Multitarget drug discovery for tuberculosis and other infectious diseases

Article abstract of DOI:10.1021/jm500131s

We report the discovery of a series of new drug leads that have potent activity against Mycobacterium tuberculosis as well as against other bacteria, fungi, and a malaria parasite the compounds are analogues of the new tuberculosis (TB) drug SQ109 (1), wh

Full text of DOI:10.1021/jm500131s

Article
pubs.acs.org/jmc
Multitarget Drug Discovery for Tuberculosis and Other Infectious
Diseases
Kai Li,†,& Lici A. Schurig-Briccio,‡,& Xinxin Feng,†,& Ashutosh Upadhyay,§ Venugopal Pujari,§
Benoit Lechartier,∥ Fabio L. Fontes,§ Hongliang Yang,§ Guodong Rao,† Wei Zhu,⊥ Anmol Gulati,†
Joo Hwan No,¶ Giovana Cintra,¶ Shannon Bogue,‡ Yi-Liang Liu,⊥ Katie Molohon,□ Peter Orlean,□
Douglas A. Mitchell,†,□,▽ Lucio Freitas-Junior,¶ Feifei Ren,# Hong Sun,# Tong Jiang,# Yujie Li,#
Rey-Ting Guo,# Stewart T. Cole,∥ Robert B. Gennis,†,‡ Dean C. Crick,§ and Eric Oldfield*,†,⊥
†Department of Chemistry, University of Illinois at Urbana−Champaign, 600 South Mathews Avenue, Urbana, Illinois 61801, United
States
‡Department of Biochemistry, University of Illinois at Urbana−Champaign, 600 South Mathews Avenue, Urbana, Illinois 61801,
United States
§Department of Microbiology, Immunology and Pathology, Colorado State University, 200 West Lake Street, Fort Collins, Colorado
80523, United States
∥
́
́ ́
Global Health Institute, Ecole Polytechnique Federale de Lausanne, Lausanne, Switzerland
⊥Center for Biophysics and Computational Biology, University of Illinois at Urbana−Champaign, 607 South Mathews Avenue,
Urbana, Illinois 61801, United States
¶Institut Pasteur Korea, Sampyeong-dong 696, Bundang-gu, Seongnam-si, Gyeonggi-do, Korea
□Department of Microbiology, University of Illinois at Urbana−Champaign, 601 South Goodwin Avenue, Urbana, Illinois 61801,
United States
▽Institute for Genomic Biology, University of Illinois at Urbana−Champaign, 1206 West Gregory Drive, Urbana, Illinois 61801,
United States
#Industrial Enzymes National Engineering Laboratory, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences,
Tianjin 300308, China
S
* Supporting Information
ABSTRACT: We report the discovery of a series of new drug
leads that have potent activity against Mycobacterium tuber-
culosis as well as against other bacteria, fungi, and a malaria
parasite. The compounds are analogues of the new tuber-
culosis (TB) drug SQ109 (1), which has been reported to act
by inhibiting a transporter called MmpL3, involved in cell wall
biosynthesis. We show that 1 and the new compounds also
target enzymes involved in menaquinone biosynthesis and
electron transport, inhibiting respiration and ATP biosyn-
thesis, and are uncouplers, collapsing the pH gradient and
membrane potential used to power transporters. The result of such multitarget inhibition is potent inhibition of TB cell growth,
as well as very low rates of spontaneous drug resistance. Several targets are absent in humans but are present in other bacteria, as
well as in malaria parasites, whose growth is also inhibited.
cell membranes. An example of this type of drug would be the
antifungal amphotericin,4 which functions by binding to
ergosterol (which is not present in human cell membranes).
A third and well-known approach is to employ combination
therapies,5 although the problems associated with finding two
new drugs active against two new targets are clearly significant.
A fourth approach is to use “multitargeting” or “polypharma-
INTRODUCTION
■
Antibiotic resistance is a public health problem that, arguably,
has the potential to destroy the efficacy of all antibiotics in the
next 10−20 years.1,2 There is, therefore, an urgent need for new
drugs, especially ones that might be more “resistance-resistant”.
One possible approach to achieving this goal is to move away
from targeting the direct killing of pathogens to inhibiting their
virulence, because this might lead to a decreased “life or death”
pressure on the organism to develop resistance.3 A second
approach would be to develop more drugs that target pathogen
Received: January 24, 2014
Published: February 25, 2014
© 2014 American Chemical Society
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cology” in which a single drug has more than one target.6,7 This
could involve “series inhibition”, in which targets would be in
the same metabolic pathway (Figure 1, left), “parallel
mutations in the mmpL3 gene and cross-resistance to 1,
although these latter effects were rather small.12 More
intriguingly, no spontaneous resistant mutants were obtained
when using 1, suggesting the possibility of multiple targets.12,13
That idea is supported by the observation that 1 also has
activity against other bacteria, e.g., Helicobacter pylori14 as well
as against the yeast Candida albicans,15 neither of which possess
the mmpL3 gene, suggesting again that other 1 targets are
harbored by these organisms and, potentially, by M. tuberculosis.
