Targeting Mycobacterium tuberculosis CoaBC through Chemical Inhibition of 4′-Phosphopantothenoyl- l -cysteine Synthetase (CoaB) Activity

Article abstract of DOI:10.1021/acsinfecdis.0c00904

Coenzyme A (CoA) is a ubiquitous cofactor present in all living cells and estimated to be required for up to 9% of intracellular enzymatic reactions. Mycobacterium tuberculosis (Mtb) relies on its own ability to biosynthesize CoA to meet the needs of the myriad enzymatic reactions that depend on this cofactor for activity. As such, the pathway to CoA biosynthesis is recognized as a potential source of novel tuberculosis drug targets. In prior work, we genetically validated CoaBC as a bactericidal drug target in Mtb in vitro and in vivo. Here, we describe the identification of compound 1f, a small molecule inhibitor of the 4′-phosphopantothenoyl-l-cysteine synthetase (PPCS; CoaB) domain of the bifunctional Mtb CoaBC, and show that this compound displays on-target activity in Mtb. Compound 1f was found to inhibit CoaBC uncompetitively with respect to 4′-phosphopantothenate, the substrate for the CoaB-catalyzed reaction. Furthermore, metabolomic profiling of wild-type Mtb H37Rv following exposure to compound 1f produced a signature consistent with perturbations in pantothenate and CoA biosynthesis. As the first report of a direct small molecule inhibitor of Mtb CoaBC displaying target-selective whole-cell activity, this study confirms the druggability of CoaBC and chemically validates this target.

Full text of DOI:10.1021/acsinfecdis.0c00904

pubs.acs.org/journal/aidcbc
Article
Targeting Mycobacterium tuberculosis CoaBC through Chemical
Inhibition of 4′-Phosphopantothenoyl‑L‑cysteine Synthetase (CoaB)
Activity
Joanna C. Evans,* Dinakaran Murugesan, John M. Post, Vitor Mendes, Zhe Wang, Navid Nahiyaan,
Sasha L. Lynch, Stephen Thompson, Simon R. Green, Peter C. Ray, Jeannine Hess, Christina Spry,
Anthony G. Coyne, Chris Abell, Helena I. M. Boshoff, Paul G. Wyatt, Kyu Y. Rhee, Tom L. Blundell,
Clifton E. Barry, III, and Valerie Mizrahi*
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ABSTRACT: Coenzyme A (CoA) is a ubiquitous cofactor present in all living cells and estimated to be required for up to 9% of
intracellular enzymatic reactions. Mycobacterium tuberculosis (Mtb) relies on its own ability to biosynthesize CoA to meet the needs
of the myriad enzymatic reactions that depend on this cofactor for activity. As such, the pathway to CoA biosynthesis is recognized
as a potential source of novel tuberculosis drug targets. In prior work, we genetically validated CoaBC as a bactericidal drug target in
Mtb in vitro and in vivo. Here, we describe the identification of compound 1f, a small molecule inhibitor of the 4′-
phosphopantothenoyl-L-cysteine synthetase (PPCS; CoaB) domain of the bifunctional Mtb CoaBC, and show that this compound
displays on-target activity in Mtb. Compound 1f was found to inhibit CoaBC uncompetitively with respect to 4′-
phosphopantothenate, the substrate for the CoaB-catalyzed reaction. Furthermore, metabolomic profiling of wild-type Mtb
H37Rv following exposure to compound 1f produced a signature consistent with perturbations in pantothenate and CoA
biosynthesis. As the first report of a direct small molecule inhibitor of Mtb CoaBC displaying target-selective whole-cell activity, this
study confirms the druggability of CoaBC and chemically validates this target.
KEYWORDS: tuberculosis, drug discovery, coenzyme A, CoaBC
which are aimed at delivering high-quality hit compounds with
novel mechanisms of action.9−14
The coenzyme A (CoA) biosynthetic pathway has attracted
attention as a source of new drug targets in a number of bacterial
pathogens,15−20 including Mtb,21 owing to its ubiquity and the
lack of sequence similarity between prokaryotic CoA biosyn-
uberculosis (TB) remains the leading cause of death from
Tan infectious disease worldwide, claiming an estimated 1.4
million lives in 2019.1 The high disease burden coupled with the
ongoing emergence and spread of strains of Mycobacterium
tuberculosis (Mtb) resistant to first- and second-line TB drugs
underscores the urgent need to develop new antitubercular
agents for the treatment of both drug-resistant and drug-
susceptible forms of the disease.2,3 In recent years, considerable
progress has been made in developing a TB drug pipeline,4
which has begun to deliver promising new drugs and drug
combinations.3,5−8 However, as attrition rates are high across
the entire TB drug pipeline there is an urgent need for
replenishment of the pipeline, starting from the earliest stage of
hit identification. This need is driving drug discovery efforts,
Received: December 23, 2020
Published: May 3, 2021
© 2021 The Authors. Published by
American Chemical Society
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Figure 1. Reaction catalyzed by CoaBC in Mtb. The CoaB domain of the bifunctional protein converts 4′-phosphopantothenate (PPA) into 4′-
phosphopantothenoyl-L-cysteine in two steps via the production of a 4′-phosphopantothenoyl-CMP intermediate. Decarboxylation of 4′-
phosphopantothenoyl-L-cysteine by the CoaC domain results in the production of 4′-phosphopantetheine (P-PantSH). CoaBC bypass occurs via
PanK-mediated phosphorylation of pantetheine (PantSH) to produce P-PantSH. PPCS, phosphopantothenoyl-L-cysteine synthetase; PPCDC,
phosphopantothenoyl-L-cysteine decarboxylase.
thesis enzymes and their eukaryotic counterparts.22 CoA serves
as a cofactor for key metabolic enzymes involved in numerous
cellular pathways, with 9% of all enzymes estimated as being
dependent on this essential cofactor.18,23 Of particular
significance in the case of Mtb is the critical role played by
CoA in the biosynthesis of lipids,21 which comprise essential
components of the cell envelope, and virulence factors,24 as well
as in the catabolism of lipids, which provides the primary source
of energy for the bacillus during infection.25 The availability of
crystal structures of a number of prokaryotic CoA pathway
enzymes has enabled structure-guided inhibitor design.26−29
However, although potent direct inhibitors of Mtb pantothenate
synthetase (PanC, Rv3602c) and pantothenate kinase (PanK,
CoaA, Rv1092c) have been developed using this approach, these
have failed to translate into lead compounds with significant
whole-cell activity against Mtb.29−32
The synthesis of CoA from its natural precursor, pantothenate
(vitamin B5, Pan), occurs in five universal enzymatic reactions,
the second and third of which are carried out by a bifunctional
CoaBC enzyme encoded by a single gene in most bacteria,
including Mtb (Rv1391).33 This enzyme comprises a C-terminal
CoaB domain with 4′-phosphopantothenoyl-L-cysteine synthe-
tase (PPCS) activity and an N-terminal CoaC domain with 4′-
phosphopantothenoyl-L-cysteine decarboxylase (PPCDC) ac-
tivity, which together catalyze the conversion of 4′-phospho-
pantothenate (PPA) into 4′-phosphopantetheine (P-PantSH)34
(Figure 1). The reaction occurs in three steps, the first two of
which are carried out by CoaB to produce a 4′-phosphopanto-
thenoyl-L-cysteine intermediate,35 which is subsequently de-
carboxylated at the cysteine moiety by CoaC to produce P-
PantSH.36 In prior work, we genetically validated CoaBC as a
drug target by demonstrating that coaBC silencing is bactericidal
in Mtb in vitro as well as during both the acute and chronic stages
of infection in mice.33 However, reports of potent and selective
small molecule inhibitors of bacterial CoaBC enzymes are
scarce, and those compounds identified have largely failed to
display significant whole-cell activity.37,38 Importantly, the
druggability of prokaryotic CoaBC enzymes was established
by the identification of the natural product CJ-15,801, which
acts as the precursor to a tight-binding CoaB (PPCS) inhibitor
with potent activity against Staphylococcus aureus.19 Further-
more, the recent identification of a small molecule inhibitor of
Mtb phosphopantetheinyl transferase (PptT),24 required for the
transfer of the P-PantSH moiety of CoA to acyl carrier proteins
(ACPs) involved in lipid production in Mtb, and the
identification of PanD as the target of the bioactive form of
pyrazinamide39,40 have validated the utility of key enzymes
involved in CoA metabolism as antitubercular drug targets.
