4-(3-Chloro-5-(trifluoromethyl)pyridin-2-yl)- N -(4-methoxypyridin-2-yl) piperazine-1-carbothioamide (ML267), a potent inhibitor of bacterial phosphopantetheinyl transferase that attenuates secondary metabolism and thwarts bacterial growth

Article abstract of DOI:10.1021/jm401752p

4′-Phosphopantetheinyl transferases (PPTases) catalyze a post-translational modification essential to bacterial cell viability and virulence. We present the discovery and medicinal chemistry optimization of 2-pyridinyl-N-(4-aryl)piperazine-1-carbothioamides, which exhibit submicromolar inhibition of bacterial Sfp-PPTase with no activity toward the human orthologue. Moreover, compounds within this class possess antibacterial activity in the absence of a rapid cytotoxic response in human cells. An advanced analogue of this series, ML267 (55), was found to attenuate production of an Sfp-PPTase-dependent metabolite when applied to Bacillus subtilis at sublethal doses. Additional testing revealed antibacterial activity against methicillin-resistant Staphylococcus aureus, and chemical genetic studies implicated efflux as a mechanism for resistance in Escherichia coli. Additionally, we highlight the in vitro absorption, distribution, metabolism, and excretion and in vivo pharmacokinetic profiles of compound 55 to further demonstrate the potential utility of this small-molecule inhibitor.

Full text of DOI:10.1021/jm401752p

Article
pubs.acs.org/jmc
Open Access on 01/22/2015
4‑(3-Chloro-5-(trifluoromethyl)pyridin-2-yl)‑N‑(4-methoxypyridin-2-
yl)piperazine-1-carbothioamide (ML267), a Potent Inhibitor of
Bacterial Phosphopantetheinyl Transferase That Attenuates
Secondary Metabolism and Thwarts Bacterial Growth
Timothy L. Foley,† Ganesha Rai,† Adam Yasgar,† Thomas Daniel,† Heather L. Baker,†
Matias Attene-Ramos,† Nicolas M. Kosa,‡ William Leister,† Michael D. Burkart,‡ Ajit Jadhav,†
Anton Simeonov,*,† and David J. Maloney*,†
†National Center for Advancing Translational Sciences, National Institutes of Health, 9800 Medical Center Drive, Rockville,
Maryland 20850, United States
‡Department of Chemistry & Biochemistry, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093,
United States
S
* Supporting Information
ABSTRACT: 4′-Phosphopantetheinyl transferases (PPTases)
catalyze a post-translational modification essential to bacterial
cell viability and virulence. We present the discovery and
medicinal chemistry optimization of 2-pyridinyl-N-(4-aryl)-
piperazine-1-carbothioamides, which exhibit submicromolar
inhibition of bacterial Sfp-PPTase with no activity toward the
human orthologue. Moreover, compounds within this class
possess antibacterial activity in the absence of a rapid cytotoxic
response in human cells. An advanced analogue of this series,
ML267 (55), was found to attenuate production of an Sfp-
PPTase-dependent metabolite when applied to Bacillus subtilis
at sublethal doses. Additional testing revealed antibacterial
activity against methicillin-resistant Staphylococcus aureus, and
chemical genetic studies implicated efflux as a mechanism for resistance in Escherichia coli. Additionally, we highlight the in vitro
absorption, distribution, metabolism, and excretion and in vivo pharmacokinetic profiles of compound 55 to further demonstrate
the potential utility of this small-molecule inhibitor.
received considerable attention for both its orthogonal
structural characteristics relative to the mammalian FAS system
and its novelty as a drug target.5 Reduction in FAS anabolic
capacity is anticipated to exert pleotropic effects on cell viability
by inhibiting the production of the primary membrane
component palmitate and halting the assembly of virulence-
determining components of the Gram-negative, Gram-positive,
and mycobacterial cell walls (lipid A, lipoteichoic acid, and
mycolic acids, respectively). Furthermore, FAS as a drug target
has been validated in vivo with chemical probes that act upon
β-ketoacyl-ACP synthase.6
Additionally, bacterial secondary metabolism contains
polyketide and nonribosomal synthases which require 4′-PP
groups from PPTases to produce metabolites needed for
bacteria to thrive in environmental and infection settings.7
Representative compounds from these pathways include
siderophores that are secreted to scavenge iron8 and the
INTRODUCTION
■
Despite the rapidity with which genomic science has enabled
the identification of genes essential to bacterial homeostasis, the
translation of these targets into pharmacological agents has
proven to be much more difficult than anticipated.1−3 Post-
translational modification (PTM) pathways represent a unique
subset of these targets since such events are scarce in
prokaryotes relative to their frequency of occurrence in
eukaryotes. In metabolism, bacterial PTMs involve the
attachment of prostheses that impart a chemical functionality
distinct from that of the 20 proteogenic amino acids. Thus,
effector enzyme function is wholly dependent upon the
installation of these groups. One such PTM is performed by
4′-phosphopantetheinyl transferase (PPTase) enzymes (Figure
1A), which install 4′-phosphopantetheinyl (4′-PP) arms to
carrier protein domains of synthase enzymes (Figure 1B).4 This
functionality serves to store and channel reactive intermediates
that would otherwise be lost to the cellular milieu by diffusion.
Pathways activated by PPTases include microbial fatty acid
synthase (FAS), a multidomain enzyme complex that has
Received: November 25, 2013
Published: January 22, 2014
© 2014 American Chemical Society
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Figure 2. Previously reported inhibitors of Sfp- and AcpS-PPTase.
targeting Sfp-PPTase (see Figure 2). Chu and co-workers
isolated SCH-538415 (Figure 2), a symmetrical and highly
planar compound, from an unidentified bacterial extract and
reported it to have moderate inhibitory activity toward AcpS.15
Prior to the initiation of our current program, we had found
that analogues of anthranilic acid-based leads originating from a
Wyeth Research program16 targeting AcpS-PPTase (e.g.,
UCSD-18ae, Figure 2) possessed no activity toward Sfp-
PPTase and had overall weak activity.17 An additional
publication from Wyeth Research came out shortly thereafter
detailing further medicinal chemistry efforts leading to Wyeth-
16 (Figure 2), which was optimized for potency toward AcpS-
PPTase (IC50 = 1.4 μM) but had limited antimicrobial
activity.18 On the basis of the limited antibacterial activity
observed for these AcpS-PPTase-specific compounds, we have
put forth a hypothesis that the presence of an Sfp-PPTase in
the bacterial genome provides an inborn mechanism of
resistance.17 Therefore, it is our presumption that a viable
antibacterial agent must simultaneously target both classes of
PPTase enzymes to effectively halt bacterial proliferation.
Toward this end, we developed a strategy to search for small-
molecule inhibitors of Sfp-PPTase.19 These efforts identified
SCH-202676 as the first compound reported to have activity
against Sfp-PPTase (Figure 2). However, compounds belong-
ing to this class are known covalent modifiers20 and possess
promiscuous activity (∼20% hit rate in PubChem assays),
which ultimately limits their utility as probe compounds.
Therefore, we conducted a high-throughput screen (HTS) in
search of additional small-molecule inhibitors of Sfp-PPTase.
Herein we describe the discovery and SAR investigation of the
2-pyridinyl-N-(4-aryl)piperazine-1-carbothioamides as best in
class small-molecule inhibitors of Sfp-PPTase. This work led to
the development of ML267 (55), a new chemical probe and
potent inhibitor of this bacterial enzyme. We demonstrate that
55 possesses the required selectivity, mechanism of action,
physicochemical properties, and cellular activities to interrogate
the role of PPTases in bacterial metabolism, and we utilize this
compound to test hypotheses about the importance of PPTase
to the function of whole bacterial cells.
Figure 1. PPTase enzymes in bacterial metabolism. (A) Bacteria utilize
synthase enzymatic pathways to assemble small carboxylic and amino
acid monomers into complex polymeric molecules, including
membrane components, virulence factors, and medicinally important
compounds. A key feature of these synthases is the presence of a post-
translational appendage to which the nascent polymer is tethered
during extension and elaboration; the attachment of this appendage is
catalyzed by PPTase enzymes. (B) PPTase enzymes utilize CoA as a
4′-phosphopantetheinyl group donor and install the functionality to
conserved serine residues within apo-CP domains of synthase
enzymes, generating biochemically active holo-CP-containing syn-
thases and the nucleotide PAP as products. PPTase = phosphopante-
theinyl transferase, CP = carrier protein, and PAP = 3′-
phosphoadenosine 5′-monophosphate.
phenolic glycolipids of Mycobacterium spp. that dampen host
defense mechanisms.9 In many cases, the capacity to
manufacture these compounds has been linked to virulence,
and disruption of synthase genes precludes the establishment of
infection.10,11 These avirulent phenotypes have been recapitu-
lated in vitro with chemical probes that target synthase
enzymes.7,12,13
Since PPTase enzymes represent the gatekeeper of these
pathways, their inhibition stands to attenuate bacterial cell
viability through direct inactivation of FAS. At the same time,
the concomitant disruption of secondary metabolism offers a
path to mitigate numerous aspects of pathogenicity. These
hypotheses have been confirmed at the genetic level, where
disruption of PPTase genes has been observed as either lethal
or severely compromising to the fitness of both Escherichia
coli14and Mycobacterium tuberculosis.11 Thus, PPTase represents
an important enzyme in bacterial metabolism and is well-
deserving of further study.