In this work, we synthesized a series of analogues of 1 in
which we varied the adamantane headgroup and the ethyl-
enediamine linker (varying the possible charge centers), Chart
1. All compounds were screened against a panel of bacteria (M.
tuberculosis, M. smegmatis, Escherichia coli, Staphylococcus aureus,
and Bacillus subtilis), two yeasts (Saccharomyces cerevisiae and
Candida albicans), a malaria parasite (Plasmodium falciparum),
and a human cell line (MCF-7), to establish antibacterial,
antifungal and antimalarial structure−activity relationships and
to assess mammalian cell toxicity. In addition, we investigated a
subset of compounds for activity against a series of putative
targets, isoprenoid biosynthesis enzymes, in addition to
investigating the effects of these compounds on respiration,
ATP synthesis, and the proton motive force (PMF).
Figure 1. Series (in the same metabolic pathway), parallel (unrelated
pathways or DNA/membrane targets), or network (series and parallel
target) inhibition.
inhibition”, in which the targets would be unrelated but an
inhibitor might mimic a common substrate or would affect, for
example, a membrane function (Figure 1, middle), or “network
inhibition”, in which many targets in series and/or in parallel
could be involved (Figure 1, right). While challenging, many
drugs that have been effective in monotherapy do in fact have
multiple targets6 while single-target drugs (many of which are
used in treating tuberculosis) are often only effective in
combination therapies.
In this work, we consider the mechanism(s) of action of the
new antituberculosis drug 1 (Chart 1), currently in phase II
clinical trials.8 This drug candidate appeared of interest because
it contains a C10 isoprenoid (geranyl) side chain together with a
strongly basic (ethylenediamine) fragment, a likely cationic
center, suggesting that it might act as a carbocation isostere for
a transition state/reactive intermediate in isoprenoid biosyn-
thesis9 and, as with other inhibitors of isoprenoid biosynthesis,
it might be involved in multitargeting.10 1 was developed in a
synthesis/screening program11 in which ∼64 000 ethylenedi-
amine analogues of the antituberculosis drug ethambutol (2)
were synthesized. 1 was ∼4× more active than any of the other
leads developed, having a minimum inhibitory concentration
(MIC) of ∼0.7−1.56 μM against M. tuberculosis (H37Rv,
Erdman, and drug-resistant strains), and insights into its mode
of action recently became available when the target of SQ109
was proposed12 to be MmpL3, a trehalose monomycolate
(TMM) transporter, an essential membrane protein that
transports TMM into the cell envelope. This conclusion was
based on the observation that several M. tuberculosis mutants
produced via serial passage with several 1-like inhibitors had
RESULTS AND DISCUSSION
■
Only One Cationic Center Is Needed for Potent
Activity in Mycobacteria. To investigate which features
contribute to the activity of 1 against a broad range of
organisms, we synthesized 1 and the 11 analogues (3−13)
shown in Chart 1 in which the ethylenediamine linker was
replaced by ethanolamine, choline, propanolamine, ethylene
glycol, or glycolic amide moieties (providing linkers with
potentially 0, 1, or 2 positive charges), as well several “head
group” analogues in which the adamantyl group was varied.
The geranyl “side chain” was kept constant. Full synthesis and
characterization details are given in the Supporting Information.
As expected, 1 had potent activity against M. tuberculosis with
an MIC of ∼0.1−0.2 μg/mL (Table 1). Interestingly, the N-
geranyl ethanolamines 3 and 13 were more potent (MIC values
as low as 0.02−0.05 μg/mL), indicating that the presence of
two nitrogens was not essential for activity. The O-geranyl
ethanolamine derivative (4) had activity similar to that of 1
(∼0.2 μg/mL), Table 1. With the N-geranyl ethanolamines (3,
13), activity was about 30-fold higher than with ethambutol
(2). The ethylene glycol (5) was far less active (50 μg/mL), as
was the glycolic amide 9 (12 μg/mL). Thus, the most active
compounds all contain one strongly basic nitrogen in the linker
region, with most activity being found with the two N-geranyl
Chart 1. Structures of Compounds Investigated
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Table 1. Inhibition by 1 and Analogues of M. tuberculosis (Mt), M. smegmatis (Ms), S. aureus (Sa), B. subtilis (Bs), E. coli (Ec), S.
cerevisiae (Sc), C. albicans (Ca), P. falciparum (Pf), and Human (MCF-7) Cell Growth
a
a
a
b
b
b
b
b
b
Mt
Ms
Sa
Bs
Ec
Sc
Ca
Pf
MCF-7
1
0.1−0.2 (0.3−0.6)
2(9.8)
3.1(9.4)
>120(364)
−
7.6(23)
−
2.8(8.5)
−
1.1(3.3)
−
3.3(10)
−
1.0(3.0)
−
6.0(18)
−
c
d
2
1(4.9)
3
0.02−0.05 (0.06−0.15)
0.19(0.57)
50(150)
1.6(4.8)
6.2(19)
>50(150)
6.2(18)
12(24)
6.2(18)
25(72)
50(198)
12(44)
12(37)
1.6(4.8)
8(24)
16(48)
2.8(8.5)
1.8(5.4)
>33(100)
>332(1000)
2.7(7.8)
15(31)
12(36)
0.79(2.4)
0.93(2.8)
7.9(24)
0.83(2.4)
0.08(0.16)
6.2(18)
3.8(11)
3.2(13)
14(51)
10(30)
4
>120(362)
>64(193)
8(23)
>66(199)
>66(199)
3.0(8.7)
4.2(8.6)
>69(200)
17(49)
4.3(13)
>66(199)
>66(198)
6.5(19)
18(37)
112(338)
73(220)
3.4(9.8)
3.2(6.5)
12(35)
5
>332(1000)
2.3(6.7)
37(76)
6
1.6(4.6)
7
0.78(1.6)
0.78(2.2)
12(34)
8(16)
8
>120(345)
>120(348)
>16(63)
32(117)
8(25)
>345(1000)
>345(1000)
>126(498)
60(220)
15(46)
>69(200)
>69(200)
44(174)
3.0(11)
3.7(11)
>69(200)
>69(200)
>50(198)
20(74)
9
9.3(27)
45(178)
6.3(23)
7.1(22)
1.3(3.9)
10
11
12
13
6.2(24)
>50(198)
17(62)
6.2(23)
6.2(19)
2.4(7.4)
1.8(5.4)
12(37)
>100(309)
2.0(6.0)
0.05(0.15)
8(24)
12(36)
0.89(2.7)
6.3(19)
a
b
c
d
MIC, μg/mL, values in parentheses are in μM. IC50, μg/mL, values in parentheses are in μM. Reference 16. Reference 17.