We recently reported the first crystal structure of the
mycobacterial CoaBC together with the first potent and
selective inhibitors of Mtb CoaB.41 However, these potent
inhibitors of CoaB activity displayed limited whole-cell activity
against Mtb due to impaired permeability/efflux and intra-
cellular metabolism.41 In this study, we confirm the druggability
of Mtb CoaBC by identifying a new CoaB inhibitor that confers
whole-cell activity against Mtb and providing evidence linking
the growth inhibitory effects of this compound on Mtb to
inhibition of CoaBC.
RESULTS
■
High-Throughput Screening to Identify Inhibitors of
Mycobacterial CoaB. High-throughput screening of 215 000
small molecules from the DDU compound library was carried
out to identify inhibitors of Mtb CoaB activity using an
adaptation of the BIOLMOL Green end-point pyrophosphate
quantification assay, as previously described.41 This compound
library predominantly comprises commercially available mole-
cules representing a wide range of chemical space that
possessing the favorable physicochemical and molecular proper-
ties required for a potential preclinical drug candidate, with a
small subset of the library (<0.5%) comprising proprietary
compounds with known phenotypic activity against a number of
various pathogens. The primary screen, which had an overall hit
rate of 0.6%, led to the identification of compound 1a, with an
IC50 of 9.9 μM against Mtb CoaB. Following confirmation of the
target-specific inhibitory activity of 1a by demonstration of its
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a
Table 1. SAR and In Vitro Profiling of Modifications to Compound 1a
a
Intrinsic clearance (Cli) using CD1 mouse liver microsomes. ND = not determined. CHI, chromatographic hydrophobicity index.
Figure 2. Target-based whole-cell screening of compounds 1i and 1f against a coaBC Tet-OFF hypomorph. The impact of ATc-mediated
transcriptional silencing of coaBC on the relative growth of a coaBC hypomorph (a,b; d,e) and wild-type Mtb H37RvMA (c) in the presence of
increasing concentrations of 1i (a), 1f (b, c), rifampicin (d), and isoniazid (e) was determined. Data are representative of the mean and SD of
independent triplicates. ATc, anhydrotetracycline; Tet-OFF, mutant whose growth is inhibited in the presence of ATc; PantS2.5, pantethine (2.5 mg/
mL); RIF, rifampicin; INH, isoniazid.
inactivity in a counter-screen designed to eliminate false positive
hits arising due to inactivation of the pyrophosphatase, a further
8 series analogues were synthesized, with the initial structure−
activity relationship (SAR) investigation focused on probing the
pharmacophore to understand the impact of the N-phenyl and
S-phenyl substituents on their Mtb CoaB activity (Table 1).
Replacement of the S-phenyl group of 1a with either 4-
methylphenyl (1b), 4-chlorophenyl (1c), or 2,4-dichlorophenyl
(1d) led to modest improvements in activity against Mtb CoaB,
while introduction of 4-methoxyphenyl (1e) led to a 4-fold
increase in potency. Conversely, introduction of a substituted
benzoxazole (1f) led to a modest reduction in potency against
Mtb CoaB (Table 1). Introduction of either a 3-chlorophenyl
(1h) or 4-chlorophenyl (1g) at the N1-position was tolerated
with respect to Mtb CoaB activity, while introduction of a
chloropyridine at this position (1i) led to a complete loss of
inhibitory activity (Table 1).
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Assessment of the pharmacological properties of the
compounds revealed that none were overly lipophilic, with all
except 1i having CHI LogD values ≤1.5, and their kinetic
solubility limits ranged between 55 and 219 μM. The
compounds showed good stability in mouse liver microsomes,
all having clearance values of ≤2.8 (mL/min)/g and therefore
meeting our internal requirement of <5 (mL/min)/g for
progression of an early hit compound. None of the compounds
showed significant cytotoxicity against HepG2 cells (IC50 > 50
μM) with the exception of 1i, which showed modest activity
(IC50 = 40 μM).
Whole-Cell Screening of Series Analogues against
Mtb In Vitro. Minimum inhibitory concentrations (MICs) of
the 9 compounds were determined for wild-type Mtb (strain
H37RvMA)42 grown in Middlebrook 7H9 media supplemented
with albumin-dextrose-catalase (ADC) and glycerol. With the
exception of 1f and 1i, none of the compounds displayed whole-
cell activity against replicating Mtb at concentrations up to 125
μM (Table 1). Although the introduction of a substituted
benzoxazole did not substantially affect the activity of 1f in the
biochemical assay, this modification resulted in a modest
improvement in whole-cell activity against Mtb (MIC = 25.9
μM) (Table 1). The introduction of a chloropyridine at the N1-
position in 1i led to complete ablation of activity in the
biochemical CoaB assay; interestingly, this resulted in
significantly improved activity against Mtb (MIC = 6.2 μM),
thus implicating another target(s) in the mechanism of
antimycobacterial action of this analogue.
Assessment of Target Selectivity in Mtb Using a coaBC
Hypomorph. As reported previously, we and others have
demonstrated the utility of conditional knockdown mutants
(hypomorphs) as tools to ascertain the target selectivity of
inhibitors in Mtb by means of target-based whole-cell screen-
ing.43−48 This approach entails assessing whether cellular
depletion of the target of interest increases the potency of
compounds with biochemical activity against that target. The
parent compound and all analogues were subjected to screening
in checkerboard assays using a coaBC Tet-OFF hypomorph33 in
which coaBC expression is under control of a tetracycline-
regulated promoter such that progressive transcriptional
silencing of coaBC occurs upon addition of increasing
concentrations of the inducer, anhydrotetracycline (ATc). The
presence of ATc had no discernible effect on the potency of 8 of
the 9 compounds against the coaBC hypomorph at inhibitor
concentrations up to 125 μM. While the lack of hyper-
sensitization of the hypomorph in the case of compound 1i
(Figure 2a) is expected given its lack of biochemical activity
against CoaB, the lack of hypersensitization of the hypomorph to
1a−1e, 1g, and 1h suggests that the inability of these
compounds to inhibit growth of Mtb is likely attributable to
compound permeation, efflux, and/or metabolism. In contrast,
progressive transcriptional silencing of coaBC in the presence of
increasing concentrations of ATc induced dose-dependent
hypersensitization of the coaBC hypomorph to 1f, resulting in
a ∼7-fold decrease in MIC in the presence of 12.5 ng/mL ATc,
an observation indicative of on-target activity in Mtb (Figure
2b). Conversely, ATc had no effect on the activity of 1f against
wild-type Mtb H37RvMA (Figure 2c), which had a MIC
equivalent to that observed for the coaBC hypomorph at low
ATc concentrations and in its absence (0−3.2 ng/mL) (Figure
2b). This result is consistent with the fact that the level of coaBC
expression in the coaBC Tet-OFF hypomorph in the absence of
ATc is similar to that observed in wild-type Mtb.33 Moreover,
ATc did not render the coaBC hypomorph more susceptible to
the first-line antitubercular drugs rifampicin and isoniazid
(Figure 2d,e) whose mechanisms of action are unrelated to
CoA biosynthesis or utilization. Therefore, coaBC transcrip-
tional silencing selectively renders Mtb hypersensitive to 1f,
suggesting that this compound mediates growth inhibition of
Mtb, at least in part, via inhibition of CoaBC activity.
Confirmation of the Target Selectivity of Compound
1f in Mtb by CoaBC Bypass. We previously confirmed the
functionality of CoaBC bypass in Mtb via phosphorylation of
pantetheine (PantSH), the reduced form of pantethine (PantS),
by the Type I PanK (CoaA) to produce P-PantSH, the product
of the CoaBC-catalyzed reaction (Figure 1).33,37,49 It therefore
follows that if 1f is acting by inhibition of CoaBC
supplementation of the growth medium with exogenous PantS
should rescue Mtb from the inhibitory effects of this compound.
Indeed, supplementation with PantS rescued the wild-type
strain from 1f toxicity (Figure 2c) and led to a 4-fold reduction
in susceptibility of the coaBC hypomorph to 1f, although rescue
of the hypomorph was not observed at the highest concentration
of 1f tested (Figure 2b). To exclude the possibility that
conversion of PantS to Pan via hydrolysis could result in
increased production of PPA, which might in turn increase the
activity of CoaBC and thereby render 1f less active against the
coaBC hypomorph, we assessed the ability of Pan to rescue
growth of this strain under conditions of maximal coaBC
silencing (i.e., at an ATc concentration of 200 ng/mL).