It is noteworthy that, in most cases, bacterial genomes
contain two structurally distinct PPTase enzymes, the AcpS-
and Sfp-PPTases. Enzymes of the former class are typically
responsible for activating FAS, while enzymes of the latter type
modify secondary metabolism synthases. Given these associa-
tions and the above-mentioned therapeutic potential of
targeting PPTases, there have been several reports by academic
and industrial researchers describing the development of small-
molecule AcpS-PPTase inhibitors, yet no reports of agents
CHEMISTRY
■
As shown in Scheme 1, compound 1 was readily synthesized by
1,1′-thiocarbonyldiimidazole-assisted coupling of commercially
available 4-methylpyridin-2-amine and 1-(3-(trifluoromethyl)-
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Through bioisosteric replacement of the thiourea, analogues
12−17 were synthesized utilizing known protocols reported for
similar compounds in the literature as shown in Scheme 2 (see
the Supporting Information for details).21−24 Phenoxycarbonyl
chloride-assisted coupling of 1-(3-(trifluoromethyl)phenyl)-
piperazine with 4-picolin-2-amine afforded the urea analogue
12. Analogue 13 was prepared by stirring the analogue 1 with
ammonium hydroxide and sodium periodate at 80 °C in a
DMF−water solvent mixture. Compounds 14 and 15, which
represent the bioisosteric replacement of the thiourea
functionality, were prepared by refluxing 1-(3-
(trifluoromethyl)phenyl)piperazine and 4-picolin-2-amine with
diphenyl N-cyanocarbonimidate (for 14) or 2-[bis-
(methylthio)methylene]malononitrile (for 15) in acetonitrile.
Scheme 1. General Procedure for the Synthesis of Analogues
1−11 and 19−21
a
a
Reagents and conditions: (a) 1,1′-thiocarbonyldiimidazole (TCDI),
CH2Cl2, 40 °C, 1 h.
phenyl)piperazine at 40 °C. The same procedure was utilized to
generate a small library of compounds (Table 1) around the 4-
methylpyridine region using differentially substituted pyridyl-
amines, 3-toluidine, and other heterocycles (in Table 1,
analogues 1−11 and 19−21).
a
Table 1. Inhibition of B. subtilis Sfp-PPTase by Analogues 1−21
a
IC50 values represent the half-maximal (50%) inhibitory concentration as determined in the HTS assay, and the experiment was performed in
b
triplicate. The term “inactive” refers to compounds with IC50 ≥ 114 μM.
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a
Scheme 2. General Procedure for the Synthesis of Analogues 12−18
a
Reagents and conditions: (a) PhOCOCl, DIPEA, DMAP, 0 °C to rt, 3 h; (b) NH4OH, NaIO4, DMF−H2O, 80 °C, 1 h; (c) acetonitrile, reflux, 16
h; (d) acetonitrile, 70 °C, 24 h; (e) xantphos, Pd2(dba)3 t-BuONa, toluene, 110 °C, 3 h; (f) S, DMF, MW, 130 °C, 0.5 h.
The thiadiazole derivative 16 was prepared utilizing xanthphos-
catalyzed amination of the 2-bromo-5-(4-(3-(trifluoromethyl)-
phenyl)piperazin-1-yl)-1,3,4-thiadiazole in the presence of
Pd2(dba)3 and cesium carbonate. The Kindler reaction of 2-
acetyl-4-picoline and 2-formyl-4-picoline with 1-(3-
(trifluoromethyl)phenyl)piperazine under microwave (MW)
conditions furnished analogues 17 and 18, respectively.
were prepared using this method followed by Boc deprotection
with trifluoroacetic acid in dichloromethane.
Despite these encouraging results, this method was
insufficient for use with several di- and trisubstituted aryl and
heterocyclic boronic acids. In these cases, Buchwald−Hartwig
amination conditions were utilized. Amination of the requisite
aryl or heteroaryl bromides was achieved using BINAP or
JohnPhos ligands in combination with Pd(OAc)2 or Pd2(dba)3
with sodium tert-butoxide or cesium carbonate in toluene. The
arylation of Boc-piperazine with aryl iodides was accomplished
using a BINOL/CuBr catalytic system in the presence of
potassium phosphate in DMF at room temperature (Scheme
3B). Subsequent Boc deprotection with trifluoroacetic acid
(TFA) in dichloromethane gave the desired free piperazin-
amines (see the Supporting Information for details). As shown
in Scheme 3C, the commercially available arylpiperazines and
compounds obtained from the above methods were treated
with various 4,6-substituted pyridin-2-amines in the presence of
1,1′-thiocarbonyldiimidazole to furnish analogues 22−56
(Table 2). To explore the SAR of the piperazine core itself,
analogues 59−67 were synthesized using 1,1′-thiocarbonyldii-
midazole-assisted thiourea synthesis (Table 3). The acyclic
analogues 57 and 58 were prepared by condensation of the 4-
picoline-2-isothiocyanate with corresponding acyclic amines as
shown in Scheme 3D.
To explore the SAR around the 3-trifluorophenyl region as
shown in Table 2, a robust synthetic method was required to
generate a library of arylpiperazine precursors, since many of
the targeted compounds were not commercially available.
Given the large number of available boronic acids, we found the
Chan-Lam reaction25,26 to be an ideal method to generate
several mono- and disubstituted arylpiperazine precursors for
preparation of analogues 27, 28, 36, and 40. However, the use
of a large excess of copper or a constant supply of oxygen gas is
necessary to regenerate the copper(II) catalyst, making this
method less compatible with parallel library synthesis. To
overcome this drawback, we used a modified version of the
Chan-Lam reaction reported by Quach et al.27 which requires
only 10 mol % copper(II) acetate but still uses an oxygen
atmosphere. To facilitate analogue synthesis, we further
modified this method by carrying out this reaction in a sealable
microwave vial. In this case, all the reactants were mixed
together, the vessel was charged with oxygen gas, and then the
vessel contents were stirred at 45 °C for 12−24 h as shown in
Scheme 3A. Several mono- and disubstituted arylpiperazines
Specifically, the synthesis of analogues 59−63, 65, and 67
was accomplished by arylation28 of the requisite Boc-protected
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a
Table 2. Inhibition of B. subtilis Sfp-PPTase by Analogues 22−56
a
IC50 values represent the half-maximal (50%) inhibitory concentration as determined in the HTS assay, and the experiment was performed in
triplicate.
amine cores with 3-trifluorophenyl iodide utilizing the 2-
isobutyrylcyclohexanone/CuI system (Scheme 4), followed by
1,1′-thiocarbonyldiimidazole-assisted coupling with 4-methyl-
pyridin-2-amine. Analogues 64 and 66 were synthesized
utilizing the general procedure outlined in Scheme 3 using
commercially available precursors 6-(trifluoromethyl)-1,2,3,4-
tetrahydroisoquinoline and 4-(3-(trifluoromethyl)phenyl)-
piperidine, respectively.
compounds that were selected for follow-up in a battery of
assays. This characterization began with retest of freshly
prepared serial dilutions of hit compounds in the Sfp-PPTase
screening assay (AID 2701) and included further activity
profiling with AcpS-PPTase (AID 602360). In addition, this set
of compounds was evaluated with Sfp-PPTase in a gel-based
phosphopantetheinylation assay to confirm biochemical inhib-
ition with an orthogonal detection format (AID 2707).19
Finally, since prior work has established the essential nature of
PPTases to bacterial viability,30−32 we profiled these com-
pounds for antibacterial activity with Bacillus subtilis HM489,
whose viability depends solely on Sfp (AID 602366).32 The
sum of these experiments identified 1 (Figure S1A, Supporting
Information) as a confirmed screening hit with inhibitory
activity against both AcpS- and Sfp-PPTases (Figure S1B) and
modest antibacterial activity against B. subtilis. We subsequently
confirmed the identity and integrity of the chemical matter by
resynthesis and reaffirmed the above findings for the
resynthesized sample.
RESULTS AND DISCUSSION
■
Discovery of N-(4-Methylpyridin-2-yl)-4-(3-
(trifluoromethyl)phenyl)piperazine-1-carbothioamide
(1) as an Inhibitor of Sfp-PPTase. We profiled the
Molecular Libraries Small Molecule Repository (at the time
totaling 311 260 compounds) for PPTase inhibitors with a
previously described activity assay for Sfp-PPTase.19 The highly
miniaturized format enabled the experiment to be conducted at
eight concentrations ranging from 3.6 nM to 114 μM following
the quantitative HTS methodology.29 Data generated by this
experiment are deposited in the public database PubChem
under assay identifier number (AID) 1490. Data analysis and
chemoinformatic filtering identified 388 structurally diverse
Compound 1 is a Reversible, Noncompetitive Inhib-
itor of Sfp-PPTase. To further characterize the biochemical
activity of 1, we first addressed the use of fluorescent substrates
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a
Scheme 3. Synthesis of Requisite Aryl/Heteroarylpiperazines and Analogues 22−58
a
Reagents and conditions: (a) Cu(OAc)2 (10 mol %), 4 Å molecular sieves, O2, CH2Cl2, 45 °C, 12−24 h; (b) TFA/CH2Cl2, rt, 1 h; (c) BINAP (10
mol %), Pd(OAc)2 (5 mol %), Cs2CO3 (1.5 equiv), toluene, 110 °C, 12−24 h; (d) JohnPhos (10 mol %), Pd2(dba)3 (5 mol %), t-BuONa (1.5
equiv), toluene, 110 °C, 2−8 h; (e) BINOL (20 mol %), CuBr (20 mol %), K3PO4 (2 equiv), DMF, rt, 12−24 h; (f) 1,1′-thiocarbonyldiimidazole
(TCDI), CH2Cl2, 40 °C; (g) DMF, 90 °C, 1 h.
in our primary and confirmatory assays. As recently highlighted,
the potential exists that the observed inhibition could be a
result of compound−label interaction that leads to an artifactual
mechanism of inhibition.33−35 To this end, we developed and
implemented a label-free biochemical assay that assessed
PPTase activity on the basis of differences in the electro-
phoretic mobility of the apo and holo states of the carrier
protein under native PAGE conditions. These experiments
revealed that 1 elicited inhibition through a mechanism
independent of the fluorescent label, with a dose-dependent
inhibition being observed in the presence of the natural CoA
and acyl carrier protein (ACP) substrates (Figure S1E,F,
Supporting Information).