ethanolamines 3 and 13 (Table 1), with 3 being ∼4−5 times
more active than that of 1.
pIC50 values for the yeasts and E. coli or B. subtilis is also
apparent (R = 0.6−0.7, Figure 2B), suggesting the possibility of
target conservation between fungi and bacteria. Because a
recognizable mmpL3 gene is absent in the fungi, these results
again indicate an alternate target or targets. These results also
lead to the idea that there could be additional targets in M.
tuberculosis, which would help explain the very low MIC values
observed and the inability to induce resistance via serial
passage, as noted by Tahlan et al.,12 although multiple-targeting
does not necessarily guarantee improved potency. The results
with the bacteria and fungi then suggested the possibility that
the growth of other organisms (protozoa) might also be
inhibited by 1 or its analogues.
To evaluate antiprotozoal activity we screened all 12
compounds (1, 3−13) against the intraerythrocytic form of
the malaria parasite, Plasmodium falciparum. As can be seen in
Table 1, 1 had a ∼1 μg/mL activity against P. falciparum, and
the three ethanolamines (3, 4, and 6) also had good activity. As
viewed on the heat map (Figure 2A), inhibition of M.
tuberculosis cell growth is strongest but is followed by P.
falciparum (in the intraerythrocytic assay) and in each case
where there is activity against P. falciparum (2, 3, 4, 6, 7), the
inhibitors (Chart 1) are expected to carry a +1 charge, as with
the best M. tuberculosis growth inhibitors. When compared to
growth inhibition results with a human cell line (MCF-7; Table
1), we see that activity against the human cells is much weaker
than against P. falciparum and, of course, against M. tuberculosis.
We calculate a therapeutic index (TI), defined as:
We then tested all 12 compounds against M. smegmatis. The
results (Table 1) show that overall activity against M. smegmatis
is less than that observed against M. tuberculosis, as can also be
seen in the “heat map” shown in Figure 2A. There is, however,
a very high correlation coefficient (R2 = 0.9, Figure 2B)
between the pIC50 (= −log10 IC50) values for M. tuberculosis
and M. smegmatis, indicating a similar mechanism of action,
leading to our use of M. smegmatis in several mechanism of
action studies, as described below.
The Cationic Inhibitors Exhibit Broad Antibacterial,
Antifungal, and Antimalarial Activity. All 12 compounds
(1, 3−13) were then tested against three other bacteria: S.
aureus, B. subtilis, and E. coli, Table 1. With S. aureus (the
methicillin-resistant S. aureus (MRSA) USA300 strain), 1 itself
had little activity; however, the N-geranyl ethanolamines 3, 12,
13 and the N-geranyl propanolamine 6 all had MIC values of
∼8 μg/mL, while the other analogues were much less active
(MIC > 32 μg/mL). A similar pattern of activity was seen
against B. subtilis, with the three ethanolamines (6, 12, 13)
exhibiting the highest levels of activity. In addition, unlike with
S. aureus, 1 itself had activity (7.6 μg/mL, Table 1). With E. coli,
1, the N-geranyl ethanolamine (3) and the N-geranyl
propanolamine (6) were all quite active, with IC50 values of
∼2−3 μg/mL, Table 1. Moreover, there was a modest
correlation (R2 = 0.5) between the M. tuberculosis (or M.
smegmatis) pMIC values and those found with E. coli, Figure
2B. These results again indicate that at least one basic amine,
most likely a cationic center, is required for best activity; plus,
there must be a target or targets other than MmpL3 in E. coli
because the mmpL3 gene is absent in this organism.
Bioinformatics searches did locate uncharacterized mmpL3-
like genes in S. aureus and B. subtilis, but it remains to be seen if
the corresponding proteins are targeted by our compounds.
We next tested all 12 compounds (1, 3−13) for activity
against S. cerevisiae and C. albicans. As can be seen in Table 1
and Figure 2A, 1 and the ethanolamines 3, 6, 11, and 13 had
activity in the 1−3 μg/mL range, with 1 and 13 being the most
potent, having an IC50 ∼ 1 μg/mL. With C. albicans, 1 was most
active, followed by the ethanolamines 3 and 13. Not
unexpectedly, there was a high correlation between the pIC50
values seen between S. cerevisiae and C. albicans (R2 = 0.8;
Table 1 and Figure 2B). A modest correlation between the
IC50(human cell line)
TI =
IC50(pathogen)
of ∼18 for 1 against P. falciparum and ∼40 for 3, while for M.
tuberculosis we find TI = 120 (1) and TI = 900 (3), suggesting
that these and related analogues may also be promising P.
falciparum drug leads. Because the human cell growth assays are
carried out in the presence of 10% fetal bovine serum (FBS),
we tested three of the most active compounds (1, 3, 13) against
E. coli in the presence or absence of 10% FBS. There was only a
1.6 0.07× increase in the IC50, meaning that, as expected,
serum binding is small and quite similar for each of these
compounds. We next sought to explore what the additional
targets for these compounds might be in cells that lack MmpL3.