Although Pan fully rescued Mtb from the growth inhibitory
effects of PanC deficiency in a panC Tet-OFF hypomorph,43 as
expected, it was unable to restore growth of the coaBC
hypomorph (Figure S1). This observation argues against the
existence of pantetheinase (vanin) activity (enzymes that
catalyze the hydrolysis of PantSH to Pan and are abundantly
present in mammalian tissues and serum50) in Mtb and, thus,
against a confounding role for PantS hydrolysis and subsequent
alteration in the physiological flux through the canonical
pathway in the PantS-mediated rescue of Mtb from 1f toxicity.
Given the propensity of Mtb hypomorphs to acquire
suppressor mutations that abrogate their ATc responsiveness,44
especially upon prolonged propagation and/or serial passage,
the coaBC hypomorph was grown to a lower cell density
(OD600) than the wild-type comparator for the use in the MIC
assays shown in Figure 2. We therefore hypothesized that the
discrepancy in PantS rescue observed between the wild-type and
coaBC hypomorph strains at the highest concentration of 1f
tested (125 μM) could be due to an inoculum effect. Consistent
with this hypothesis, the MICs of 1f in the presence of PantS
(2.5 mg/mL) were found to be 83.3 μM and 77.2 μM for the
wild-type and the coaBC hypomorph, respectively, when the
cultures used to inoculate the MIC plates were at an OD600 of
0.2, but increased to >125 μM when the inoculating cultures
were at a higher cell density (OD600 = 0.6). Importantly, the
inhibitory activity of 1f against the wild-type strain was
unaffected by the density of inoculating culture (MIC = 25.9
μM, assessed at OD600 = 0.2 or 0.6), and the presence of ATc did
not confer any non-specific growth inhibitory effects at the lower
cell density (Figure S2). Taken together with the ATc dose-
dependent hypersensitization of the coaBC hypomorph, these
results support the conclusion that the antibacterial activity of 1f
is mediated through inhibition of CoaBC.
Elucidation of the Mode of Inhibition and Selectivity
of Compound 1f. To assess the mode of inhibition of CoaBC
by 1f, competition experiments were carried out between the
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Figure 3. CoaBC inhibition by compound 1f. Dose response profile for MtbCoaBC (a) and HCoaB (b). Lineweaver−Burk plots showing the effect of
varying concentrations of compound 1f in the presence of varying concentrations of CTP (c), PPA (d), and L-cysteine (e). Data are representative of
the mean and SD of independent triplicates.
compound and the three substrates of CoaB (PPA, CTP, and L-
cysteine) using the EnzChek coupled enzyme assay, as
previously described.41 We first confirmed that compound 1f
was inactive on the coupled reporter enzymes at 100 μM. The
IC50 for this compound using the EnzChek assay was
determined to be 24.3 μM against the Mtb CoaBC, comparable
to that observed in the high-throughput screen (Figure 3a). To
evaluate the selectivity of compound 1f, the Mtb enzyme was
substituted with human CoaB in the EnzChek coupled enzyme
assay. A very similar dose−response profile was observed with an
IC50 value of 23.4 μM, suggesting poor selectivity of this
compound for the Mtb enzyme (Figure 3b).
Mtb CoaB exhibited an apparent Km of 27.4 2.6, 45.4 5.3,
and 29.8 3.0 μM for CTP, PPA, and L-cysteine, respectively
(Figure S3). Inhibition experiments demonstrated that 1f
displays uncompetitive inhibition for PPA, with an αKi of 19.0
0.6 μM, and noncompetitive inhibition for CTP and L-
cysteine, with Ki values of 10.1 1.4 μM and 22.7 7.6 μM,
respectively (Figure 3c−e), consistent with the compound
binding preferentially in the presence of PPA. This is in contrast
to our previous observation with allosteric inhibitors of CoaBC,
which bound preferentially in the presence of all three
substrates.41 We therefore decided to employ a different
approach to further validate these findings. Using differential
scanning fluorimetry (DSF), the melting temperature of CoaBC
in the absence of natural ligands was determined to be 46 °C
(Table 2; Figure S4). Evaluation of the effect of 1f on the melting
temperature of CoaBC in the presence of the three substrates
resulted in a substantial shift in melting temperature only when
PPA was present, regardless of the presence or absence of other
substrates and products (Table 2), thus further confirming that
binding of compound 1f to CoaB is dependent on the presence
of PPA (Table 2). The negative melting shift observed in the
presence of PPA and L-cysteine is consistent with a mixed
inhibition profile for L-cysteine.
Table 2. Effect of Compound 1f on the Melting Temperature
of Mtb CoaBC in the Presence of the CoaB Substrates CTP,
PPA, and L-Cysteine
a
Thermal shift relative to CoaBC (°C)
b
Substrates (1 mM)
Substrate
Substrate + 1f (2.5 mM) ΔTm
No substrate
CTP
CMP
−
5.5
1.5
1.5
5.5
2.5
0
1
PPA
3.5
10.5
7
L-cysteine
0.5
1.5
1
CMP + L-cysteine
CMP + PPA
CMP + L-cysteine + PPA
L-cysteine + PPA
CTP + PPA
CTP + L-cysteine
1.5
4.5
4.5
4.5
11.8 0.8
5.2 0.3
2.5
10.5
11
1.5
18.8 0.3
5.2 0.3
1
7
6.5
−3
7
0
a
CoaBC melting temperature under the assay conditions used is 46
°C. Data are representative of three independent triplicates. Where
b
SD is not indicated, SD = 0. ΔTm represents the difference in
melting temperature in the presence and absence of compound 1f
under each condition.
temperature of Mtb CoaBC suggests that PPA is able to bind
to the Mtb CoaB in the absence of CTP (Table 2). In Escherichia
coli, binding of PPA to CoaB causes movements in the protein
that close the PPA binding site, thereby limiting access to the
CTP binding site52 (Figure S5). Mechanistically, this triggers
the formation of a dead-end complex in which PPA and
compound 1f bind to the free enzyme, preventing CTP binding
and thereby blocking catalysis. This accounts for the inhibition
profile of compound 1f, which is very close to competitive
inhibition in the case of CTP. A crystal structure of CoaB with 1f
bound could not be obtained, but the observed mode of
inhibition together with known movements that occur in the
protein after PPA binding (Figure S5), with a flexible loop
covering the PPA binding site upon binding of this substrate,52
Although it was previously reported that binding of PPA to
Enterococcus faecalis CoaB occurs after CTP binding,51 the
ability of PPA alone to substantially shift the melting
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Figure 4. Metabolic impact of exposure of wild-type Mtb H37RvMA to 1f and 2b at 5 μM and 50 μM. Relative metabolite abundances (based on ion
intensities) are indicated in heat map format, where relative abundance refers to the change in the abundance of a given metabolite in the presence of
varying concentrations of 1f and 2b as compared to an untreated (DMSO) control. Data are log2 transformed, with columns representing individual
treatments, as indicated, and vertical clustering by rows revealing groups of metabolites exhibiting similar responses to drug exposure. Data are
representative of independent triplicates and were parsed by uncentered Pearson’s correlation with centroid linkage clustering and rendered using the
image generation program Java TreeView (http://jtreeview.sourceforge.net/).
suggests a different binding mode for 1f with distinct properties
from the previously reported allosteric inhibitors.41
reassembly of CoA via recycling of its P-PantSH moiety from
holo-ACPs in the presence of ATP, as has been observed in E.