To better understand the mechanism through which 1
inhibits the target, we evaluated the effects that a varying
substrate concentration had on potency. In these experiments,
which utilized an HPLC assay and operated under an initial-
rates regimen, 1 exhibited a near constant IC50 of 300 nM when
CoA or apo-ACP acceptor substrates were increased to
concentrations 50- and 100-fold above their Km values,
respectively (Figure S1C,D, Supporting Information). This
trend is consistent with a noncompetitive mechanism of
inhibition36 and indicates that the compound binds to the free
enzyme, enzyme−substrate complex, or ternary complex with
similar affinities at a location other than the active site: a form
of allosteric modulation.
Supporting Information), while the latter was found be an
irreversible inhibitor, a result that is consistent with other
reports of the compound’s reactivity (Figure S2B).20 Parallel
evaluation of our primary hit 1 (Figure S1G, Supporting
Information) in the same manner revealed completely
reversible inhibition of Sfp, with enzymatic activity restored
upon dilution of the enzyme−1 solution in a compound-free
buffer (orange data symbols, Figure S1G), relative to a buffer
containing a constant concentration of the compound (green
data symbols, Figure S1G).
Structure−Activity Relationship Studies of 1. After
confirming the activity of 1, we planned systematic structural
modifications to the various regions of the molecule in an effort
to establish tractable SAR. Our first area of exploration was the
western 2-aminopyridine moiety as shown in Table 1. Removal
of the 4-Me group (2) resulted in only a slight drop in potency,
whereas addition of electron-withdrawing groups such as 4-CF3
(4) and 4-C(O)OMe (7) resulted in a significant loss of
activity. Moreover, removal of the pyridine nitrogen (5) or
changing the position of the pyridine (6) also led to inactive
compounds. Thus, it appears that the pyridine nitrogen needs
to be adjacent to the thiourea motif to maintain Sfp-PPTase
inhibitory activity. Conversely, modification with electron-
donating groups, such as addition of an extra methyl group
adjacent to the nitrogen (3), 4-OMe (8), and other
heterocycles (10 and 11), led to compounds with activity
comparable to that of 1.
Finally, we probed the reversibility of inhibition with a
rescue-by-dilution experiment that assessed the recovery of
enzymatic activity after a large (100-fold) dilution from
conditions producing 90% inhibition of enzymatic activity.37
These experiments included control compounds PAP and
With initial SAR explorations of the western region of the
molecule complete, we then focused our attention on the
thiourea moiety. As shown in Table 1, replacement of the
thiourea was not well tolerated with the urea (12), guanidine
(13), and thiadiazole (16) all being inactive. Attempts to
replace the thiourea structural motif with bioisosteres such as
cyanoguanidine (14) or a 1,1-dicyanoketene group (15) also
led to complete loss of activity. Methylation of the nitrogen
SCH-202676, the latter of which was discovered during assay
19
optimization as an active compound from LOPAC1280
.
Inhibition by the former proved to be reversible, as anticipated
by the fact that it is one of the reaction products (Figure S2A,
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(19) or changing the nitrogen from being a part of a ring to
being exocyclic (20) resulted in loss of potency. Interestingly,
replacement of the 2-amino group with a methylene spacer
(17) retained some activity toward Sfp-PPTase albeit much
reduced, with an IC50 value of 5.8 μM. As such, these data
suggested that the thiourea moiety was clearly the most optimal
group for the desired activity. However, the thiourea
functionality is often considered an undesirable structural
feature, primarily due to its potential in forming toxic
metabolites via oxidation to the S-oxide or sulfinic acid
(typically P450 or FMO-catalyzed), followed by GSH-trapping.
These covalent GSH adducts could ultimately lead to oxidative-
stress-induced cell death.38 Therefore, upon completion of the
SAR efforts, we planned to investigate the propensity of this
undesired reaction to occur with our lead compound(s) (vide
infra).
Table 3. Inhibition of B. subtilis Sfp-PPTase by Analogues
57−67
a
Given that the 4,6-dimethyl substitution in 3 displayed
potency comparable to that of the 4-methylpyridine moiety of
1, we decided to further investigate both pyridine derivatives
when exploring the SAR of the eastern phenyl region. In these
efforts, a large number of analogues were synthesized, and
representative compounds are shown in Table 2. Overall,
substitution of the pendant phenyl group was very well
tolerated, with most of the analogues 22−44 having
comparable activity. The general trend suggested that
electron-withdrawing groups are preferred, with most of these
analogues exhibiting submicromolar potency. The analogues
which showed the most potent activity included 29 (R = 3,5-
CF3), 40 (R = 3-CF3-4-Cl), 41 (R = 2-CF3-4-Cl), and 43 (R =
3,4,5-Cl). In contrast, compounds in which electron-donating
substituents were incorporated (e.g., 24 (R = 3-NMe2) and 34
(R = 3,5-OMe)) had weaker potency with IC50 values of 5.8
and 2.0 μM, respectively. These data suggest that this region of
the molecule may provide a handle for modulating the ADME
properties (e.g., solubility) in future SAR efforts given the
general tolerance for structural modifications. With analogues
45−56 (Table 2), we aimed to investigate more significant
structural changes to this region, including removal of the aryl
group (45) or replacing it with smaller groups such as a methyl
group (46), and both changes significantly diminished the
potency (IC50 = 14.5 and 6.5 μM respectively). Addition of a
methylene spacer between the aryl group and piperazine (48)
was not well tolerated (IC50 = 11.5 μM). However,
incorporation of a sulfonamide moiety (49) resulted in potency
comparable to that of 1. Replacement of the aryl group with a
thiadiazole (50) resulted in a ∼2-fold drop in potency.
In an effort to improve the overall solubility of the molecule,
various nitrogen-containing heterocyles were synthesized
(compounds 51−56). Gratifyingly, this change was tolerated
and led to the discovery of analogue 55, which was ultimately
determined to be our probe molecule (ML267) after all
compound attributes such as potency, in vitro ADME
properties, and antibacterial activity were taken into consid-
eration (vide infra). Notably, compound 55 possesses a 4-OMe
group in contrast to the 4,6-dimethyl derivative, which led to a
2−3-fold improvement in potency (IC50 = 0.81 μM (54) vs
IC50 = 0.29 μM (55)) but also contributed to improvement in
other attributes mentioned above. We had synthesized and
tested a small set of analogues containing the 4-methoxypyr-
idine combined with the best arylpiperazines in the right-hand
side of the molecule. These analogues showed similar or less
potency compared to compound 55 and exhibited a consistent
SAR trend (data not shown).
a
IC50 values represent the half-maximal (50%) inhibitory concen-
tration as determined in the HTS assay, and the experiment was
performed in triplicate. The term “inactive” refers to compounds with
IC50 ≥ 114 μM.
b
Scheme 4. Synthesis of Requisite Aryl/Heteroarylpiperazines
and Analogues 59−63, 65, and 67
a
a
Reagents and conditions: (a) 2-isobutyrylcyclohexanone, Cs2CO3,
CuI, DMF, 70 °C, 2−10 h; (b) TFA, DCM, rt, 1 h; (c) 1,1′-
thiocarbonyldiimidazole (TCDI), CH2Cl2, 40 °C.
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Our final area of SAR exploration was modification of the
piperazine linker, as shown in Table 3. A literature search
uncovered Troviridine [1-(5-bromopyridin-2-yl)-3-(2-(pyridin-
2-yl)ethyl)thiourea] (also known as LY300045-HCl), a
thiourea-containing small molecule that was originally
developed by Eli Lilly as a non-nucleoside reverse transcriptase
inhibitor but later was found to have antimicrobial properties.39
Similar to our lead scaffold, Troviridine contains a 2-
aminopyridine attached to the thiourea moiety; however,
instead of a piperazine, it contains an acyclic linker. Therefore,
we replaced the piperazine core with two acyclic linkers (57
and 58); however, these compounds lost all activity. Despite
this, we were encouraged that a compound with a structure
comparable to that of our lead had advanced into human trials.
Moreover, the inactivity of “Troviridine-like” analogues 57 and
58 assured us that our compound has a divergent mechanisms
of action, particularly in reference to the antimicrobial activity.
Additional SAR explorations of the piperazine motif included
varying the ring size to smaller, larger, and fused ring systems.
