. coli.29, 30 To evaluate activity against
CdFabK, phenylimidazole compounds were screened against purified enzyme to
determine percent inhibition at 10 M. Compounds yielding over 50% inhibition were
further screened to determine on-target 50% inhibitory concentrations (IC50). Compounds
were also screened at 10 M against purified FabI from Staphylococcus aureus (SaFabI)
to directly compare activity profiles.
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We confirmed the phenylimidazole compounds inhibit CdFabK in a dose-
dependent manner. All compounds except 1a, 1c, 1d, and 1h showed greater than 50%
inhibition of CdFabK at 10 M. IC50s ranged from 7.35 M with 1g to 2.86 M with 1e,
with the series prototype, 1b, showing an IC50 of 3.31 M. Resulting Hill coefficients
from IC50 logistic curve fits for 1b, 1e, and 1g were 0.901 (95% Confidence Interval (CI)
= 0.761-1.064), 0.8594 (95% CI = 0.6449-1.134), and 1.014 (95% CI = 0.7417-1.365),
respectively. These values are not significantly different from unity (Hill slope of 1.000),
suggesting these compounds display normal inhibitory behavior and do not bind
cooperatively, aggregate, form micelles, or demonstrate problematic insolubility. It also
indicates the enzyme’s active sites function independently, despite CdFabK being a
functional dimer.27, 28
All compounds showed IC50s against SpFabK in the sub-micromolar range, with
1b resulting in an IC50 of 67 nM, roughly 49-fold better as compared to CdFabK.
Likewise, 1e and 1g resulted in respective IC50s of 78 and 85 nM against SpFabK,
illustrating roughly 87- and 37-fold better respective IC50s as compared to CdFabK.
While activity of 1b against SpFabK was more potent than against CdFabK, it was still
roughly 30 times less potent against SpFabK in our hands than reported elsewhere.29 As
the monomer enzyme concentration used here (50 nM) was lower than that used in
previous reports (about 60 nM), the reason for this discrepancy is not readily apparent.
FabI was confirmed to be resistant to the phenylimidazole compounds as both 1b and
AG-205 showed little inhibitory effect against SaFabI with percent inhibition values well
below 50% (22.40 and 18.22%, respectively) at 10 M.
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The target specificity profile of CdFabK was examined via activity in the
presence of triclosan—a well-known, potent, slow-binding inhibitor of the FabI
isozyme.47 To verify our own methods, triclosan was confirmed to be a potent inhibitor of
SaFabI, with nearly 90% inhibition at 10 M. Like SpFabK, CdFabK proved to be
resistant to triclosan at 100 M and IC50 values were not determined.
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Phenylimidazole compound 1b is competitive for NADH and uncompetitive for the
enoyl substrate against CdFabK
To elucidate the modality of inhibition of the phenylimidazole compounds against
CdFabK, we analyzed the inhibitory activity of 1b against purified enzyme in the
presence of varying concentrations of NADH while holding Cro-CoA constant, and
likewise in the presence of varying concentrations of Cro-CoA while holding NADH
constant. Both non-linear fits and Lineweaver-Burk plots were analyzed. The best non-
linear fits for each substrate suggest that 1b acts as a competitive inhibitor of CdFabK
with respect to NADH (R2 = 0.91) and an uncompetitive inhibitor with respect to Cro-
CoA (R2 = 0.90). The Lineweaver-Burk plots for each substrate corroborate these
findings (Figure 3).
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Figure 3. Characterization of Inhibitory Activity and Mechanism of 1b against
CdFabK. (A) Sigmoidal plots demonstrating dose response curves for the inhibition of
CdFabK by two different phenylimidazole analogues, 1b (black line with dots) and 1g
(blue line with triangles). (B) Sigmoidal plots as dose response curves for the inhibition
of CdFabK by 1b with different concentrations of Cro-CoA, 150 µM Cro-CoA (black
line with dots) and 325 µM Cro-CoA (red line with squares). (C) Lineweaver-Burk plot
showing competitive inhibition of CdFabK binding NADH by 1b. CdFabK was
incubated with varying concentrations of 1b and NADH at a fixed concentration of Cro-
CoA (325 µM). Concentrations of 1b were 33 µM (closed circle), 11 µM (square), 3.67
µM (triangle), 1.22 µM (upside-down triangle), 0.4 µM (diamond), and 0 µM (open
circle). (D) Lineweaver-Burk plot showing uncompetitive inhibition of CdFabK binding
Cro-CoA by 1b. CdFabK was incubated with varying concentrations of 1b and Cro-CoA
at a fixed concentration of NADH (150 µM). Concentrations of 1b were 33 µM (closed
circle), 11 µM (square), 3.67 µM (triangle), 1.22 µM (upside-down triangle), 0.4 µM
(diamond), and 0 µM (open circle).