coli.54
Metabolic Consequences of Exposure of Mtb to
Compound 1f. The metabolic derangement resulting from
exposure of Mtb to 5 μM or 50 μM of either 1f or the potent
allosteric CoaB inhibitor 2b (IC50 = 0.08 μM)41 (Figure 4) for
24 h was determined, as previously described.53 Unlike 1f, which
is active against Mtb (MIC = 25.9 μM), compound 2b did not
display significant whole-cell activity (MIC > 250 μM).41
Interestingly, however, both compounds elicited similar
alterations in the abundance of metabolites specifically
associated with Pan and CoA biosynthesis as compared to
untreated cells (Figure 4; Figure S6). Exposure to both
compounds produced a distinct metabolomic signature
involving metabolites related to the CoA pathway, which was
largely consistent with that observed following transcriptional
silencing of coaBC in Mtb.33 Specifically, there was substantial
accumulation of dephospho-CoA, the immediate precursor of
CoA, and notable depletion of the CoA thioesters propionyl-
CoA, succinyl-CoA, and malonyl-CoA relative to untreated
bacilli (Figure 4; Figure S6). The latter effect, which was dose-
dependent in the case of 1f, is consistent with the reduced levels
of malonyl- and acetyl-CoA observed upon transcriptional
silencing of coaBC.33 Unlike the CoA depletion observed upon
coaBC silencing,33 no detectable change in the level of CoA was
observed in Mtb treated with either compound 1f or 2b under
the conditions tested (Figure 4). Notably, however, while
analogous alterations in the levels of all other metabolites were
observed after 1.5 days of coaBC silencing, depletion of CoA was
only observed in the coaBC hypomorph at day 3. This may
reflect the longevity of this cofactor in Mtb, mediated by the
Finally, the fate of the compounds upon exposure to Mtb was
followed by monitoring their intracellular accumulation. Both 1f
and 2b were found to accumulate intracellularly in Mtb in a
dose-dependent manner, but the intracellular abundance of 1f
was substantially greater than 2b at both concentrations tested
(Figure S7). To exclude the possibility that this could be due to
inherent or bacterial-induced differences in the ionization
efficiencies of these compounds, standard curves of 1f and 2b
spiked into bacterial extracts were generated. The comparable
ionization efficiencies observed for both compounds (Figure
S8) further support our observation that the intracellular
abundances of these compounds vary substantially, corroborat-
ing our previous observation that the phenotypic divergence
between these compounds is likely due to the rapid
biotransformation of 2b.41,55,56 It is therefore reasonable to
conclude that the comparatively low intracellular level to which
compound 2b accumulates in Mtb over 24 h is sufficient to
induce metabolomic perturbations but does not reach the
threshold level required to manifest in a growth phenotype
through inhibition of CoaBC.
DISCUSSION
■
CoA is a ubiquitous cofactor across all domains of life. The
sequence divergence between prokaryotic and eukaryotic
components of the CoA biosynthesis pathway22 makes it a
potentially attractive source of novel drug targets in pathogens
that include Mtb.21 This notion has been reinforced by the
identification of PanD as the target of pyrazinoic acid, the
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bioactive form of the first-line antitubercular drug pyrazina-
mide,39,40 and the discovery of an inhibitor of Mtb PanK
requiring activation by the monooxygenase, EthA.57 However,
efforts to identify inhibitors of enzymes in the CoA pathway of
Mtb by target-based approaches have largely stalled at the level
of whole-cell activity. Using hypomorphs of Mtb as tools to
assess the target selectivity of small-molecule inhibitors of PanC
and PanK, we43 and others31 concluded that these failures might
be explained, at least in part, by the relative invulnerability of the
corresponding targets.33 The identification of CoaBC as a
bactericidal target in the CoA biosynthesis pathway in Mtb33
therefore prompted us to focus our efforts on this bifunctional
enzyme. This led to the first reported crystal structure of the
bifunctional CoaBC from M. smegmatis and identification of two
distinct chemical scaffolds that potently inhibit the CoaB
domain of Mtb CoaBC.41 Although neither inhibitor showed
significant whole-cell activity against Mtb, these promising
biochemical and structural data reinforced the potential of
CoaBC as a target and encouraged us to seek alternative
inhibitory scaffolds.
A high-throughput screen for inhibitors of the CoaB activity of
Mtb CoaBC yielded a primary screening hit, 1a, with activity
against the enzyme in the low micromolar range (IC50 = 9.9
μM); however, this compound was inactive against replicating
Mtb (MIC > 125 μM). Limited exploration around compound
1a led to the identification of an analogue, 1f, which showed
similar activity against the enzyme (IC50 = 15.6 μM) but
displayed modest whole-cell inhibitory activity against Mtb
(MIC = 25.9 μM). Importantly, the observed ATc-dependent
hypersensitization of a coaBC hypomorph33 to this compound
suggested that it engages CoaBC in whole Mtb cells. This
conclusion is supported by two further lines of evidence: first,
supplementation of the culture medium with PantS, which
specifically enables CoaBC bypass, rescued Mtb from 1f toxicity,
thus establishing a direct link between CoaBC engagement and
growth inhibition. Second, 1f elicited a metabolomic profile with
distinctive features consistent with CoA pathway disruption and
reminiscent of that observed upon transcriptional silencing of
coaBC, as evidenced by accumulation of dephospho-CoA and
dose-dependent depletion of the thioesters, propionyl-,
succinyl-, and malonyl-CoA, which are involved in multiple
cellular processes. However, unlike transcriptional silencing of
coaBC for 3 days, exposure of Mtb to 1f for 24 h had no
discernible impact on the level of CoA. While this may reflect the
longevity of this cofactor,54 it is worth noting that genetic
inhibition of a target (via its depletion within the cell) is an
imperfect surrogate for chemical inhibition of that target,44,47,58
which complicates direct comparison of the metabolic
consequences of transcriptional silencing vs. drug treatment.
As noted above, the substantial accumulation of dephospho-
CoA and lack of accumulation of PPA following exposure of Mtb
to compounds 1f and 2b as well as in response to coaBC
silencing were not readily predicted, consistent with our
assertion that the impact of CoaBC inhibition is more complex
than alteration of its specific substrate and product levels
alone.33 PanK, which catalyzes the first committed step in the
conversion of Pan to CoA, is considered the rate-limiting
enzyme in the CoA biosynthesis pathway59 and is regulated by
feedback inhibition by CoA and its thioesters.60 However, Mtb
CoaD is also regulated by feedback inhibition by CoA, as well as
by dephospho-CoA, the product of its own reaction,27,61−63 and
we recently reported that CoA and several acyl-CoAs are
additionally capable of inhibiting CoaB activity in Mtb.41
Together, these observations suggest that the regulatory
mechanisms mediating CoA biosynthesis in Mtb are complex
and incompletely understood. As such, it is interesting to
speculate that the depletion of ATP observed following exposure
of Mtb to compounds 1f and 2b (Figure 4) may contribute to
the observed accumulation of dephospho-CoA by reducing the
catalytic efficiency of the ATP-dependent Mtb CoaE,64 which
catalyzes the final step in the pathway by conversion of
dephospho-CoA to CoA. Additionally, Mtb CoaE is compet-
itively inhibited by CTP, for which it has an affinity similar to
that of its substrate, dephospho-CoA.64 Under physiological
conditions, CoaE is predominantly found in an inactive trimeric
form, which is converted to an active monomeric form in the
presence of dephospho-CoA.65 However, due to overlap of the
dephospho-CoA and CTP binding sites, binding of CTP to
CoaE completely abolishes its enzymatic activity by preventing
conversion to the active form despite the presence of
dephospho-CoA, with 100 μM CTP resulting in a greater than
60% decrease in CoaE activity.65 Given that 1f displays
noncompetitive (but very close to competitive) inhibition for
CTP (Ki = 10.1 1.4 μM) and that Mtb CoaB exhibits an
apparent Km of 27.4 2.6 μM for CTP, the inhibitory activity of
1f may cause a transient increase in intracellular CTP pools, thus
limiting the catalytic efficiency of CoaE and contributing to the
observed accumulation of dephospho-CoA. This hypothesis is
further supported by the observation that Mtb CoaBC can bind
PPA in the absence of CTP, triggering the formation of a dead-
end complex in which PPA and 1f are bound to the free enzyme,
thereby preventing the binding of CTP. Notably, this mode of
binding, with both 1f and 2b showing uncompetitive inhibition
for PPA, could also account for the lack of accumulation of this
CoaBC substrate observed following exposure of Mtb to both of
these compounds for 24 h. Importantly, however, despite the
induction of these unpredictable metabolomic alterations, the
comparable signatures observed following exposure of Mtb to 1f
or 2b and upon transcriptional silencing of coaBC provide
further evidence in support of the target selectivity of these
compounds.