The smaller rings showed similar activity (60), whereas larger
rings (67) or the substituted piperazine (62) had reduced
potency. The fused pyrrolidine motif (65) had comparable
potency (IC50 = 0.73 μM), but the solubility was markedly
reduced. Interestingly, when the nitrogen linked to thiourea is
exocyclic (e.g., 59), all activity is lost, whereas the piperazine
nitrogen attached to an aryl ring is not essential (e.g., 66, IC50 =
0.81 μM).
and this profiling not only reveals potential toxicity of the
parent compound, but also its metabolites. While this method is
certainly not to be considered a thorough investigation of
toxicity, it provided us a means to quickly profile numerous
compounds. Notably, despite the generally favorable activity of
the original compound 1, it demonstrated moderate toxicity
toward this cell line, whereas most other compounds were
inactive. Finally, as part of our initial biological activity profile,
we sought to further characterize the compounds for
antibacterial activity using the B. subtilis HM489 strain.32 This
strain contains sfp as the only locus encoding a functional
PPTase gene product, making the allele essential to viability of
this organism. These experiments revealed that we had modest
inhibitors of bacterial growth that generally tracked with SAR in
the biochemical assay. This important finding is exemplified by
urea derivative 12, which was inactive against Sfp/AcpS-PPTase
and lacked activity in the antibacterial assays (Table 4). While
the antibacterial activity of these compounds was modest in
these high-throughput assays, in subsequent antibacterial
studies using more traditional methods of minimum inhibitory
concentration (MIC) determination, we found the compounds
to be generally more potent (vide infra). After careful analysis
of the data in Table 4, and profiling of select compounds for
their in vitro ADME properties, 55 emerged as the compound
with the best balance of properties. In these profiling studies,
55 demonstrated dual activity toward the bacterial Sfp- and
AcpS-PPTase targets (Figure S4, Supporting Information),
presenting IC50 values of 290 nM and 8.1 μM, respectively.
Furthermore, we profiled top compounds for activity with the
human PPTase, an important antitarget. While we observed
inhibition of this enzyme with PAP and SCH202676, 55
exhibited no inhibition at concentrations up to 125 μM (Figure
S4B, lower panel). Comparison of these data to the inhibition
observed in the same biochemical assay for Sfp-PPTase (Figure
S4B, upper panel) indicated the selectivity index, the ratio of
inhibition observed for the bacterial target relative to the
human enzyme, to be greater than 500-fold.
Initial SAR explorations around compound 1 led to a
thorough understanding of the structural features that are
required for activity. Thus, we then sought to more thoroughly
characterize the best Sfp-PPTase inhibitors for their activity
against AcpS-PPTase and antibacterial activity. Additionally, we
profiled this panel for activity with the human PPTase, an
important antitarget, and cytotoxicity with human HepG2 cells
(Table 4). As mentioned above, our primary target of interest
a
Table 4. Biological Activity Profile of Select Compounds
Compound 55 Possesses Specific Gram-Positive-
Targeted Bactericidal Activity. To evaluate the antibacterial
spectrum of activity possessed by 55, we assembled a panel of
microorganisms which included B. subtilis strains HM489, 168,
and OKB105, which possess all possible PPTase genotype
combinations sfp+/acpS−, sfp−/acpS+, and sfp+/acpS+, respec-
tively, in a strain 168 isogenic background, as well as E. coli K12
and the laboratory strain BW25113. With respect to human
pathogens, the panel contained Pseudomonas aeruginosa
ATCC9028, a Gram-negative organism of clinical importance,
as well as three strains of methicillin-sensitive and community-
acquired methicillin-resistant Staphylococcus aureus
(ATCC6538, USA300, and USA500). Finally, the panel also
contained fluconazole-sensitive and -resistant strains of Candida
albicans as archetypical fungal pathogens (ATCC90028 and
ATCC96901, respectively). Compound 55 displayed a
spectrum of activity that targets Gram-positive organisms,
where, in addition to inhibiting the growth of B. subtilis at a
concentration of 1.7 μg/mL, it also thwarted the growth of
both methicillin-sensitive and -resistant strains of S. aureus in a
similar concentration range [Figure 3, where the growth
indicator resazurin (purple) is reduced to resorufin (pink) by
metabolically active microorganisms].’ No growth inhibition
was observed for the Gram-negative organisms E. coli and P.
aeruginosa, a result that was investigated further (vide infra), or
for fungal organisms. The finding of null activity with
cytotoxicity
(HepG2), IC50
(μM)
B. subtilis
(HM489) IC50
(μM)
Sfp-PPTase AcpS-PPTase
compd IC50 (μM)
IC50 (μM)
1
55
12
22
41
53
54
63
0.78
0.29
inactive
0.41
0.58
0.73
0.86
0.81
4.6
14
9.8
8.1
inactive
7.0
9.3
inactive
10.2
10.2
16.2
18.6
12.9
inactive
9.4
inactive
inactive
15.8
18.6
13.2
36.2
13.2
30
inactive
a
Sfp-PPTase and AcpS-PPTase IC50 values were determined using the
HTS assay. Compound toxicity toward HepG2 cells was assessed by
measuring the cellular ATP content using a luciferase-coupled ATP
quantitation assay (CellTiter-Glo, Promega Corp., Madison, WI).
Antibacterial activity against B. subtilis (HM489) was accessed by
measuring the cellular ATP content using a luciferase-coupled ATP
quantitation assay (Bac-Titer Glo, Promega Corp.).
was Sfp-PPTase, yet activity against AcpS-PPTase is desirable
for maximal antimicrobial activity. As shown in Table 4, most
compounds display activity toward AcpS-PPTase, but the IC50
values were generally 5−10-fold less potent. To assess acute
cytotoxicity, we chose to profile the top compounds using
HepG2 (hepatocellular carcinoma) cells since hepatotoxicity
has been reported for some thiourea-containing compounds,
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eukaryotic microbes was not discouraging, as total coverage of
the panel would have been taken as nonspecific inhibition of
growth and could be considered suspicious for an antibacterial
candidate.2
We followed up on this finding with a study of bacterial cell
membrane integrity, as Gram-positive whole-cell experiments
can be convoluted by nonspecific damage to the lipid bilayer.
We employed the differential nucleic acid staining of B. subtilis
168 bacterial cells by Syto9 and propidium iodide in the
BacLight LIVE/DEAD microscopy assay, which utilizes green
and red staining to differentiate healthy and compromised cells,
respectively.40 Chloramphenicol (CAM) and cetyltrimethylam-
monium bromide (CTAB), two compounds that possess
minimum inhibitory concentration (MIC) values similar to
that of 55 (∼2 μg/mL), were included as controls to serve as
known specific and nonspecific agents, respectively. Testing of
these agents indicated a rapid compromise of bacterial cell
integrity for CTAB after 15 min of incubation, where similar
testing of chloramphenicol and 55 revealed continued cellular
viability over the short incubation period at concentrations that
exceeded MIC by 10-fold (Figure 4A). Therefore, we
concluded that compound 55 does not elicit antibacterial
activity through the nonspecific disruption of the Gram-positive
membrane and likely acts on an intracellular target.
Figure 3. Spectrum of antibacterial activity possessed by 55. Broth
dilution experiments were performed to assess the microbicidal activity
of 55 with different organisms. In these experiments, growth is
detected with the indicator resazurin, a purple dye which is reduced to
the pink compound resorufin by metabolically active organisms. Wells
corresponding to the MIC are outlined in red. The antibacterial
activity of 55 was restricted to Gram-positive organisms, represented
here by strains of B. subtilis and S. aureus, with no growth inhibition
observed for Gram-negative bacteria or fungi.
With this information, we further characterized the
antibacterial activity of 55 to discern whether the compound
merely halted proliferation of bacteria or possessed bactericidal
activity. The results of these experiments, summarized in Table
S1 (Supporting Information), demonstrated that the minimum
bactericidal concentration (MBC) of 55 fell within a range of
1×−4× the MIC in S. aureus and B. subtilis. The generally
Figure 4. Compound 55 does not disturb bacterial membranes and is expelled by efflux pumps in Gram-negative bacteria. (A) Microscopy
experiments using the exclusion of propidium iodide (PI) were conducted to evaluate the effects of 55 on the integrity of the membrane in B. subtilis.
In these, Syto9 stains DNA of all bacteria green, while PI can only penetrate compromised bacterial cells. CAM, CTAB, and 55 were evaluated at 1×,
4×, and 10× their MIC values, and the number of observed cells was counted. Chloramphenicol (CAM), a non-membrane-active antibiotic, and
cetyltrimethylammonium bromide (CTAB), a known membrane-disrupting agent, were included as controls for the experiment. (B) Disruption of
tolC, the primary efflux pump of E. coli, induced a 55-sensitive phenotype in broth dilution experiments, where concentrations as low as 12 μM
inhibited bacterial growth (red-outlined well). (C) Building upon the results of (B), checkerboard synergy experiments were conducted with the
AcrAB-TolC inhibitor phenylarginine-β-napthamide (PAβN) and demonstrate an effective chemical knockout strategy to induce a 55-sensitive
phenotype in wild-type E. coli. Wells containing no metabolically active bacteria are observed as purple in this experiment, and wells corresponding to
the lowest concentration of PAβN capable of synergizing with 55 to halt bacterial proliferation at each concentration of 55 are outlined in red.