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diphosphate synthase (GGPPS); S. aureus and E. coli
undecaprenyl diphosphate synthases (UPPSs), S. aureus
farnesyl diphosphate synthase (FPPS), and human GGPPS.
In essentially all cases, IC50 values were ≥50 μM. The exception
was human GGPPS, which was inhibited by 1 (the only
compound with two basic groups) with a 4.5 μM IC50. These
enzymes are all so-called cis or trans-“head-to-tail” prenyl-
transferases18 and the presence of the two (as opposed to one)
hydrophobic domains (in addition to the cationic center) might
not be required for enzyme inhibition. There are, however,
other prenyl transferases that might be targeted in which two
hydrophobic domains, together with a carbocation center,
would better mimic transition states/reactive intermediates.
These would include the so-called “head-to-head” prenyl
transferases, as well as some of the enzymes involved in
quinone biosynthesis. There are demonstrated or putative
head-to-head prenyl transferases in M. tuberculosis (Rv3397c),
M. smegmatis, (Mycsm_04912), S. aureus (CrtM), B. subtilis
(YisP), S. cerevisiae (squalene synthase, SQS), and C. albicans
(SQS) and in humans (SQS), but no homologous proteins can
be found by standard BLAST searches in P. falciparum. The
products (where known) of these enzymes vary, and not all are
essential for survival in vitro. Nevertheless, we tested a subset of
compounds (1, 3, 4) for activity against either SaCrtM or
human SQS, finding only weak activity (∼100 μM) in all cases.
These results support the notion that the head-to-head prenyl
transferases are unlikely cell growth inhibition targets of our
compounds in these organisms.
The other obvious candidates are the enzymes involved in
quinone biosynthesis (Scheme 2) or quinone utilization. We
thus next investigated the two quinone biosynthesis enzymes,
MenA and MenG, both of which are likely to utilize cationic
transition states/reactive intermediates during catalysis. MenA
(EC 2.5.1.74, 1,4-dihydroxy-2-naphthoate polyprenyl trans-
ferase) catalyzes the isoprenylation of 1,4-dihydroxy-2-naph-
thoic acid by long chain isoprenoid diphosphates,19 Scheme 2,
and MenA is of interest as an M. tuberculosis drug target.20−22 In
an initial set of experiments we tested three potent M.
tuberculosis and M. smegmatis growth inhibitors (1, 3, and 13) in
the M. smegmatis MenA (MsMenA) membrane fragment
inhibition assay described previously,20−22 finding IC50 values
of ∼6 μM (Table 2). Typical dose−response curves are shown
in Figure S1A, Supporting Information. While this assay
revealed only modest activity, the observation that MenA
activity was in fact inhibited by the three most potent inhibitors
is a potentially important one because this inhibition might be
expected to inhibit electron transfer/ATP synthesis, of
particular importance in nonreplicating/persister cells,22 and
to contribute to cell growth inhibition beyond that seen with
MmpL3 inhibition alone. What was also of interest was that 1
had similar activity (IC50 = 9 μM, in the same assay as used
here22) against MenA to that we reported previously with Ro
48-8071, a lipophilic amine that decreases menaquinone
biosynthesis and blocks M. tuberculosis as well as M. smegmatis
cell growth. These growth inhibition effects with Ro 48-8071
(as well as the inhibition of respiration) were reported
previously to be reversed in both organisms by addition of
400 μM menaquinone-4 (MK-4) or phylloquinone to the
medium.22
Figure 2. Inhibition of cell growth for M. tuberculosis (Mt), M.
smegmatis (Ms), S. aureus (Sa), B. subtilis (Bs), E. coli (Ec), S. cerevisiae
(Sc), C. albicans (Ca), P. falciparum (Pf), and a human cell line (MCF-
7) by 1, 3−13. (A) Heat map. Red = strong inhibition; yellow =
moderate inhibition; green = weak/no inhibition. (B) Correlation R
values for cell growth inhibition pIC50 (= −log10 IC50, μM) or pMIC =
(−log10 MIC, μM) between all systems investigated.
Possible Protein Targets for SQ109 and Its Ana-
logues. The general patterns of activity seen with the
compounds described above have some similarities across the
diverse organisms investigated in that at least one cationic
center, or perhaps more importantly a protonatable nitrogen, is
required for activity. In M. tuberculosis, 1 is thought to act by
inhibiting MmpL3, a TMM transporter,12 although as noted by
Tahlan et al., other targets could also be involved. This seems
quite likely because in most cases these other organisms lack
mmpL3 or a clearly identifiable orthologue, and do not utilize
TMM, as is also the case with H. pylori.14 Given that protonated
geranylamines might be good isosteres for transition states or
reactive intermediates in enzymes involved in isoprenoid
biosynthesis (Scheme 1), we investigated if 1 could inhibit
any of the following enzymes: M. tuberculosis cis-farnesyl
diphosphate synthase (Rv1086); M. tuberculosis cis-decaprenyl
diphosphate synthase (Rv2361); P. vivax geranylgeranyl
A second possible target is MenG (EC 2.1.1.163, 2-
polyprenyl-1,4-naphthoquinone methyltransferase) which car-
ries out the S-adenosylmethionine (SAM)-dependent methyl-
ation of demethylmenaquinone (the product of the MenA
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a
Scheme 1. Several Reactions of Interest in Isoprenoid Biosynthesis in the Systems Investigated in This Study
a
The enzymes in red were tested for inhibition by 1. cis-FPPS and trans-FPPS, UPPS, and DPPS are not inhibited by 1 but CrtM is, and CrtM-1
structures have been reported (PDB ID 4EA1, 4EA2) and serve as models for MenA inhibition.