The lack of correlation between CoaBC inhibition and whole-
cell activity observed for the other compounds investigated in
this study provides yet another example of the profound
challenges of target-based TB drug discovery.3 In this regard, the
chemical validation of CoaBC resulting from the identification
of 1f as a compound that can penetrate Mtb, evade metabolism,
and engage its cellular target, thereby inhibiting Mtb growth,
represents a significant advance and provides a platform for
further drug discovery efforts. Although 1f lacks selectivity with
respect to the human enzyme, ongoing efforts to exploit
additional chemical properties of these molecules are underway,
with the aim of identifying compounds that not only show
selectivity for Mtb CoaB but also demonstrate more potent
inhibition of biochemical and phenotypic activity. The finding
that 1f uncompetitively inhibits the mycobacterial CoaB,
binding preferentially in the presence of PPA, presents a mode
of inhibition distinct from those previously described41 and
highlights the versatility of Mtb CoaB as a target that is
potentially amenable to chemical inhibition via multiple
approaches and by a variety of diverse chemical scaffolds.
METHODS
■
Compound Synthesis. Solvents and reagents were
purchased from commercial suppliers and used without further
purification. Dry solvents were purchased in sure sealed bottles
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stored over molecular sieves. Reactions using microwave
irradiation were carried out in a Biotage Initiator microwave.
Normal phase thin layer chromatography (TLC) was performed
using precoated silica plates (Kieselgel 60 F254, BDH) with
visualization via UV light (UV254/365 nm) or ninhydrin
solution. Flash chromatography was performed using Combi-
flash Companion Rf (Teledyne ISCO) and prepacked silica gel
columns (Teledyne ISCO). Mass-directed preparative high
performance liquid chromatography (HPLC) separations were
performed using an Agilent 1260 Infinity system equipped with
diode array detector (DAD) and mass detector, using Waters
Sunfire C18 OBD Prep Column, 100 Å, 5 μm, 19 mm × 100 mm
with SunFire C18 Prep Guard Cartridge, 100 Å, 10 μm, 19 mm
× 10 mm; using water (solvent A) and methanol (solvent B). 1H
NMR spectra were recorded on a Bruker Avance DPX 500
spectrometer (1H at 500.1 MHz, 13C at 125 MHz, 19F at 470.5
MHz), or a Bruker Avance DPX 400 (1H at 400 MHz).
Chemical shifts (δ) are expressed in ppm, using the residual
solvent as the internal reference in all cases. Signal splitting
patterns are described as singlet (s), doublet (d), triplet (t),
quartet (q), multiplet (m), broad (br), or a combination thereof.
Coupling constants (J) are quoted to the nearest 0.1 Hz.
High resolution mass spectrometry (HRMS) was carried out
using a Bruker Daltonics MicrOTOF mass spectrometer run in
positive mode. LC-MS analysis and chromatographic separation
were conducted using (i) Bruker Daltonics MicrOTOF mass
spectrometer or an Agilent Technologies 1200 series HPLC
connected to an Agilent Technologies 6130 quadrupole LC/
MS, where both instruments were connected to an Agilent diode
array detector, (ii) Bruker MicrOTOF II focus ESI mass
spectrometer connected in parallel to a Thermo Dionex
Ultimate 3000 RSLC system with diode array detector, (iii)
Advion Expression mass spectrometer connected to a Thermo
Dionex Ultimate 3000 HPLC with diode array detector, using
either a Waters XBridge column (50 mm × 2.1 mm, 3.5 μm
particle size) or a Waters X-select column (30 mm × 2.1 mm, 2.5
μm particle size) with a gradient of 5−90% acetonitrile/water +
0.1% formic acid, or (iv) Shimadzu LCMS-2020 single
quadrupole liquid chromatograph mass spectrometer (LC/
MS) system equipped with a Hypersil gold column 50 mm × 2.1
mm 1.9 μm pore size. All assay compounds had a measured
purity of ≥95% as determined using these analytical LC-MS
systems.
by column chromatography (SiO2, 0−100% ethyl acetate/
hexane) to give the title compound (41 mg, 0.13 mmol, 46.7%
yield) as a yellow solid.
1H NMR (500 MHz, DMSO-d6) δ 12.13 (s, 1H), 7.74 (d, J =
7.6 Hz, 2H), 7.48 (dd, J = 8.0, 8.0 Hz, 2H), 7.29 (t, J = 7.3 Hz,
1H), 7.11 (d, J = 8.1 Hz, 2H), 7.00 (d, J = 7.8 Hz, 2H), 2.24 (s,
3H), 2.09 (s, 3H). HRMS ES+ 297.1043, C17H14N2OS requires
297.1056.
Compound 1c, 4-(4-Chlorophenyl)sulfanyl-5-methyl-2-
phenyl-pyrazol-3-ol. To a stirred solution of 5-methyl-2-
phenyl-pyrazol-3-ol (55 mg, 0.32 mmol) in acetonitrile (3
mL) were added 4-chlorobenzenethiol (68.5 mg, 0.48 mmol)
and sodium hydroxide (15.1 mg, 0.38 mmol), and the reaction
was heated at 80 °C for 18 h. The reaction mixture was
concentrated in vacuo to give a crude residue, which was purified
by column chromatography (SiO2, 0−100% ethyl acetate/
hexane) to give the title compound (25 mg, 0.07 mmol, 23.5%
yield) as a yellow solid.
1H NMR (500 MHz, DMSO-d6) δ 12.30 (s, 1H), 7.75 (d, J =
7.6 Hz, 2H), 7.50−7.46 (m, 2H), 7.35 (d, J = 8.5 Hz, 2H), 7.29
(t, J = 7.4 Hz, 1H), 7.10 (d, J = 8.5 Hz, 2H), 2.12 (s, 3H). HRMS
ES+ 317.0488, C16H14N2OSCl requires 317.0509.
Compound 1d, 4-(3,4-Dichlorophenyl)sulfanyl-5-methyl-
2-phenyl-pyrazol-3-ol. To a stirred solution of 5-methyl-2-
phenyl-pyrazol-3-ol (55 mg, 0.32 mmol) in acetonitrile (3 mL)
were added 3,4-dichlorobenzenethiol (77.1 mg, 0.43 mmol) and
sodium hydroxide (15.1 mg, 0.38 mmol), and the reaction was
heated at 80 °C for 18 h. The reaction mixture was concentrated
in vacuo to give a crude residue, which was purified by column
chromatography (SiO2, 0−100% ethyl acetate/hexane) to give
the title compound (58 mg, 0.16 mmol, 54.6% yield) as a yellow
solid.
1H NMR (500 MHz, DMSO-d6) δ 12.38 (s, 1H), 7.77−7.75
(m, 2H), 7.64 (d, J = 2.3 Hz, 1H), 7.51−7.48 (m, 2H), 7.37−
7.29 (m, 2H), 6.78 (d, J = 7.0 Hz, 1H), 2.13 (s, 3H). HRMS ES+
351.0122, C16H13N2OSCl2 requires 351.0120.
Compound 1e, 4-(4-Methoxyphenyl)sulfanyl-5-methyl-2-
phenyl-pyrazol-3-ol. To a stirred solution of 5-methyl-2-
phenyl-pyrazol-3-ol (55 mg, 0.32 mmol) in acetonitrile (3
mL) were added 4-methoxybenzenethiol (66.4 mg, 0.47 mmol)
and sodium hydroxide (15.1 mg, 0.38 mmol), and the reaction
was heated at 80 °C for 18 h. The reaction mixture was
concentrated in vacuo to give a crude residue, which was purified
by column chromatography (SiO2, 0−100% ethyl acetate/
hexane) to give the title compound (25 mg, 0.07 mmol, 24%
yield) as a yellow solid.
Compound 1a, 4-Phenylsulfanyl-5-methyl-2-phenyl-pyr-
azol-3-ol. To a stirred solution of 5-methyl-2-phenyl-pyrazol-3-
ol (50 mg, 0.29 mmol) in acetonitrile (3 mL) were added
benzenethiol (47.4 mg, 0.43 mmol) and sodium hydroxide (13.8
mg, 0.34 mmol), and the reaction was heated at 80 °C for 18 h.
The reaction mixture was concentrated in vacuo to give a crude
residue, which was purified by column chromatography (SiO2,
0−100% ethyl acetate:hexane) to give the title compound (58
mg, 0.20 mmol, 68.0% yield) as a yellow solid.
1H NMR (500 MHz, DMSO-d6) δ 12.10 (m, 1H), 7.73 (d, J =
7.6 Hz, 2H), 7.49−7.45 (m, 2H), 7.27 (t, J = 7.3 Hz, 1H), 7.09
(d, J = 8.3 Hz, 2H), 6.89 (d, J = 8.8 Hz, 2H), 3.71 (s, 3H), 2.11
(s, 3H). HRMS ES+ 313.0983, C17H17N2O2S requires 313.1005.