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Figure 5. Inhibition of surfactin production by 55. (A) Surfactin 68 is produced by a nonribosomal peptide synthetase pathway in B. subtilis that
contains 24 distinct functional domains, represented as cubes. Seven of these are CP domains that require post-translational activation by Sfp-
PPTase, and the monitoring of the fermentative yield provides a means to assess the blockade of PPTase-dependent processes in the bacterial cell.
(B) Culture time course experiments demonstrate that sublethal concentrations of 55 attenuate surfactin production by B. subtilis OKB105. (C)
Culture density data are plotted for the culture time course experiment in (B) and demonstrate a modest effect of 55 on the growth of the cultures,
indicating that the compound did not impede their ability to reach a terminal density.
accepted criteria of defining antibacterial activity as cidal is a
ratio of MBC to MIC less than or equal to 4.41 By this
definition, 55 exhibited bactericidal activity in all cases tested.
Efflux of Compound 55 by the AcrAB−TolC Complex
Induces a Resistant Phenotype in E. coli. The limited
spectrum of activity possessed by 55 was surprising, since our
biochemical testing indicated that the compound was active
with AcpS in the direct enzymatic assay. This led us to
hypothesize that the Gram-negative outer membrane was the
source of this complication, as it presents a formidable barrier
possessing both low penetrability and an active efflux
mechanism.42 In E. coli, a tripartite complex of the TolC−
AcrA−AcrB gene products constitutes the primary efflux pump
and source of resistance through this mechanism.43 Thus, we
tested E. coli BW25113 and JW0451, the wild-type strain and
an efflux-defective mutant that contains a lesion in the acrB
locus, respectively,44 for susceptibility to 55. We anticipated
that active transport out of the cell by the AcrAB−TolC system
would manifest as an observed hypersusceptibility of the ΔacrB
strain, while insensitivity would indicate cellular penetration or
another mechanism as the source of resistance. For these
experiments, we observed a clear differential susceptibility
between the two strains (Figure 4B), where disruption of the
acrB locus rendered the organism susceptible to 55 at
concentrations as low as 12.5 μg/mL. These findings indicated
that efflux is the primary mechanism of resistance in this
organism and that 55 is capable of penetrating the Gram-
negative bacterial cell.
wild-type strain BW25113 at the top concentrations tested, but
offered pronounced growth inhibition in combination (Figure
4C). The fractional inhibition concentration index, a ratio of
the potency of the combined test articles relative to that of the
singular components,46 was estimated to be less than 0.25, a
finding strongly indicative of synergism, although we were not
able to precisely determine this parameter since neither
compound elicited growth inhibition at the top concentrations
tested.
Compound 55 Attenuates Surfactin Production in B.
subtilis. To further understand the on-target engagement of
PPTase enzymes within the bacterial cell, we exploited the
association of Sfp-PPTase with secondary metabolism in B.
subtilis. The lipopeptide surfactin 68 is produced by a
nonribosomal peptide synthetase pathway, diagrammed in
Figure 5A. This canonical megasynthase complex contains 24
functional domains, 7 of which are 4′-PP-accepting carrier
protein domains. A singly missed 4′-PP transfer event will
disrupt processivity in the megasynthase and render the
complex incapable of producing the metabolite. As such, this
system represents a strategy to study target engagement by 55
in the bacterial cell through monitoring of the fermentative
yield of 68. Toward this end, we conducted time course
experiments for cultures of B. subtilis in the presence of
increasing concentrations of 55, where we periodically
measured both bacterial growth and surfactin titer over 28 h
(Figure 5B,C). Given the potent antibacterial activity of 55
against this organism, delicate selection of test conditions was
required to supply high enough concentrations of compound to
elicit a response but also provide for normal bacterial growth.
In these experiments, cultures treated with 55 at concentrations
of 0.53 or 1.1 μg/mL resulted in 33% and 41% reductions in
surfactin titer relative to a vehicle control after 28 h of culture,
To further validate the above findings using a chemical
genetic model, we tested 55 for synergistic activity with the
broad-spectrum efflux pump inhibitor phenylarginine-β-naph-
thamide (PAβN).45 In these experiments, neither compound
alone was found to possess antibacterial activity against the
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a
Table 5. In Vitro ADME Profile of 55
microsomal stability
compd
aq solubility (pH 7.4)
cLogP
3.35
(T1/2, min)
CYP inh (3 μM) (%)
25 (2D6) 20 (3A4)
PAMPA
1122
plasma stability (mouse) (%)
100 (at 2 h)
55
20 μM
40 (mouse)
>30 (rat)
a
Aqueous solubility (PBS buffer), mouse liver microsome (MLM) stability, CYP2D6/3A4 inhibition, and plasma stability were determined at
Pharmaron Inc. All other studies were conducted at NCATS. The microsomal stability data represent the stability in the presence of NADPH.
Compound 55 showed no degradation without NADPH present over a 1 h period. Dextromethorphan and 6β-hydroxytestosterone were the
substrates used for the CYP2D6 and CYP3A4 inhibition studies, respectively. PAMPA represents passive permeability measured as 1 × 10−6 cm/s.
a
Table 6. In Vivo PK Profile of 55
compd
route
T1/2 (h)
Cmax (ng/mL)
AUCinf (h·ng/mL)
Vd (L/kg)
MRT (h)
3.4
clearance (mL/min/kg)
7.2
P/B
55
iv
2.4
2.0
4114
6983
1.5
2.5
2.1
ip
17633
68860
a
All experiments were conducted at Pharmaron Inc. using male CD1 mice (6−8 weeks of age). Data were collected in triplicate at eight time points
over a 24 h period. 55 was formulated as a solution (5% DMSO and 10% Solutol in H2O). For iv, dosed at 3 mg/kg. For ip, dosed at 30 mg/kg.
MRT = mean residence time (the time for elimination of 63.2% of the iv dose). P/B = plasma to brain ratio [AUClast(plasma)/AUClast(brain)].
respectively (Figure 5B). These reductions in titer were
accompanied by modest extensions in the culture lag phase,
although continued culture revealed that 55 had a null effect on
both the doubling time in the log phase and final cellular
densities (Figure 5C). This dose-dependent attenuation of
surfactin titer in cultures which accumulated similar quantities
of biomass is consistent with engagement of Sfp-PPTase by 55
inside living bacteria.
bioactivation and did not show any GSH adducts in follow-
up studies (Table S3, Supporting Information).
Having demonstrated a favorable in vitro ADME profile of
55, we next sought to investigate the in vivo PK profile in
support of testing this compound in proof of concept (POC)
antibacterial animal models (vide infra). As shown in Table 6,
55 was administered to CD1 mice via both iv and ip routes at 3
and 30 mg/kg doses, respectively, using a solution formation
consisting of 5/10/85 (w/w/w) DMSO, Solutol, and water.
Compound 55 exhibits favorable systemic exposure levels
(AUCinf = 68860 h·ng/mL) that are 40−50-fold higher than the
MIC values against S. aureus strains at 30 mg/kg dosing.
Moreover, the compound has a reasonable T1/2 (2.0 h), and
low clearance (7.2 mL/min/kg) results in an exposure (total
drug concentration, not free drug concentration) that exceeds
the in vitro antibacterial MIC value for over 8 h. The
compound also efficiently crosses the BBB and thus could be
potentially used for bacterial infections which reside in the
brain. Furthermore, dosing of 55 at these concentrations did
not result in any adverse clinical observations over the 24 h
period, which seems to indicate that no acute toxicity is to be
expected at the doses used for in vivo POC studies.
As discussed above, 55 exhibits antibacterial activity against
MRSA and has a desirable ADME/PK profile, and thus, it was
poised to be tested in vivo. For these studies, we profiled a
panel of clinical isolates to assess the susceptibility of various
genotypes (Table S2, Supporting Information), and this
revealed similar potencies (within 3-fold) for all strains tested.