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Phenylimidazole 1b, 1e, 1f, and 1g selectively inhibited C. difficile growth over other
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key gut flora
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Activities of four phenylimidazole derivatives 1b, 1e, 1f, and 1g were tested
against C. difficile and representative gut flora (Table 2). Tests were done in the presence
and absence of the efflux pump inhibitor verapamil, as 1b was shown to be susceptible to
efflux in the strain C. difficile CD630.25 The anti-C. difficile MICs of 1b, 1e, 1f, and 1g
ranged from 4-8 M and 2-4 M, in the absence and presence of verapamil. In general,
the MICs of the compounds only differed by 2-fold. While vancomycin’s anti-C. difficile
activity (0.4-1.6 M) was superior, it also inhibited the growth of the representative gut
flora, whereas the phenylimidazole compounds did not, even up to 128 M.
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Discussion
We report here the kinetic evaluation of CdFabK, the biochemical activity of a
series of phenylimidazole analogues, and microbiological data suggesting these
compounds’ selective antibacterial activity against C. difficile over several other
prominent gut organisms. The compounds display promising, low micromolar CdFabK
inhibitory activity without significantly affecting the in vitro growth of important gut
organisms. CdFabK biochemical activity assays confirmed the inhibitory activity of all
phenylimidazole analogues against CdFabK, with several showing potent, low
micromolar activity. This data further demonstrates the enzyme’s druggability and the
potential of the phenylimidazole compounds as leads for developing a novel series of
narrow-spectrum anti-C. difficile drug candidates. As the imidazole and thiazole-urea
moieties in the first-generation inhibitor 1b were predicted in modeling studies to
establish three hydrogen bond interactions, and thus play a significant role in FabK
binding, initial structure activity relationship (SAR) and physicochemical exploration
during druggability validation was focused on the 4-bromo-phenyl region of the molecule
(Figure 4, Scheme 1). The observed and calculated solubility of 1b is poor (average
solubility 0.36 M via Biomeck and 0.01 M via LC/MS). Therefore, in this work, a new
expanded set of substituents with different lipophilic and electronic properties was
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introduced in the tail region to further probe existing SAR as well as enhance solubility.
Specifically, six new compounds 1c-1h, along with two known compounds 1a-b, were
synthesized (Scheme 1) and subsequently evaluated and, while chemical modifications
did not substantially increase potency, IC50 values indicate inhibitory activity was not
abolished despite crucial solubility enhancement via modification at this region. This is
illustrated by comparing 1b (4-bromophenyl, cLogP 4.56 and IC50 3.31 M) and 1g (4-
cyanophenyl, significantly improved cLogP 3.15, similar IC50 7.35 M). Because the
target enzyme’s natural, in vivo function involves the binding of relatively lipophilic fatty
acid precursor substrates, this was a noteworthy discovery that will serve as an important
structural basis for future inhibitor design and synthesis. While 1e and 1f showed MIC
values similar in potency to 1b and 1g, increased cLogP values (4.6 and 4.8, respectively)
for both compounds preclude them from further assessment.
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Figure 4. FabK Inhibitors. A. AG-205 hit compound shown docked into CdFabK
model. Hydrogen bonds indicated by yellow dashes. B. 2D ligand interaction diagram of
AG-205 bound to CdFabK active site. C. phenylimidazole inhibitor 1b shown docked
into CdFabK model. D. 2D ligand interaction diagram of 1b bound to CdFabK active
site. See Supporting Information for Materials and Methods.
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A high degree of primary sequence identity and active site similarity exists
between CdFabK and SpFabK (58% overall identity, 74% overall similarity, and 1% gaps
with 2 missing loops on SpFabK, PDB 2Z6J; 100% and 91% active site identity within 3
Å and 4Å from bound inhibitors, respectively, Figure S1; residues shown interacting
with modeled inhibitors are 100% conserved between CdFabK and SpFabK, Figure 4).