Compound 1f, 4-(5-Chloro-1,3-benzoxazol-2-yl)sulfanyl-
5-methyl-2-phenyl-pyrazol-3-ol. To a stirred solution of 5-
methyl-2-phenyl-pyrazol-3-ol (75 mg, 0.43 mmol) in acetoni-
trile (3 mL) were added 5-chloro-1,3-benzoxazole-2-thiol
(119.8 mg, 0.65 mmol) and sodium hydroxide (20.7 mg, 0.52
mmol), and the reaction was heated at 80 °C for 18 h. The
reaction mixture was concentrated in vacuo to give a crude
residue, which was purified by column chromatography (SiO2,
0−100% ethyl acetate:hexane) to give the title compound (15
mg, 0.04 mmol, 9.3% yield) as a yellow solid.
1H NMR (500 MHz, DMSO-d6) δ 12.19 (s, 1H), 7.75 (d, J =
7.6 Hz, 2H), 7.48 (dd, J = 7.9, 7.9 Hz, 2H), 7.32−7.26 (m, 3H),
7.15−7.07 (m, 4H), 2.13 (s, 3H). HRMS ES+ 283.0895,
C16H15N2OS requires 283.0900.
Compound 1b, 4-(4-Methylphenyl)sulfanyl-5-methyl-2-
phenyl-pyrazol-3-ol. To a stirred solution of 5-methyl-2-
phenyl-pyrazol-3-ol (50 mg, 0.29 mmol) in acetonitrile (3
mL) were added sodium 4-methylbenzenethiolate (42 mg, 0.29
mmol) and sodium hydroxide (13.8 mg, 0.34 mmol), and the
reaction was heated at 80 °C for 18 h. The reaction mixture was
concentrated in vacuo to give a crude residue, which was purified
1H NMR (500 MHz, DMSO-d6) δ 12.10 (s, 1H), 7.81−7.79
(m, 2H), 7.68−7.65 (m, 1H), 7.53−7.39 (m, 3H), 7.33−7.26
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(m, 2H), 2.07 (s, 3H). HRMS ES+ 358.0378, C17H13N2O2SCl
requires 358.0411.
trile (3 mL) were added 4-chlorothiophenol (99.3 mg, 0.69
mmol) and sodium hydroxide (22 mg, 0.55 mmol), and the
reaction was heated at 80 °C for 18 h. The reaction mixture was
concentrated in vacuo to give a crude residue, which was purified
by column chromatography (SiO2, 0−100% ethyl acetate/
hexane) to give the title compound (96 mg, 0.26 mmol, 55.9%
yield) as a yellow solid.
Compound 1g, 2-(4-Chlorophenyl)-4-(4-chlorophenyl)-
sulfanyl-5-methyl-pyrazol-3-ol. Step 1, 2-(4-Chlorophenyl)-
5-methyl-pyrazol-3-ol. To a stirred solution of 4-chlorophe-
nylhydrazine hydrochloride (275 mg, 1.54 mmol) in acetic acid
(3 mL) was added ethyl-3-oxobutanoate (200 mg, 1.54 mmol),
and the reaction was heated at reflux for 3 h. The solvent was
removed in vacuo to give a crude residue, which was purified by
column chromatography (SiO2, 0−100% ethyl acetate/hexane)
to give the title compound (115 mg, 0.5 mmol, 32.2% yield),
which was used without further purification. LRMS (ESI) m/z
[MH+] = 209.1.
1H NMR (500 MHz, DMSO-d6) δ 12.43 (s, 1H), 7.82−7.78
(m, 2H), 7.56−7.53 (m, 2H), 7.36−7.33 (m, 2H), 7.12−7.08
(m, 2H), 2.13 (s, 3H). LRMS (ESI) m/z [MH+] = 352.1.
High-Throughput Screening of Small Molecule Libra-
ries. A library of 215 000 small molecules from the DDU
compound collection was screened to identify potential
inhibitors of Mtb CoaB at 30 μM using an adaptation of the
BIOMOL Green (Enzo Life Sciences) pyrophosphate quanti-
fication assay, as previously described.41 Briefly, decreased
production of phosphate following addition of a pyrophospha-
tase was indicative of reduced pyrophosphate production by
CoaB and, hence, inhibition.
Step 2, 2-(4-Chlorophenyl)-4-(4-chlorophenyl)sulfanyl-5-
methyl-pyrazol-3-ol. To a stirred solution of 2-(4-chlorophen-
yl)-5-methyl-pyrazol-3-ol (93 mg, 0.43 mmol) in acetonitrile (3
mL) were added 4-chlorothiophenol (96.7 mg, 0.67 mmol) and
sodium hydroxide (21.4 mg, 0.54 mmol), and the reaction was
heated at 80 °C for 18 h. The reaction mixture was concentrated
in vacuo to give a crude residue, which was purified by column
chromatography (SiO2, 0−100% ethyl acetate/hexane) to give
the title compound (70.3 mg, 0.19 mmol, 42.7% yield) as a
yellow solid.
Counter-screening of Primary Screen Hits. Counter-
screens carried out to exclude false positive compounds were
performed in 50 μL reactions containing 0.5 U/mL
pyrophosphatase and 2 μM pyrophosphate in 100 mM Tris-
HCl, pH 7.6, buffer containing 1 mM MgCl2 and 1 mM TCEP
for 2 h at room temperature in 384-well microplates (Greiner
Bio-One). Control reactions were performed in the absence of
the pyrophosphatase. The reactions were terminated by
addition of 50 μL BIOMOL Green (Enzo Life Sciences), and
product formation was measured by absorbance (650 nm) using
a PHERAstar microplate reader (BMG Labtech) following
incubation for 20 min. Liquid dispensing was performed using a
Thermo Scientific Matrix Wellmate, and data were processed
and analyzed using ActivityBase (IDBS).
Pharmacological Profiling of Compounds. Compounds
were profiled for HepG2 cytotoxicity, mouse liver microsomal
clearance, and aqueous solubility, as previously described.66
Bacterial Strains and Growth Conditions. The coaBC
Tet-OFF hypomorph used in this study was constructed as
previously described,33 and derived from the virulent, PDIM-
producing parental strain, Mtb H37RvMA.42 All strains were
routinely grown in Difco Middlebrook 7H9 broth (BD)
supplemented with Middlebrook albumin-dextrose-catalase
(ADC) enrichment (BD), 0.2% glycerol (Sigma-Aldrich), and
0.05% Tween-80, unless otherwise indicated. Hygromycin and
kanamycin were used at concentrations of 50 and 25 μg/mL,
and pantethine (Sigma-Aldrich) was included at 2.5 mg/mL
where required. In order to repress the expression of target genes
in cells expressing revTetR, cultures were routinely grown in the
absence of supplementation to OD600 ≈ 0.2 prior to dilution in
Middlebrook 7H9 broth containing the ATc inducer at
concentrations up to 200 ng/mL in order to transcriptionally
silence coaBC. To avoid inactivation of the inducer, all cultures
containing ATc were incubated in the dark, and exposure of the
cultures to light was minimized.
1H NMR (500 MHz, DMSO-d6) δ 12.43 (s, 1H), 7.82−7.78
(m, 2H), 7.56−7.53 (m, 2H), 7.36−7.33 (m, 2H), 7.12−7.08
(m, 2H), 2.13 (s, 3H). HRMS ES+ 351.0120, C16H13N2O2SCl2
requires 351.0121.
Compound 1h, 2-(3-Chlorophenyl)-4-(4-chlorophenyl)-
sulfanyl-5-methyl-pyrazol-3-ol. To a stirred solution of ethyl
2-chloro-3-oxobutanoate (45.9 mg, 0.28 mmol) in DMF (1 mL)
were added 4-chlorobenzenethiol (40.3 mg, 0.28 mmol) and
DIPEA (58.3 μL, 0.34 mmol); the reaction was stirred at room
temperature for 1 h and then heated at 100 °C for 9 h. The
solvent was removed in vacuo to give a crude residue, which was
dissolved in acetic acid (0.7 mL). To this (3-chlorophenyl)-
hydrazine hydrochloride (50 mg, 0.28 mmol) and sodium
acetate (25 mg, 0.31 mmol) were added, and the reaction
mixture stirred at room temperature for 1 h followed by heating
at 100 °C for 12 h.