a
Scheme 2. Menaquinone Biosynthesis of MK-3
a
MK-8,9 are the abundant species in cells. MenA forms demethylmenaquinol (DMK), which spontaneously oxidizes to demethylmenaquinone.
DMK is the substrate of MenG.
reaction). As with MenA, the MenG reaction is inhibited by 1
and the potent ethanolamine analogues 3, 13 (Table 2, and
Figure S1B,C, Supporting Information), with IC50 in the 6−13
μM range. Unlike the C-alkylations with prenyl diphosphates,
the MenG reaction uses SAM (as a C1 source), and Mg2+ is not
required. With 1 binding to MenG, the cationic center in the
inhibitor might mimic a cationic transition state/reactive
intermediate, although another possibility is that the cationic
center simply mimics the SAM S-methyl sulfonium group.
Thus, both MenA and MenG are inhibited in vitro by 1 and its
analogues, which can be expected to supplement MmpL3-based
inhibition in the mycobacteria, as well as provide alternative
targets in some of the organisms that lack the mmpL3 gene.
Moreover, inhibition of two sequential targets (series
inhibition) in a biosynthetic pathway can often be quite
effective because the product of the first reaction is the
substrate for the second reaction.23
growth (and, within experimental error, of E. coli cell growth,
Table 1). We additionally found that there was a moderate
correlation between E. coli cell growth inhibition and EcMenA
inhibition with an R2 = 0.43 (using pIC50 = −log10 IC50, both
values in μM) values, suggesting that MenA inhibition may be
involved in cell growth inhibition. As described below, the
experimental vs predicted E. coli cell growth inhibition
correlation increased to R2 = 0.77 with the incorporation of a
second experimental parameter, ΔpH collapse.
The structure of MenA is not known, but it is predicted to be
a transmembrane protein containing about nine α-helices, as
shown in Figure 3A.24 Using modern structure prediction
programs such as Phyre225 that are secondary-structure based,
MenA is predicted (Figure 3B) to adopt basically all the same
α-helical folds as found in farnesyl diphosphate synthase and
CrtM (the S. aureus dehydrosqualene synthase) but where one
N- and two C-terminal helices (transmembrane helices 1, 8,
and 9 in Figure 3A) are not modeled, Figure 3B. A total of 198
residues (68%) are, however, modeled at a predicted >90%
accuracy, and the predicted structure has the closest similarity
to the crystal structure of farnesyl diphosphate synthase from
Methylococcus capsulatus (PDB ID 3TS7), although remarkably
We next used an expressed E. coli MenA (hereafter EcMenA)
detergent-based assay to obtain inhibition data for all 12
inhibitors, Table 2, and Figure S2, Supporting Information.
Interestingly, the most potent inhibitor was 3 (IC50 = 400 nM),
and 3 was also the most potent inhibitor of M. tuberculosis cell
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transferase enzyme that is known to utilize a carbocation
mechanism, consistent with the experimental observation that
only cationic species inhibit MenA.
Table 2. Enzyme, Respiration, and PMF (ΔpH, Δψ)
Inhibition Results
Mycobacterium smegmatis
Escherichia coli
Menaquinone Rescue Experiments. We next measured
the activity of 1 against both actively growing (M. tuberculosis
H37Rv) and nonreplicating (streptomycin-starved M. tuber-
culosis 18b)28 mycobacteria, using a resazurin microplate
reduction assay (REMA; Figure 4). 1 had a MIC of 0.15 μg/
mL against actively replicating H37Rv in this assay, as expected.
It also displayed activity against the nonreplicating streptomy-
cin-starved 18b strain (where MmpL3/TMM transport is
presumably not involved because there is no cell growth), and
the effects of 1 on both strains were affected by MK-4 addition
(Figure 4). In the H37Rv aerobic assay, the MIC shifted from
0.15 μg/mL in the absence of MK-4 (Figure 4A) to ∼1 μg/mL
when the medium was supplemented with 1 mM menaquinone,
consistent with a role for 1 in inhibiting quinone biosynthesis
and/or electron transport. As noted above, a remarkably similar
effect was seen previously with Ro 48-8071, another lipophilic
amine, at 400 μM MK-4,22 for both M. tuberculosis and M.
smegmatis. The effect of 1 against nonreplicating (streptomycin-
starved 18b) bacteria, as seen by REMA as a decrease in
fluorescence (lack of resazurin reduction to the highly
fluorescent red resorufin) above a 1 concentration of 1 μg/
mL was also blocked by MK-4 addition (Figure 4B). The
activity of 1 against nonreplicating (streptomycin-starved 18b)
cells was confirmed by plating and counting CFU after 7 days
of drug exposure (Figure 4C) with normal 7H9 medium or
with 7H9 medium containing 1 mM MK-4. As can be seen in
Figure 4C, 1 at 1 μg/mL had essentially no effect on
(nonreplicating) bacterial activity in the presence of MK-4
and only a small effect in the absence of MK-4. However, at 10
μg/mL 1, while there was again a small effect on activity in the
presence of MK-4, cell activity in the absence of MK-4 was
reduced by ∼4 log units, consistent with a role for 1 in blocking
respiration and hence ATP synthesis.