Despite this, the phenylimidazole compounds showed greater activity against SpFabK
than CdFabK (sub-micromolar vs. low micromolar, respectively). The compounds, along
with triclosan, were tested against both FabK and FabI to determine comparative FAS-II
inhibition profiles. The compounds potently inhibited FabK but lacked activity against
FabI, while triclosan potently inhibited FabI but lacked activity against FabK. The reason
for the potency disparity of phenylimidazoles against the two similar FabK enzymes is
not readily apparent. The mode of inhibition for the phenylimidazoles was reported to be
competitive inhibition of SpFabK binding NADH and uncompetitive of SpFabK binding
Cro-CoA,28 and confirmed here to be the same against CdFabK (Figure 2) and therefore
not the cause for potency dissimilarity. Evaluation at incorrect Kmapp values of substrate
and cofactor was another potential cause for dissimilar potencies, therefore a substrate
inhibition model was considered, increasing the Kmapp of Cro-CoA to 327 M, up from
176 M seen with a standard Michaelis-Menten model. After re-testing 1b against
CdFabK with 325 M Cro-CoA, the IC50 dropped from 3.31 M to 1.27 M. While this
resulted in an IC50 closer to that observed against SpFabK, it still represents a roughly 19-
fold increase and, therefore, also fails to fully explain the discrepancy. A final
consideration is the possibility that the unique requirement of C. difficile for branched
chain fatty acids may impact the substrate requirements of CdFabK, making a branched
enoyl substrate more appropriate than Cro-CoA for compound screening, or even
introducing the possibility of additional medicinal chemistry modifications to be made to
the phenylimidazole compounds suited specifically to CdFabK.
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With the continued use of broad-spectrum antibiotics, the increasingly
diminishing spectrum of useful anti-CDI antibacterial agents, and the increased incidence
of new pathogenic strains, the need for novel antibacterial agents targeting C. difficile
continues to outpace their discovery. As demonstrated in these studies, a notably
attractive target for antibacterial development resides in the bacterial FAS II pathway,
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which is structurally dissimilar from the mammalian fatty-acid synthesis (FAS I)
pathway.48 FAS II also contains a number of dissimilar, independent enzymes and
enzyme homologues collectively responsible for the synthesis of important bacterial fatty
acid precursors and, therefore, appears inherently well-suited for selective targeting.25, 49
Additionally, the pathway has now been well-validated as being essential in both Gram-
negative bacteria and a number of Gram-positive bacteria, even in the presence of
exogenous fatty acids. Via direct comparison here of the phenylimidazole compounds’
IC50 values against different purified enoyl-ACP reductase enzymes from different
organisms, the selective druggability of CdFabK can be confirmed. Furthermore, analysis
of in vitro MIC values of selected compounds against a panel of key gut microbes in the
presence and absence of an efflux pump inhibitor further illustrates this. As such, the
selective inhibition of CdFabK represents a particularly promising pathway for future
narrow-spectrum anti-difficile development. In vivo efficacy, toxicity, and dysbiosis
studies in animal models of phenylimidazoles and derivatives will be indispensable for
continued development and, therefore, are anticipated for future studies. Furthermore, as
more information pertaining to the human microbiome continues to surface, an ongoing
analysis of the distribution of current and novel FAS-II isozymes across the increasing
number of known gut microbes will be of critical importance. Such analyses will guide
an evolving understanding of the extent of antibacterial specificity and overall promise
that targeting FabK truly offers.
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Materials and Methods
Chemical synthesis. Compounds 1a-h were synthesized by following literature
procedures with modifications (Scheme 1).25, 29, 30, 50, 51 In brief, amine intermediates 5
were synthesized from benzyl(cyanomethyl)carbamate 2 in 3 steps. Firstly, 2 was treated
with sodium methoxide in methanol for 8 h, followed by the addition of ammonium
chloride and stirring for 3 days to afford amidine 3 in 91% yield as an HCl salt. Amidine
HCl salt 3 was refluxed with a substituted 2-bromoacetophenone and potassium
carbonate in THF/water (10:1) for 18 h to afford imidazole derivatives 4.30 The Cbz
group was removed by refluxing 4 in water and HCl 50 to provide amine intermediates 5
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in 27-69% yields. The carboxamide intermediate 9 was synthesized in two steps from
commercially available starting materials. Firstly, 2-mercaptopyridine 6 and 2-amino-5-
bromothiazole monohydrobromide 7 were stirred with K2CO3 in DMF at 80ºC for 1.5 h
under nitrogen atmosphere to afford 8 in 67% yield after flash column chromatography.
Compound 8 was reacted with 1,1’-carbonyldiimidazole (CDI) 51 in DCM to afford
intermediate 9 in 74% yield after filtration and washing solid with DCM. Finally,
compounds 1a-g were obtained by reacting 9 with substituted amine 5 in chloroform and
triethylamine for 1 h. The reactions were filtered, diluted with CHCl3 (50 mL), washed
with water (50 mL × 3), and concentrated to give 1a-g in pure form and 30-89% yields
after trituration with DCM/diethyl ether (1:1) (Scheme 1). Compound 1h was obtained in
excellent yield via the demethylation of 1d using BBr3 in DCM. Complete general
chemistry and procedures, reagents and conditions, characterization data with 1H and 13C
NMR spectra, as well as HPLC chromatograms for phenylimidazole compounds can be
found in the Supporting Information.