With this information, we pursued an in vivo POC study that
utilized the MRSA-USA500 strain (BK2395) since our
compound had demonstrated favorable in vitro activity against
it and it has demonstrated suitable virulence in mouse models
of sepsis.48 Unfortunately, in this experiment, 55 did not
increase survival compared to the vehicle control, while
vancomycin treatment completely protected animals from
morbidity (Figure S6, Supporting Information). These findings
led us to investigate the source of discrepancy between our in
vitro and in vivo results. To rule out the rapid development of
resistance in vivo, bacteria were harvested from the kidneys of
mice treated with the vehicle or 55. For each crude isolate, five
representative strains were isolated. We then determined MIC
values for these strains in parallel along with the parental
MRSA-USA500 strain and observed identical susceptibility of
all strains to 55. These results indicated that neither passage of
the organism in mice nor challenge with 55 in vivo led to
While our preliminary studies involved some characterization
of the in vitro ADME properties, after selection of 55 as a lead
compound, a more detailed analysis was obtained. As shown in
Table 5, 55 has a modest solubility in PBS buffer (pH 7.4) of
20 μM, which is approximately 60 times higher than the
recorded IC50 value (290 nM). Moreover, 55 displayed stability
to mouse and rat liver microsomes with T1/2 values of 49.5 and
>30 min, respectively. The compound was completely stable
(measured for 1 h) in the absence of the NADPH cofactor,
suggesting a CYP-mediated oxidation event. Passive perme-
ability was assessed using the PAMPA assay, and very favorable
permeability was observed (1122 × 10−6 cm/s). Next, we
wanted to examine whether the probe compound inhibits
specific cytochrome P450 enzymes 2D6 and 3A4 as these two
isoforms account for the metabolism of approximately 80% of
drugs.47 To assess the potential for CYP inhibition and drug−
drug interactions (DDIs), we looked at the effect of
coincubation of our compound(s) at 3 μM with known CYP
substrates (2D6 with dextromethorphan and 3A4 with 6β-
hydroxytestosterone). We found that 55 exhibits modest CYP
inhibition of 25% and 20% for 2D6 and 3A4, respectively. To
further elucidate the potential for CYP liabilities, follow-up IC50
values and testing of a more expansive representation of CYP
isoforms will be required. However, we also investigated CYP
inhibition for other analogues, and inhibition was inconsistent
across the series, so we are confident that these liabilities could
be addressed through targeted structural modifications. Given
the potential instability of the thiourea moiety in aqueous and
biological media,38 we were encouraged that 55 showed no
signs of degradation in aqueous media at a wide pH range (2−
9), buffers (PBS and HEPES) (Figure S7, Supporting
Information), and mouse plasma (Table 5). In addition, the
thiourea functionality is a known structural alert that can cause
toxicity due to formation of reactive metabolites under
physiological conditions (vide supra). However, our probe
compound 55 and analogue 41 are not susceptible to
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reduced susceptibility to the compound. Realizing that the
antibacterial activity of compounds can be attenuated in the
presence of whole blood and/or serum, we repeated the MIC
determinations of 55 in the presence of calf serum (20%, v/v)
in cation-adjusted Mueller Hinton II broth and observed a
significant shift in potency (>57 μM vs 3.4 μM). These data,
along with subsequent analysis via equilibrium dialysis, revealed
that 55 displays a high degree (98.6%) of plasma protein
binding (PPB), and thus, the lack of activity is likely a
consequence of the lipophilic nature of this compound. While
disappointing, other groups have shown the ability to
successfully modulate the lipophilicity of the lead compound
through the strategic placement of polar moieties to improve
the antibacterial activity in vivo. In fact, a group of researchers
at Pfizer recently reported improved in vivo efficacy of their
lead antibacterial candidate by lowering PPB via lowering
cLogP.49 As described above, the eastern region of the molecule
is highly tolerant to structural modifications (Table 2), which
could be exploited to modulate the cLogP and thus hopefully
improve activity in vivo. In addition, we plan on conducting
more extensive PK studies to look at tissue distribution and free
drug concentration, which may guide not only a future
medicinal chemistry effort, but also aid the design of our in
vivo proof of concept models.
Over the course of this work, a heated debate has emerged as
to the suitability of the FAS as a drug target.50−54 It appears
that, in some Gram-positive pathogens, FAS is dispensable
when bacteria are propagated in the presence of serum, which is
a nutrient source rich in fatty acids. Further study has found
that the results may not be accurately predictive for even closely
related bacteria, and further investigation of this phenomenon
is warranted to tease apart the details with finer resolution.
Nonetheless, this discrepancy provides another mechanism
through which bacterial tolerance to 55 may be occurring in
vivo, and such a mechanism may confound the advancement of
in vitro PPTase-targeting inhibitors through in vivo POC
studies of staphylococcal septicemia. However, this does not
speak to the role that PPTase plays in the production of
virulence factors and elaborate cell wall components, especially
in the case of Mycobacterium spp., where recent findings have
confirmed the essential nature of both AcpS- and Sfp-
PPTases.55 These organisms depend on the concerted effort
of FAS and PKS systems for the assembly of mycolic acids, and
these PPTase dependencies have been substantiated in rodent
models of infection.56 Thus, PPTase inhibitors remain a target
worthy of pursuit and may find a more immediate applicability
as tools to pharmacologically probe the role of PPTases in
mycobacterial pathogenesis.
The critical role of this PTM in bacterial metabolism has
been studied by others, but to date these efforts have not
produced compounds with appreciable antibacterial activity.
This limited success can likely be attributed to the inability of
these programs to consider the presence of an Sfp-PPTase in
addition to their primary target, AcpS-PPTase. This secondary
enzyme is capable of compensating for loss of the AcpS
locus32,57,58 and thus provides a direct mechanism through
which bacterial organisms may develop resistance through
simple transcriptional/translational upregulation.17 To address
this issue, we have pursued the development of a chemical
probe with dual PPTase inhibitory activity.
Here we have detailed the development and characterization
of 55, a novel small molecule that possesses these character-
istics. We have demonstrated the qualities of this chemical
probe and evaluated it according to the benchmarks suggested
by the community.59 The features of 55 can be summarized
according to the five principles of a quality chemical probe as
follows: (i) We have performed detailed molecular profiling with
relevant molecular targets, with observed potencies of 290 nM
and 8.1 μM against the primary development target Sfp-PPTase
and bacterial orthologue AcpS, respectively, which is contrasted
by null activity with the human enzyme. In the context of an
antibacterial discovery campaign, where the end goal is the
inhibition of bacterial growth in man, this indicates a selectivity
index of >500-fold with respect to the human orthologue. (ii)
With respect to the mechanism of action, we demonstrate that
the chemotype inhibits Sfp-PPTase independent of a
fluorescent label through an allosteric mechanism that is
noncompetitive with substrates and is rapidly reversible. (iii)
Regarding the identity of the active species, synthetic/medicinal
chemistry efforts verified the structure, purity, stability, and
chemical tractability of the chemotype. In addition, a
structurally similar but inactive control was identified which
was devoid of AcpS/Sfp-PPTase inhibitory activity as well as
antibacterial activity. (iv) To demonstrate the utility of 55 as a
probe, we confirmed the ability of a dual-specific PPTase
inhibitor to thwart the growth of bacteria in the absence of a
rapid cytotoxic response. Additionally, 55 demonstrated activity
against clinically relevant microorganisms, where it stifled the
growth of methicillin-resistant S. aureus. Expanding on these
findings, we also identified the primary mechanism of resistance
in E. coli to be efflux by the AcrAB−TolC system and
demonstrated a chemical genetic approach to mitigate this issue
and extend the spectrum of antibacterial activity to include
Gram-negative organisms. We recognize that optimizing a
chemotype to circumvent efflux is a formidable task and is
exacerbated in clinically relevant Gram-negative pathogens
where efflux pathways are more complex than in the model
organism E. coli.60 Beyond these assessments of antimicrobial
activity, the ability of 55 to attenuate the production of
surfactin, a metabolite dependent on the biochemical
functionality of Sfp-PPTase (Figure 5B), strongly indicates
that 55 is acting on-target inside the bacterial cell. (v) Finally, in
conjunction with the spirit and principles of the scientific
community and the probe characteristic of availability, we will
provide samples of 55 freely upon request.
CONCLUSION
■
PPTase enzymes catalyze an essential PTM that activates the
assembly of fatty acid, polyketide, and nonribosomal peptides.
Metabolites from all three of these classes are necessary for
bacterial cell viability and virulence. As a result, inhibition of
these pathways has received attention as an attractive approach
to the development of new antimicrobial compounds. Rather
than targeting the machinery involved in the production of a
single metabolite, we have chosen to investigate the inhibition
of this PTM as it represents a unifying feature between these
pathways. PPTase inhibitors may leverage this overlap to
downregulate the production of key cellular components and
virulence-determining factors.
EXPERIMENTAL PROCEDURES
■
General Methods for Chemistry. All air- or moisture-sensitive
reactions were performed under positive pressure of nitrogen with
oven-dried glassware. Anhydrous solvents such as dichloromethane,
N,N-dimethylformamide (DMF), acetonitrile, methanol, and triethyl-
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amine were purchased from Sigma-Aldrich. Preparative purification
was performed on a Waters semipreparative HPLC system. The
column used was a Phenomenex Luna C18 (5 μm, 30 × 75 mm) at a
flow rate of 45 mL/min. The mobile phase consisted of acetonitrile
and water (each containing 0.1% trifluoroacetic acid). A gradient of
10−50% acetonitrile over 8 min was used during the purification.
Fraction collection was triggered by UV detection (220 nm).
Analytical analysis was performed on an Agilent LC/MS system
(Agilent Technologies, Santa Clara, CA). Method 1: A 7 min gradient
of 4−100% acetonitrile (containing 0.025% trifluoroacetic acid) in
water (containing 0.05% trifluoroacetic acid) was used with an 8 min
run time at a flow rate of 1 mL/min. A Phenomenex Luna C18 column
(3 μm, 3 × 75 mm) was used at a temperature of 50 °C. Method 2: A
3 min gradient of 4−100% acetonitrile (containing 0.025% trifluoro-
acetic acid) in water (containing 0.05% trifluoroacetic acid) was used
with a 4.5 min run time at a flow rate of 1 mL/min. A Phenomenex
Gemini Phenyl column (3 μm, 3 × 100 mm) was used at a
temperature of 50 °C. Purity determination was performed using an
Agilent diode array detector for both method 1 and method 2. Mass
determination was performed using an Agilent 6130 mass
available 2,4-dimethylpyridin-2-amine: LC/MS retention time t1
(method 1, 7 min) = 5.384 min and t2 (method 2, 3 min) = 3.247
min; 1H NMR (400 MHz, DMSO-d6) δ 7.75 (dd, J = 7.1, 2.5 Hz, 2H),
7.68−7.59 (m, 1H), 7.23 (s, 1H), 6.85 (s, 1H), 4.01 (d, J = 5.6 Hz,
4H), 2.93 (q, J = 4.6, 3.7 Hz, 4H), 2.40 (s, 3H), 2.29 (s, 3H); HRMS
(ESI) m/z (M + H)+ calcd for C19H21ClF3N4S 429.1122, found
429.1133.