The solvent was removed in vacuo to give a crude residue,
which was dissolved in DMSO and filtered, and the filtrate was
subjected to HPLC purification to give the title compound (26.7
mg, 0.076 mmol, 27.2% yield) as a pink solid.
1H NMR (400 MHz, DMSO-d6) δ 12.54 (s, 1H), 7.85 (s,
1H), 7.79−7.76 (m, 1H), 7.51 (t, J = 8.2 Hz, 1H), 7.36−7.32
(m, 3H), 7.13−7.09 (m, 2H), 2.12 (s, 3H). LRMS (ESI) m/z
[MH+] = 351.0.
Compound 1i, 2-(4-Chlorophenyl)-4-(4-chlorophenyl)-
sulfanyl-5-methyl-pyrazol-3-ol. Step 1, 2-(5-Chloro-2-pyrid-
yl)-5-methyl-pyrazol-3-ol. To a stirred solution of (5-chloro-2-
pyridyl)hydrazine (220.6 mg, 1.54 mmol) in acetic acid (3 mL)
was added ethyl-3-oxobutanoate (200 mg, 1.54 mmol), and the
reaction was heated at reflux for 3 h. The solvent was removed in
vacuo to give a crude residue, which was purified by column
chromatography (SiO2, 0−100% ethyl acetate/hexane) to give
the title compound (242 mg, 1.1 mmol, 71.3% yield) as an off-
white solid.
MICs and Checkerboard Assays for Assessing Target
Selectivity. The minimum inhibitory concentrations (MICs)
of the compounds were determined using the microbroth
dilution assay and growth inhibition was quantitatively
determined using Alamar Blue as a measure of fluorescence
output, as previously described.46 Briefly, 2-fold serial dilutions
of each compound were inoculated with a suspension of Mtb
H37RvMA42 or the coaBC hypomorph33 at cell densities of
1H NMR (500 MHz, DMSO-d6) δ 12.03 (s, 1H), 8.46 (d, J =
2.6 Hz, 1H), 8.40 (s, 1H), 8.02 (dd, J = 2.6, 9.0 Hz, 1H), 5.16 (s,
1H), 2.17 (s, 3H). LRMS (ESI) m/z [MH+] = 210.1
Step 2, 2-(4-Chlorophenyl)-4-(4-chlorophenyl)sulfanyl-5-
methyl-pyrazol-3-ol. To a stirred solution of 2-(5-chloro-2-
pyridyl)-5-methyl-pyrazol-3-ol (96 mg, 0.46 mmol) in acetoni-
∼105 CFU/mL (OD600 = 0.6) and ∼104 CFU/mL (OD600
=
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0.2), respectively, to a final volume of 100 μL in a U-bottom 96-
well microtiter plate and incubated at 37 °C for 10 days. A
volume of 10 μL of Alamar Blue solution was then added to each
well, and the plates were incubated at 37 °C for a further 24 h.
Fluorescence as an indication of growth was measured using a
SpectraMax i3x multimode microplate reader (Molecular
Devices) in bottom-reading mode with excitation at 544 nm
and emission at 590 nm. All data are representative of
independent triplicates.
for a further 24 h. Metabolite extraction and LC-MS-based
metabolomic profiling were carried out as previously
described.33 All data obtained by metabolomics were the
average of independent triplicates.
Mycobacterium tuberculosis Compound Uptake. Com-
pound uptake was determined using a filter culture system, as
previously described.41,53 Briefly, Mtb was grown on nylon
Durapore 0.22 μm membrane filters placed on top of
Middlebrook 7H11 agar plates supplemented with 0.2% glycerol
and 10% OADC for 1 week at 37 °C to expand the biomass. The
membranes were then placed atop a reservoir containing
Middlebrook 7H9 broth with or without compound, such that
the underside of the bacteria-laden filters was in direct contact
with the media. Following 24 h of incubation at 37 °C, which we
previously determined to be pre-lethal for most frontline
drugs,11 the filters were plunged into 40:40:20 methanol:ace-
tonitrile:water precooled to −20 °C, and cells were lysed using a
bead beater. Cell lysates were mixed with equal volumes of 50%
acetonitrile and 0.2% formic acid, and drug accumulation was
measured using mass spectrometry in positive and negative ion
mode, as previously described.53 Relative drug levels were
quantified by comparison with standard curves generated from
bacterial lysates spiked with compound, using the method of
standard addition.67 Internal standards were routinely included
with each sample run, and data were additionally normalized to
sample protein biomass.
Protein Purification and Enzymatic Assays. E. coli
BL21(DE3) containing a pET28aSUMO-CoaBC construct or
a human CoaB construct with a cleavable N-terminal 6×His tag
was grown to mid-exponential growth phase (OD600 = 0.6−0.8)
in 2×YT media supplemented with kanamycin (30 μg/mL) at
37 °C. Overexpression of the enzymes was induced by the
addition of 0.5 mM IPTG and incubation at 18 °C for 18−20 h.
Both enzymes were purified, and enzymatic assays were
performed using the EnzChek pyrophosphate assay kit (E-
6645) (Life Technologies) except in the case of L-cysteine
competition, where a CMP quantification assay was used, as
previously described.41 Briefly, the IC50 for compound 1f was
determined in 100 mM Tris, pH 7.5, 1 mM TCEP, 2% DMSO, 1
mM MgCl2, 200 μM MSEG, 0.03 U/mL inorganic pyrophos-
phatase, 1 U/mL purine nucleoside phosphorylase, with 32 nM
Mtb CoaBC, 125 μM CTP, 125 μM PPA, 500 μM L-cysteine,
and variable concentrations of 1f (2−256 μM). In the assays
with human CoaB, an enzyme concentration of 1 μM was used.
The inhibition of the coupling enzymes by 1f was assessed, and
the compound was inactive against these enzymes at the tested
range of concentrations. Kinetic parameters and competition
assays for CTP and PPA were determined under the same
conditions but with variable concentrations of CTP (31.25 μM,
62.5 μM, 125 μM, 250 μM, and 500 μM) with 125 μM PPA and
500 μM L-cysteine or variable concentrations of PPA (31.25 μM,
62.5 μM, 125 μM, 250 μM, and 500 μM) with 125 μM CTP and
500 μM L-cysteine.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acsinfecdis.0c00904.
■
sı
Effect of exogenous supplementation with pantothenate
on the growth of CoaBC- and PanC-deficient Mtb, lack of
effect ATc on the susceptibility of Mtb H37RvMA to
compound 1f when assessed at low cell density, Mtb
CoaBC saturation curve fit for Michaelis−Menten model,
DSF melting profile of CoaBC, movement of a flexible
loop covering PPA binding site, impact of exposure of
Mtb H37Rv to 1f and 2b on acyl-CoA species and CoA
pathway metabolites, total intracellular accumulation of
compound 1f and 2b, and standard curves showing
comparable ionization efficiencies of compounds 1f and
2b spiked into bacterial extracts (PDF)
Kinetic parameters and competition assays for L-cysteine were
determined in 1 mM MgCl2, 100 mM Tris, pH 7.5, 1 mM TCEP,
2% DMSO, 32 nM Mtb CoaBC, 125 μM CTP, 125 μM PPA,
and variable concentrations of L-cysteine (31.25 μM, 62.5 μM,
93.75 μM, 125 μM, and 250 μM). All reactions were carried out
in at least triplicate. IC50, Michaelis−Menten, and inhibition
constants were calculated using GraphPad Prism.
Differential Scanning Fluorimetry. Differential scanning
fluorimetry (DSF) was used to assess inhibitor binding to the
CoaB domain of CoaBC. The assay was performed in a 96 well
plate format using a CFX Connect (Bio-Rad). Each well
contained a solution of 5 μM M. tuberculosis CoaBC, 25 mM
Tris, pH 8.0, 150 mM NaCl, 1 mM MgCl2, 2.5% DMSO, 5×
Sypro Orange. Substrates or products were added individually to
each well at a final concentration of 1 mM in the presence and
absence of 2.5 mM 1f. Combinations of substrates and products
(CMP and L-cysteine; CTP and L-cysteine; CMP and PPA; CTP
and PPA; CMP, PPA, and L-cysteine; PPA and L-cysteine) each
at a final concentration of 1 mM were also tested in the presence
or absence of 2.5 mM 1f. Unfolding of CoaBC in the absence of
any substrate or product was measured in parallel. All data are
representative of independent triplicates.