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Enzyme expression and purification. The CdFabK and SpFabK enzymes were
expressed and purified to homogeneity and confirmed as reported elsewhere.25 SaFabI
was produced in high yield and purity as reported elsewhere in the literature.52 SaFabI
was confirmed via PAGE gel analysis and enzyme activity assay analysis in an Implen
NP80 nanophotometer as described below (West Lake Village, CA).
FAS-II biochemical enzyme assays. All compounds were dissolved in DMSO at
concentrations of 10 mM, then further diluted in pure DMSO to the required
concentrations. Both CdFabK and SpFabK assays were conducted via the following
protocol: Reactions were carried out at 25C in FabK Assay Buffer (100 mM HEPES pH
8.0, 500 mM NH4Cl, 10% glycerol, and 0.125 mg/mL -Globulins; 10% DMSO) with
150 M Cro-CoA and 150 M NADH. Compounds were incubated in three-fold
dilutions (ranging from 100 M to 5 nM for CdFabK and 33 M to 1.7 nM for SpFabK)
in the presence of 50 nM target enzyme. Compounds and enzyme were incubated
together for 10 minutes before the Cro-CoA substrate was added and the reaction was
started immediately afterward via the addition of NADH. NADH fluorescence (340
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nm/460 nm) was measured in a Biotek Synergy H1 microplate reader (Winooski, VT) in
a final volume of 100 L in Greiner Bio-One™ 384-Well µClear™ Bottom Polystyrene
Microplates (Monroe, NC) via 20 second intervals for 10 minutes to evaluate the rate of
reaction.
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SaFabI assays were conducted at 25 C in FabI Assay Buffer (50 mM MES pH
5.5, 150 mM NaCl, 10 mM EDTA, and 2% DMSO) with 300 M NADPH and 1 mM
Cro-CoA. Compounds were incubated at 10 M with 500 nM SaFabI for 20 minutes.
Reactions were started via the addition of NADPH. NADPH absorbance was measured
(340 nm) every 10 seconds for 10 minutes in a final volume of 100 L to evaluate the
rate of reaction.
FabK IC50 calculations and kinetics. Starting at three minutes, linear slopes were
measured for three additional minutes and used to determine the reaction rates.
Measurements were conducted in duplicate and IC50s were calculated via GraphPad
Prism 7.0d (La Jolla, CA) using four-parameter logistic (Hill) curve analysis using the
equation Y = Bottom + (Top – Bottom)/[1+10^((LogIC50 – X)*HillSlope)], where X is
logarithm of dose and Y is response. Kinetics with respect to cofactor and substrates were
also assessed via GraphPad Prism 7.0d comparing both Michaelis-Menten and substrate
inhibition models.
FabI activity. Starting at two minutes, linear slopes were measured for three minutes and
used to determine the reaction rates. Measurements were conducted in duplicate and
percent inhibitions were calculated against un-inhibited enzyme.
MICs in select gut flora. The strains used in this study are listed in Table 2. The
susceptibility tests to various compounds were performed in microtiter 96-well plate as
described previously.25 The MIC tests were performed in BHI broth for C. difficile, C.
perfringens and B. ovatus, whereas Bifidobacterium broth (Himedia) was used for B.
breve strains. These strains were grown at 37ºC in a Don Whitley A35 anaerobic
chamber. Susceptibility testing against S. pyogenes was performed in aerobic growth
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conditions in BHI-TY and Muller Hinton broth, respectively. Susceptibility tests were
performed in presence or absence of the efflux pump inhibitor verapamil (50 µg/ml).
MICs are reported as the lowest concentration of compound that inhibited bacterial
growth of respective test organisms.
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Acknowledgements
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We thank Y. Li, X. Fu, and L. Yang at St. Jude Children’s Research Hospital for ADME
studies on compound 1b. Table of Contents figure created with BioRender. Molecular
graphics and analyses (Figure 1) performed with UCSF Chimera, developed by the
Resource for Biocomputing, Visualization, and Informatics at the University of
California, San Francisco, with support from NIH P41-GM103311.
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Funding Sources
We are grateful to the National Institute of Allergy and Infectious Diseases of the
National Institutes of Health for funding (R21AI126755). This research was also made
possible by the American Foundation for Pharmaceutical Education, Pre-Doctoral Award
in Pharmaceutical Sciences to JAJ and by the Center for Pediatric Experimental
Therapeutics (CPET) via financial support to JAJ as a CPET scholar.
Supporting Information
Supporting information, including a sequence alignment comparing CdFabK to SpFabK;
as well as the methods pertaining to the sequence alignment, general chemistry synthesis
and procedure for phenylimidazole compounds, spectral analysis of phenylimidazole
compounds and HPLC purity, homology modeling of C. difficile FabK, and molecular
docking and modeling of C. difficile FabK inhibitors, is available online: This material is
available free of charge via the internet at http://pubs.acs.org.
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