Δψ
ΔpH
e
a
a
b
c
d
entry MenA MenG respiration
collapse
MenA
collapse
1
4.8
13
58
0.5
55
31
3.3
0.4
1.8
4.2
1.9
1.0
5.8
16
0.8
0.8
1.0
15
3
4
15
4
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
8
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
5.7
36
78
5
600
150
50
6
4.8
0.5
330
1.1
18
7
51
8
150
150
150
150
130
44
12
9
280
9500.0
2500.0
140.0
0.3
7.4
4.7
6.7
7.1
0.8
10
11
12
13
4.6
4.6
3.3
3.2
a
IC50 in μM, M. smegmatis membrane fraction (Figure S1, Supporting
Information). IC50 in μM, from methylene blue reduction assay
b
c
(Figure S3, Supporting Information). IC50 in μM, from DisC3(5)
d
assay (Figure S7, Supporting Information). IC50 in μM, expressed E.
e
coli MenA (Figure S2, Supporting Information). . IC50 in μM,
measured with E. coli IMVs (Figures S4 and S5, Supporting
Information). N.D.: not determined
there is only a 10% residue identity. The first and second
aspartate-rich motifs essential for Mg2+ binding and catalysis in
FPPS and CrtM are located in very similar regions in the
MtMenA model, as shown in the superposition with CrtM in
Figure 3C (orange spheres = conserved Asps in EcMenA
model; blue spheres = Asp-rich motif in CrtM). This then
suggests, based on the 1−CrtM X-ray structure,26 the binding
sites for 1 (pink) shown in Figure 3C. The two Asp-rich
domains in MenA are also highly conserved, as shown by a
SCORECONS27 analysis (Table S1, Supporting Information).
Although only a computational prediction, it is of interest that
the highest scoring Phyre2 prediction is found with a prenyl
Figure 3. (A) Transmembrane helices predictions for MtMenA. (B) Transmembrane helices in Phyre2 model of MtMenA (helices S1, S8, and S9
from A are not modeled). Orange: Asp-rich motifs. (C) MenA model (cyan) and CrtM (green, PDB: 4EA1, N- and C-terminal helices are removed).
Blue: Asp-rich motifs in CrtM. CrtM structure contains SQ109 (two conformers), shown as magenta spheres.
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Figure 4. Menaquinone rescue experiments. (A) Aerobic M. tuberculosis H37Rv growth inhibition in the presence of increasing MK-4
concentrations, measured by REMA. (B) As in A but with nonreplicating M. tuberculosis 18b. (C) M. tuberculosis 18b cells were plated after 7 days of
drug exposure with (gray) or without (black) MK-4. Colony forming unit counts were assessed after one month of incubation. Concentrations are in
μg/mL.
Respiration, TMM, and the PMF. The results described
above show that 1 has activity against not only the two
mycobacteria (M. tuberculosis and M. smegmatis) but also a
range of other bacteria, fungi, and a protozoan, each of which
lack a bioinformatically recognizable mmpL3 gene. In M.
tuberculosis and M. smegmatis, inhibition of MenA/MenG would
inhibit respiration, resulting in a decrease in ATP biosynthesis.
This could help explain how 1 increases the level of TMM
given that MmpL3 is a TMM transporter of the RND family of
efflux pumps, many of which are powered by the PMF.
Restated, 1 might exhibit an indirect action upon the TMM
transporter by removing its “power source” (the proton motive
force), in addition to directly binding to, and inhibiting, the
transporter. This indirect action could be accomplished in one
of two ways: (1) blocking respiration (by depletion of
menaquinone by inhibition of MenA or MenG or by directly
inhibiting a component of the electron transport chain); (2) a
direct effect on the PMF (Δψ + ΔpH, where Δψ is the
membrane potential and ΔpH, the pH gradient). The
possibility of the involvement of the PMF is suggested from
the results of a number of studies in which lipophilic bases (e.g.,
amiodarone, local anesthetics, and NSAIDS29−31) can act as
uncouplers. In addition, there could also be multidrug targeting
affecting MmpL3 (or efflux pumps32), MenA, MenG, and the
PMF, which would be expected to produce potent inhibition of
cell growth/respiration/ATP synthesis, as well as a low rate of
resistance.
growth inhibition and the pIC50 (= −log10 IC50, IC50 in μM) for
inhibition of whole cell respiration inhibition (R2 = 0.55) for all
12 compounds (Tables 1 and 2).