N-(4,6-Dimethylpyridin-2-yl)-4-(5-(trifluoromethyl)pyridin-2-yl)-
piperazine-1-carbothioamide Trifluoroacetate (53). This compound
was prepared following the above general procedure starting from
commerically available precursors 2,4-dimethylpyridin-2-amine and 1-
(5-(trifluoromethyl)pyridin-2-yl)piperazine: LC/MS retention time t1
(method 1, 7 min) = 4.615 min and t2 (method 2, 3 min) = 3.114 min;
1H NMR (400 MHz, DMSO-d6) δ 8.45 (d, J = 2.5 Hz, 1H), 7.85 (dd, J
= 9.2, 2.6 Hz, 1H), 7.29 (s, 1H), 6.96 (d, J = 9.5 Hz, 2H), 4.10−3.99
(m, 4H), 3.79 (dd, J = 6.5, 4.0 Hz, 4H), 2.44 (s, 3H), 2.33 (s, 3H);
HRMS (ESI) m/z (M + H)+ calcd for C18H21F3N5S 396.1464, found
396.1469.
4-(3-Chloro-5-(trifluoromethyl)pyridin-2-yl)-N-(4,6-dimethylpyri-
din-2-yl)piperazine-1-carbothioamide Trifluoroacetate (54). This
compound was prepared following the above general procedure
starting from commerically available precursors 2,4-dimethylpyridin-2-
amine and 1-(3-chloro-5-(trifluoromethyl)pyridin-2-yl)piperazine:
LC/MS retention time t1 (method 1, 7 min) = 5.113 min and t2
1
spectrometer with electrospray ionization in the positive mode. H
NMR spectra were recorded on Varian 400 MHz spectrometers.
Chemical shifts are reported in parts per million with undeuterated
solvent (DMSO-d6 at 2.49 ppm) as the internal standard for DMSO-d6
solutions. All of the analogues tested in the biological assays have
purity greater than 95% on the basis of both analytical methods. High-
resolution mass spectrometry was recorded on an Agilent 6210 time-
of-flight LC/MS system. Confirmation of the molecular formula was
accomplished using electrospray ionization in the positive mode with
the Agilent Masshunter software (version B.02).
General Procedure for the Synthesis of N-(Aryl/heteroaryl)-
4-arylpiperazine-1-carbothioamides. A mixture of substituted
pyridin-2-amine (1 equiv) and 1,1′-thiocarbonyldiimidazole (TCDI)
(1.05 equiv) in dichloromethane (2 mL/mmol) was stirred for 15 min
at room temperature. To the clear yellow solution was added
arylpiperazine (1.1 equiv), and the reaction mixture was stirred at 40
°C for 1 h. The solvent was evaporated, and the crude product was
taken up in 2 mL of DMSO and purified via reversed-phase
chromatography to give the products as TFA salts (the details and
characterization for all the compounds is depicted in the Supporting
Information). Characterization data for representative compounds are
given below.
1
(method 2, 3 min) = 3.287 min; H NMR (400 MHz, DMSO-d6) δ
8.58 (d, J = 2.1 Hz, 1H), 8.23 (d, J = 2.1 Hz, 1H), 7.25 (s, 1H), 6.93
(s, 1H), 4.06 (dd, J = 6.5, 3.6 Hz, 4H), 3.61 (dd, J = 6.6, 3.7 Hz, 4H),
2.43 (s, 3H), 2.31 (s, 3H); HRMS (ESI) m/z (M + H)+ calcd for
C18H20ClF3N5S 430.1075, found 430.1089.
4-(3-Chloro-5-(trifluoromethyl)pyridin-2-yl)-N-(4-methoxypyri-
din-2-yl)piperazine-1-carbothioamide Trifluoroacetate (ML267)
(55). This compound was prepared following the above general
procedure starting from commerically available precursors 4-
methoxypyridin-2-amine and 1-(3-chloro-5-(trifluoromethyl)pyridin-
2-yl)piperazine: LC/MS retention time t1 (method 1, 7 min) = 4.88
1
min and t2 (method 2, 3 min) = 3.04 min; H NMR (400 MHz,
DMSO-d6) δ 8.56 − 8.61 (m, 1H), 8.16−8.26 (m, 2H), 7.15 (s, 1H),
6.83 (s, 1H), 4.05−4.13 (m, 4H), 3.88 (s, 3H), 3.58−3.67 (m, 4 H);
13C NMR (101 MHz, DMSO-d6) δ 180.45, 168.50, 159.01, 153.62,
143.08, 136.44, 136.41, 124.78, 122.09, 119.61, 118.77, 118.44, 107.22,
102.47, 102.37, 56.43, 56.29, 48.06, 47.56; HRMS (ESI) m/z (M +
H)+ calcd for C17H18ClF3N5OS 432.0867, found 432.0856.
N-(4-Methylpyridin-2-yl)-4-((3-(trifluoromethyl)phenyl)amino)-
piperidine-1-carbothioamide Trifluoroacetate (63). A mixture of 4-
methylpyridin-2-amine (0.1 g, 0.925 mmol, 1 equiv) and TCDI (0.165
g, 0.925 mmol, 1.0 equiv) in dichloromethane (3 mL) was stirred for
15 min at room temperature. To the clear yellow solution was added
commercially available N-(3-(trifluoromethyl)phenyl)piperidin-4-
amine (0.226 g, 0.925 mmol, 1 equiv), and the resulting solution
was stirred at 40 °C for 1 h. The solvent was evaporated, and the crude
product was taken up in 2 mL of DMSO and purified via reversed-
phase chromatography to give the product as a TFA salt: LC/MS
retention time t1 (method 1, 7 min) = 4.954 min and t2 (method 2, 3
min) = 3.025 min; 1H NMR (400 MHz, DMSO-d6) δ 8.21 (d, J = 5.4
Hz, 1H), 7.39 (d, J = 1.6 Hz, 1H), 7.31−7.22 (m, 1H), 7.05−7.00 (m,
1H), 6.91−6.85 (m, 2H), 6.83−6.77 (m, 1H), 5.50 (br s, 1H), 4.54 (d,
J = 13.2 Hz, 2H), 3.68 (tt, J = 9.4, 3.9 Hz, 1H), 3.43 (ddd, J = 13.7,
11.1, 2.8 Hz, 2H), 2.35 (s, 3H), 2.07−1.89 (m, 2H), 1.43 (dtd, J =
13.6, 10.2, 3.8 Hz, 2H); HRMS (ESI) m/z (M + H)+ calcd for
C19H22F3N4S 395.1512, found 395.1510.
N-(4-Methylpyridin-2-yl)-4-(3-(trifluoromethyl)phenyl)piperazine-
1-carboxamide Trifluoroacetate (12). To a mixture of 4-methylpyr-
idin-2-amine (0.5 g, 4.62 mmol, 1 equiv), (i-Pr)2NEt (0.970 mL, 5.55
mmol, 1.2 equiv), and DMAP (0.113 g, 0.925 mmol, 0.2 equiv) in
dichloromethane (25 mL) was added phenyl carbonochloridate (0.664
mL, 5.09 mmol, 1.1 equiv) at 0 °C, and the reaction was allowed to stir
at room temperature for 1 h. To the resulting clear solution was added
1-(3-(trifluoromethyl)phenyl)piperazine (1.13 mL, 6.01 mmol, 1.3
equiv), and the reaction mixture was stirred for an additional 2 h at
room temperature. Volatiles were removed by forced air, and the
N-(4-Methylpyridin-2-yl)-4-(3-(trifluoromethyl)phenyl)piperazine-
1-carbothioamide Trifluoroacetate (1). This compound was
prepared following the above general procedure starting from
commerically available 4-methylpyridin-2-amine and 1-(3-
(trifluoromethyl)phenyl)piperazine: LC/MS retention time t1 (meth-
1
od 1, 7 min) = 4.91 min and t2 (method 2, 3 min) = 3.13 min; H
NMR (400 MHz, DMSO-d6) δ 10.09 (s, 1H), 8.22 (d, J = 5.4 Hz,
1H), 7.53−7.42 (m, 2H), 7.33−7.18 (m, 2H), 7.16−7.07 (m, 1H),
7.03 (d, J = 5.4 Hz, 1H), 4.08 (t, J = 5.2 Hz, 4H), 3.40 (dd, J = 6.5, 3.9
Hz, 4H), 2.36 (s, 3H); 13C NMR (101 MHz, DMSO-d6) δ 181.16,
158.49, 153.14, 150.95, 130.53, 130.49, 130.22, 126.22, 120.70, 119.17,
118.96, 115.15, 111.29, 111.25, 48.49, 47.41, 21.38; HRMS (ESI) m/z
(M + H)+ calcd for C18H20F3N4S 381.1355, found 381.1341.