AUTHOR INFORMATION
Corresponding Authors
■
Joanna C. Evans − MRC/NHLS/UCT Molecular
Mycobacteriology Research Unit & DST/NRF Centre of
Excellence for Biomedical TB Research & Wellcome Centre for
Infectious Diseases Research in Africa, Institute of Infectious
Disease and Molecular Medicine and Department of
Pathology, Faculty of Health Sciences, University of Cape
Town, Observatory 7925, South Africa; orcid.org/0000-
0002-4139-9527; Email: Joanna.Evans2503@gmail.com
Valerie Mizrahi − MRC/NHLS/UCT Molecular
Mycobacteriology Research Unit & DST/NRF Centre of
Excellence for Biomedical TB Research & Wellcome Centre for
Infectious Diseases Research in Africa, Institute of Infectious
Disease and Molecular Medicine and Department of
Pathology, Faculty of Health Sciences, University of Cape
Town, Observatory 7925, South Africa; orcid.org/0000-
0003-4824-9115; Email: Valerie.Mizrahi@uct.ac.za
Metabolite Extraction and Metabolomic Profiling.
Mtb-laden filters used for metabolomic analyses were generated
as previously described53 and incubated at 37 °C for 4 days to
expand biomass. Mtb-laden filters were then transferred onto a
fresh 7H10 agar plate with or without 1f and 2b at 5 and 50 μM
1675
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Authors
pubs.acs.org/journal/aidcbc
Article
Complete contact information is available at:
https://pubs.acs.org/10.1021/acsinfecdis.0c00904
Dinakaran Murugesan − Drug Discovery Unit, College of Life
Sciences, University of Dundee, Dundee DD1 5EH, Scotland,
U.K.
John M. Post − Drug Discovery Unit, College of Life Sciences,
University of Dundee, Dundee DD1 5EH, Scotland, U.K.
Vitor Mendes − Department of Biochemistry, University of
Cambridge, Cambridge CB2 1GA, U.K.; orcid.org/0000-
0002-2734-2444
Zhe Wang − Department of Microbiology and Immunology,
Weill Cornell Medical College, New York 10065, United States
Navid Nahiyaan − Department of Microbiology and
Immunology, Weill Cornell Medical College, New York 10065,
United States
Sasha L. Lynch − MRC/NHLS/UCT Molecular
Mycobacteriology Research Unit & DST/NRF Centre of
Excellence for Biomedical TB Research & Wellcome Centre for
Infectious Diseases Research in Africa, Institute of Infectious
Disease and Molecular Medicine and Department of
Pathology, Faculty of Health Sciences, University of Cape
Town, Observatory 7925, South Africa
Stephen Thompson − Drug Discovery Unit, College of Life
Sciences, University of Dundee, Dundee DD1 5EH, Scotland,
U.K.
Simon R. Green − Drug Discovery Unit, College of Life Sciences,
University of Dundee, Dundee DD1 5EH, Scotland, U.K.;
orcid.org/0000-0001-5054-4792
Peter C. Ray − Drug Discovery Unit, College of Life Sciences,
University of Dundee, Dundee DD1 5EH, Scotland, U.K.
Jeannine Hess − Yusuf Hamied Department of Chemistry,
University of Cambridge, Cambridge CB2 1EW, U.K.
Christina Spry − Yusuf Hamied Department of Chemistry,
University of Cambridge, Cambridge CB2 1EW, U.K.;
orcid.org/0000-0002-8156-7070
Anthony G. Coyne − Yusuf Hamied Department of Chemistry,
University of Cambridge, Cambridge CB2 1EW, U.K.;
orcid.org/0000-0003-0205-5630
Chris Abell − Yusuf Hamied Department of Chemistry,
University of Cambridge, Cambridge CB2 1EW, U.K.;
orcid.org/0000-0001-9174-1987
Helena I. M. Boshoff − Tuberculosis Research Section,
Laboratory of Clinical Immunology and Microbiology,
National Institute of Allergy and Infectious Disease, National
Institutes of Health, Bethesda, Maryland 20892, United
States; orcid.org/0000-0002-4333-206X
Paul G. Wyatt − Drug Discovery Unit, College of Life Sciences,
University of Dundee, Dundee DD1 5EH, Scotland, U.K.;
orcid.org/0000-0002-0397-245X
Kyu Y. Rhee − Department of Microbiology and Immunology,
Weill Cornell Medical College, New York 10065, United States
Tom L. Blundell − Department of Biochemistry, University of
Cambridge, Cambridge CB2 1GA, U.K.
Clifton E. Barry, III − MRC/NHLS/UCT Molecular
Mycobacteriology Research Unit & DST/NRF Centre of
Excellence for Biomedical TB Research & Wellcome Centre for
Infectious Diseases Research in Africa, Institute of Infectious
Disease and Molecular Medicine and Department of
Pathology, Faculty of Health Sciences, University of Cape
Town, Observatory 7925, South Africa; Tuberculosis Research
Section, Laboratory of Clinical Immunology and Microbiology,
National Institute of Allergy and Infectious Disease, National
Institutes of Health, Bethesda, Maryland 20892, United States
Author Contributions
J. C. Evans and S. L. Lynch designed and performed the
microbiology experiments. D. Murugesan, J. M. Post, S.
Thompson, S. R. Green, P. C. Ray, and C. Spry developed and
performed the high-throughput screening. V. Mendes and J.
Hess designed and performed the kinetic experiments. J. Hess
and C. Spry synthesized 4′-phosphopantothenate. Z. Wang and
N. Nahiyaan performed the metabolomic and compound uptake
experiments. J. C. Evans, V. Mendes, S. Thompson, S. R. Green,
P. C. Ray, A. G. Coyne, C. Abell, H. I. M. Boshoff, P. G. Wyatt, K.
Y. Rhee, T. L. Blundell, C. E. Barry III, and V. Mizrahi managed
the project. J. C. Evans and V. Mizrahi wrote the manuscript. All
authors approved the manuscript.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by HIT-TB (OPP1024021) and
SHORTEN-TB (OPP1158806) grants from the TB Drug
Accelerator program of the Bill and Melinda Gates Foundation
(to C. E. Barry III, K. Y. Rhee, and V. Mizrahi), the South African
Medical Research Council, the National Research Foundation of
South Africa, and an International Research Scholar’s grant from
the HHMI (to V. Mizrahi) and, in part, by the Intramural
Research Program of NIAID (H. I. M. Boshoff and C. E. Barry
III). J. Hess was financially supported by the Swiss National
Science Foundation (SNSF Early PostDoc Mobility Fellowship,
P2ZHP2_164947) and the Marie Curie Research Grants
Scheme, EU H2020 Framework Programme (H2020-MSCA-
IF-2017, ID: 789607). C. Spry was funded in part by a NHMRC
Overseas Biomedical Fellowship (1016357) and in part by the
Bill and Melinda Gates Foundation HIT-TB (OPP1024021). T.
L. Blundell is funded by the Wellcome Trust (Wellcome Trust
Investigator Award 200814_Z_16_Z: RG83114). CoaBC
screening was funded by a MRC-CinC (Grant No.
MC_PC_14099). We thank Alex Cookson, Kirsty Cookson,
Emma Gutcher, and Desiree Zeller for assistance with
compound logistics.
ABBREVIATIONS
■
ATc, anhydrotetracycline; CMP, cytidine-5′-monophosphate;
CoaB, 4′-phosphopantothenoyl-L-cysteine synthetase (PPCS);
CoaC, 4′-phosphopantothenoyl-cysteine decarboxylase
(PPCDC); CTP, cytidine-5′-triphosphate; DSF, differential
scanning fluorimetry; EthA, monooxygenase; Mtb, Mycobacte-
rium tuberculosis; Pan, pantothenate (vitamin B5); PanC,
pantothenate synthetase; PanD, aspartate decarboxylase; PanK
(CoaA), type I pantothenate kinase; PantS, pantethine; PantSH,
pantetheine; PPA, 4′-phosphopantothenate; P-PantSH, 4′-
phosphopantetheine; PptT, phosphopantetheinyl transferase;
TCEP, tris(2-carboxyethyl)phosphine; PDIM, phthiocerol
dimycocerosate; OADC, oleic-albumin-dextrose-catalase
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