These results suggest the possibility of a direct effect on
electron transport (because the effects observed are rapid: tens
of minutes), blocking respiration, consistent with the MK-4
rescue experiments. The nature of the target or targets involved
are beyond the scope of this current study, but we did carry out
preliminary experiments with 1 against a series of dehydro-
genases by monitoring the reduction of the artificial electron
acceptor MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-
tetrazolium bromide). We used an M. smegmatis membrane
preparation and a variety of substrates including NADH
(measuring both Complex I and alternative NADH dehydro-
genases, NDH-2), deamino-NADH (measuring Complex I
activity but not NDH-2), succinate (measuring succinate
dehydrogenase), malate (measuring quinone-dependent malate
dehydrogenase), and lactate (measuring lactate dehydrogen-
ase). IC50 values for 1 were in general ∼30 μg/mL, the
exception being malate dehydrogenase (IC50 = 10 μg/mL),
results that suggest that more than one-electron-transfer
protein may be involved, in cells, with the inhibitors mimicking
quinone substrates.
Uncoupler Effects in Membrane Vesicles and in Cells.
The results presented above show that 1 and its analogues
inhibit MenA, MenG, electron transfer proteins, and respiration
and that MK-4 can rescue cell growth or activity, the relatively
high IC50s for enzyme inhibition/respiration (when compared
to cell growth inhibition) being suggestive of multiple targeting.
What is of particular interest about all of the results described
above is that they seem to point in one direction: respiration,
offering a possible explanation for the previous observation that
TMM accumulates (with 1), in M. tuberculosis, due to MmpL3
inhibition. This MmpL3 inhibition could be due to direct
binding or a more indirect effect on the PMF/ATP synthesis.
To test the hypothesis that 1 and its analogues might collapse
the PMF, we first used E. coli inverted membrane vesicles
(IMVs), essentially as described by Haagsma.33 The results
Respiration and Electron Transport. In earlier work,21,22
we showed that several MenA inhibitors, analogues of Ro 48-
8071, blocked respiration in M. tuberculosis and M. smegmatis
(as evidenced by inhibiting the reduction of methylene blue),
that there was a correlation between cell growth inhibition and
respiration inhibition,22 and that the effects of the inhibitors
could (at least in part) be reversed by adding MK-4 at the 400
μM level. We thus next tested all compounds for their effects
on methylene blue reduction, in M. smegmatis (Figure S3,
Supporting Information), finding that there was a moderate
correlation between pMIC (= −log10 MIC, MIC in μM) for cell
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obtained with SF6847, one of the most potent uncouplers
known, are shown in Figure 5A and indicate a very rapid
We found similar PMF effects in intact M. smegmatis cells in
which there was a collapse in Δψ (positive outside), as
measured by using DisC3(5) fluorescence, Figure S7,
Supporting Information. Addition of 1 or the potent analogues
to M. smegmatis cells resulted in an immediate increase in
DisC3(5) fluorescence, indicating a collapse of Δψ. As can be
seen in Figure S7B, Supporting Information, 1 collapses the
membrane potential in a dose-dependent manner with an
“EC50” of ∼20 μg/mL. The EC50 for one of the most potent
cell growth inhibitors 13 was ∼15 μg/mL.
In addition to these investigations of Δψ collapse in intact
cells, we investigated ΔpH collapse in intact M. smegmatis cells,
using 31P NMR spectroscopy. Phosphorus NMR chemical
shifts are sensitive indicators of local pH values.34 As can be
seen in Figure S8A, Supporting Information, the 31P NMR
chemical shift of phosphate inside M. smegmatis is ∼0.35 ppm
downfield from external Pi, and from these chemical shifts, the
internal and external pH values can be determined: results are
shown in Figure S8B, Supporting Information. There is a ΔpH
= 0.26 (inside more basic) in wild type M. smegmatis cells, but
this pH gradient is collapsed by the uncoupler CCCP (m-
chlorophenylcarbonyl cyanide phenylhydrazone), by the
antiporter nigericin, and by 1 and 13, while as expected, the
K+ ionophore valinomycin has no effect. The effects of 1 and
the other potent analogue thus leads to collapse of Δψ as well
as ΔpH in both inverted vesicles and in whole cells. This
collapse in the PMF, in cells, can be expected to result in an
inhibition of ATP synthesis, as is indeed found experimentally
(Figure 5D) with the potent lead, 3. In addition, the collapse of
the proton motive force is expected to inhibit activity of the
MmpL3/TMM transporter.
Figure 5. (A) ΔpH collapse in E. coli IMVs by a known uncoupler
SF6847. (B) ΔpH collapse in E. coli IMVs by 1 and 5. (C) Effects of 1
and analogues on Δψ in M. smegmatis cells. (D) Effects of 3 on ATP
biosynthesis of effect in M. smegmatis cells.
(seconds) collapse in ΔpH, as reported by Haagsma et al.33
The same effect was seen with 1 and analogues that have potent
activity in cell growth inhibition, while inactive (diether/amide)
analogues (e.g., 5) had no effect, Figure 5B, Figures S4 and S5,
Supporting Information. Similar results were obtained with
both E. coli and M. smegmatis vesicles. The effects on the
collapse in ΔpH were seen in vesicles in which the pH gradient
was driven by either ATP hydrolysis or by electron transport in
the presence of succinate or NADH. Using Oxonol VI as a
probe, we also found that Δψ in E. coli IMVs (positive inside)
was collapsed (Figure S6A) by the same compounds, and there
was a correlation between the collapse of the membrane
potential and the collapse in ΔpH (using ACMA fluorescence;
Table 2; R2 = 0.79, Figure S6B, Supporting Information). This
is consistent with these lipophilic cations acting as proto-
nophores, carrying protons across the membrane lipid bilayer,
with only compounds containing a basic nitrogen supporting
the uncoupling activity.