N-(4-Methylpyridin-2-yl)-4-(2-(trifluoromethyl)phenyl)piperazine-
1-carbothioamide Trifluoroacetate (22). This compound was
prepared following the above general procedure starting from
commerically available 4-methylpyridin-2-amine and 1-(2-
(trifluoromethyl)phenyl)piperazine: LC/MS retention time t1 (meth-
1
od 1, 7 min) = 4.993 min and t2 (method 2, 3 min) = 3.215 min; H
NMR (400 MHz, DMSO-d6) δ 8.18 (d, J = 5.3 Hz, 1H), 7.76−7.57
(m, 3H), 7.46−7.34 (m, 2H), 6.96 (d, J = 5.1 Hz, 1H), 4.09−3.95 (m,
4H), 2.93 (m, 4H), 2.32 (s, 3H); HRMS (ESI) m/z (M + H)+ calcd
for C18H20F3N4S 381.1355, found 381.1361.
4-(4-Chloro-2-(trifluoromethyl)phenyl)-N-(4,6-dimethylpyridin-2-
yl)piperazine-1-carbothioamide Trifluoroacetate (41). This com-
pound was prepared following the above general procedure starting
from intermediate 1-(4-chloro-2-(trifluoromethyl)phenyl)piperazine
(see the Supporting Information for the synthesis) and commerically
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residue was dissolved in DMF and purified via reversed-phase
chromatography to give 12 as a TFA salt: LC/MS retention time t1
(method 1, 7 min) = 4.43 min and t2 (method 2, 3 min) = 3.095 min;
1H NMR (400 MHz, DMSO-d6) δ 10.21 (s, 1H), 8.17 (d, J = 5.9 Hz,
1H), 7.54−7.39 (m, 2H), 7.29−7.18 (m, 2H), 7.17−7.05 (m, 2H),
3.71−3.63 (m, 4H), 3.31−3.29 (m, 4H), 2.40 (s, 3H); 13C NMR (101
MHz, DMSO-d6) δ 158.27, 153.81, 150.90, 150.53, 140.63, 130.07,
130.04, 129.76, 119.59, 119.03, 115.02, 114.70, 111.33, 111.29, 47.41,
43.50, 21.45; HRMS (ESI) m/z (M + H)+ calcd for C18H20F3N4O
365.1584, found 365.1590.
C. albicans ATCC 90028 (fluconazole-sensitive), and C. albicans
ATCC 96901 (fluconazole-resistant).
For the acquisition of images for Figures 3D and 4C, resazurin was
employed as an indicator to assist in visualization in a method similar
to that of Sarker et al.63 A 10 μL volume of sterile aqueous resazurin
solution (0.7% w/v) was added to the wells of test plates resulting
from the protocol above. After further incubation 4 h at 37 °C, the
plates were then imaged with an Epson photoscanner.
ASSOCIATED CONTENT
* Supporting Information
■
Methods. Sfp- and AcpS-PPTase qHTS Assays. The assay was
performed in 50 mM HEPES·Na (pH 7.6), 10 mM MgCl2, 0.01%
Nonidet P-40, and 0.01% BSA. A 3 μL volume of each reagent,
consisting of buffer (in columns 3 and 4 as a negative control) and Sfp-
or AcpS-PPTase (in columns 1, 2, and 5−48, final concentration of 15
or 100 nM, respectively) were dispensed into a 1536-well Greiner
black solid-bottom plate. Compounds (23 nL) were transferred via a
Kalypsys pintool equipped with a 1536-pin array. The plate was
incubated for 15 min at room temperature, followed by the addition of
1 μL of substrate (final concentrations for rhodamine−CoA and BHQ-
2-YbbR were 5 and 12.5 μM, respectively) to start the reaction. The
plate was then centrifuged at 1000 rpm for 15 s, and the fluorescence
intensity was recorded on a ViewLux high-throughput charge-coupled
device (CCD) imager (Perkin-Elmer) using standard BODIPY optics
(525 nm excitation and 598 nm emission). The plate was then
incubated for 30 or 60 min (Sfp or AcpS, respectively), and a second
read on the ViewLux was performed. The fluorescence intensity
difference over the 30 or 60 min period (Sfp or AcpS, respectively)
was used to calculate the respective reaction rate for each well. All
screening operations were performed on a fully integrated robotic
system (Kalypsys Inc., San Diego, CA) as described elsewhere. Plates
containing DMSO only (instead of compound solutions) were
included approximately every 50 plates throughout the screen to
monitor any systematic trend in the assay signal associated with
reagent dispenser variation or a decrease in enzyme specific activity.
PPTase Gel Assay. A DMSO solution of confirmed hits (0.5 μL)
was added to a 1.33× enzyme solution (15 μL, containing 26.6 nM
Sfp, 66 mM HEPES·Na, 13.3 mM MgCl2, 0.0133% NP-40, and
0.133% BSA, pH 7.6). After a 10 min incubation, the enzymatic
reaction was initiated by the addition of a 4× substrate solution (4 μL,
containing 50 μM rhodamine−CoA and 50 μM apo-actinorhodin−
ACP). The reactions were terminated after a 30 min incubation at
room temperature by the addition of a 2× quench solution (20 μL,
containing 4 M urea, 25 mM EDTA, and 0.004% phenol red, pH 8.0).
Samples were separated under native conditions on a 20%
polyacrylamide gel using standard Laemmli conditions. Following
the run, the gels were imaged with a Chemi-Doc Plus imager (Bio-
Rad, Hercules, CA), and the band intensity was quantified using the
ImageJ software package. Pixel density values were normalized to
control wells and fit with the four-parameter Hill equation using in-
house tools. PubChem AID 602362.
Minimum Inhibitory Concentration Determination. Methods for
MIC determination were made in accordance with standards put forth
by the National Clinical Laboratory Standards Institute detailed in
documents M07-A861 and M27-A262 for bacterial and fungal species,
respectively. Briefly, organisms were maintained on a solid medium.
Innoculum was prepared from overnight liquid cultures of bacteria or
by suspending 2 day old colonies in RPMI 1640 medium. Test articles
dissolved in DMSO (1 μL) were added to sterile medium [50 μL,
cation-adjusted Mueller Hinton II broth for bacteria (BD BBL,
Franklin Lakes, NJ) or RMPI 1640 for fungi (Invitrogen Corp.,
Carlsbad, CA)] followed by innoculum prepared in the same medium
(50 μL, containing ∼1 × 103 cfu) and incubated at 30−37 °C for 16−
20 h (48 h for fungi). The plates were visually inspected for microbial
growth, and the first well containing no visible microbial growth was
scored as the MIC. Strains evaluated in this manner included B. subtilis
168, B. subtilis HM489,32 E. coli K12, P. aeruginosa ATCC 9028, S.
aureus ATCC 6538 (methicillin-sensitive), S. aureus ATCC BAA-1717
(community-acquired methicillin-resistant strain USA300-HOU-MR),
S
Additional supplemental figures, experimental procedures, and
spectroscopic data (1H NMR, LC/MS, and HRMS) for
representative compounds. This material is available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Authors
*E-mail: asimeono@mail.nih.gov.
*E-mail: maloneyd@mail.nih.gov.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
T.L.F., G.R., A.Y., T.D., H.L.B., M.A.-R., W.L., A.J., A.S., and
D.J.M. were supported by the intramural research program of
the National Center for Advancing Translational Sciences and
the Molecular Libraries Initiative of the National Institutes of
Health Roadmap for Medical Research (Grant
U54MH084681). N.M.K. and M.D.B. were supported by
NIH Grants R21AI090213 and R01GM094924. We thank Sam
Michael, Michael Balcom (deceased), and Richard Jones for
automation support, Paul Shinn and Danielle van Leer for the
assistance with compound management, Edward H. Kerns and
Kimloan Nguyen for determination of in vitro ADME
properties, Elizabeth Fernandez, Christopher Leclair, and
Heather Baker for assistance with compound purification and
processing, Mohamed Marahiel (Philipps-Universitat Mar-
̈
burg), the Bacillus Genomic Stock Center, and The National
BioResource Project of the Japanese National Institute for
Genetics for bacterial strains, Udo Oppermann (University of
Oxford) for the human PPTase expression plasmid, and Victor
Torres (Anti-infectives Screening Core, New York University)
for MIC studies on clinical isolates of MRSA and conducting
the mouse model of staphylococcal septicemia.
ABBREVIATIONS USED
■
PPTase, 4′-phosphopantetheine transferase; Sfp, PPTase
involved in surfactin production; AcpS, PPTase involved in
FAS, acyl carrier protein synthase; CoA, coenzyme A; NRPS,
nonribosomal peptide synthethase; PKS, polyketide synthetase;
FAS, fatty acid synthetase; CA-MRSA, community-acquired
methicillin-resistant Staphylococcus aureus; SAR, structure−
activity relationship; HTS, high-throughput screen; MIC,
minimum inhibitory concentration; ADME, absorption, dis-
tribution, metabolism, and excretion; ip, intraperitoneal; iv,
intravenous; mpk, milligrams per kilogram; RLMs, rat liver
microsomes; MLMs, mouse liver microsomes; POC, proof of
concept; BBB, blood−brain barrier; PAMPA, parallel artificial
membrane permeability assay; NADPH, nicotinamide adenine
dinucleotide phosphate; CYP, cytochrome P450; PBS,
phosphate-buffered saline; PaβN, phenylarginine β-naphthyla-
mide; DDI, drug−drug interaction; PPB, plasma protein
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