Quantitative Models for Cell Growth Inhibition. Any
quantitative analysis of cell growth inhibition based on enzyme
inhibition or another property (e.g., ΔpH collapse) is of course
challenging, but it should be possible to use the multidescriptor
approach described previously35 with, in this case, no purely
mathematical descriptors being required. We thus use eq 1:
pIC50(cell) = a × pIC50(A) + b × pIC50(B) + c
(1)
where pIC50(A) is −log10 (IC50, A) for enzyme or property A
(such as MenA inhibition), and B is a second property, e.g.,
ΔpH collapse. We show by way of examples in Figures 6A,B,
three-dimensional plots for E. coli cell growth inhibition: log
IC50 = f (MenA, ΔpH), and for M. smegmatis: log MIC = f
(respiration, Δψ), where we find correlation coefficients of R2 =
Figure 6. Experimental (red circles) and computed (colored plane) results for cell growth inhibition based on eq 1. (A) E. coli cell growth inhibition
predicted using MenA, ΔpH collapse (IC50s in μM, R2 for the model = 0.77). (B) M. smegmatis growth inhibition using respiration (methylene blue
assay) and Δψ collapse (MIC and IC50s in μM, R2 for the model = 0.64).
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0.82 (E. coli) and R2 = 0.72 (M. smegmatis) values for the
experimental-versus-predicted cell growth inhibition results. In
previous work we investigated correlations between cell and
enzyme activity (pIC50) assays in 10 diverse systems finding35
on average an R2 = 0.32 for the 10 cell/enzyme correlations,
similar to the R2 = 0.43 we find for the MenA alone/E. coli cell
growth inhibition correlation. Incorporation of the second
parameter (the percentage of ΔpH collapse) increases the R2 to
0.77, suggesting the importance of multitargeting, in E. coli. The
correlation is worse for M. smegmatis due, perhaps, to the
omission of an MmpL3 term, expected to be particularly
important in the mycobacteria.
Are There Other MenA-like Targets? The results
presented above show that E. coli MenA is inhibited by 1 and
its analogues and that there is a correlation between MenA
inhibition and cell growth inhibition (in M. smegmatis),
suggesting that diverse MenAs may be inhibited by these
compounds. However, this result is perhaps surprising in that in
E. coli, MenA is not an essential gene for aerobic growth
because UbiA can be used in aerobic respiration. One
possibility is that EcUbiA might also be inhibited by 1 (and
its analogues). While we have not yet investigated this
experimentally, what we have found is that MtMenA, EcMenA,
and EcUbiA are all predicted (using the Phyre2 program) to
have an FPPS/GGPPS-like structure.
In all three cases the structures are predicted to contain a
central FPPS/GPPS-like catalytic domain (comprising ∼2/3 of
the overall amino acid sequence) that has close similarity to the
same two (soluble) prenyl synthases: Methylococcus capulatus
FPPS and Lactobacillus brevis GGPPS. Predicted sequence
identity investigations have shown that MenA and UbiA have
moderate homology,36,37 but correlations with FPPS and
GGPPS were not made in those studies because the actual
sequence identities are very low, about 10−16%. However,
secondary-structure-based algorithms do permit accurate
structure predictions, even when residue identities are low.
These previous bioinformatics studies also demonstrated that
another class of proteins, protoporphyrin IX farnesyl trans-
ferases (e.g., heme o synthase), have significant sequence
homology to the MenA/UbiA proteins, and all three classes of
proteins are Mg2+-dependent prenyl transferases. Once again,
the structure of heme o synthase is not known but is predicted
to be another nine-helix transmembrane protein with a central
FPPS/GGPPS-like core, suggesting that MenA, UbiA, and
protoporphyrin IX farnesyl transferases are all likely to be
inhibited by 1 and related systems.
MmpL3 and MmpL11 as Targets. MmpL3 is thought to
be a target for 1 (and other diverse inhibitors12,13,38,39),
blocking TMM transport. It has also been shown that MmpL3
together with a related protein, MmpL11, is associated with
heme uptake.40,41 The X-ray structures of MmpL3 and
MmpL11 have not been reported. However, both are
membrane proteins and are predicted to have 11−12
transmembrane helices.24 Using the Phyre2 program,25 we
find with MmpL3 that 653 residues (69% coverage) are
predicted with 100% confidence to have the structure shown in
Figure 7A and with MmpL11, 642 residues (66% coverage) are
predicted with 100% confidence to have the structure shown in
Figure 7B. Both structures are very similar to those found in
cation efflux pumps such as CusA (PDB ID 3ko7) and
multidrug efflux pumps such as the acriflavin resistance protein
B (AcrB; PDB ID 1oy8), although the C-terminus (∼1/3 of the
total protein) is not modeled in either MmpL3 or MmpL11.
Figure 7. Molecular models for MmpL3, MmpL11. (A) Phyre2
structure predictions for MmpL3. (B) Phyre2 structure predictions for
MmpL11. (C) Phyre2 predictions showing hydrophobic residues
(white/gray) and their proposed relation to the membrane for
MmpL3. (D) Phyre2 predictions showing hydrophobic residues
(white/gray) and their proposed relation to the membrane for
MmpL11. (E) Structure of representative M. tuberculosis growth
inhibitors that are thought to target MmpL3 and (F) sites of resistance
mutations (blue spheres) in MmpL3.
The transmembrane hydrophobic domains are shown in Figure
7C,D (in white/light orange). 1 as well as several other
inhibitors12,13 (Figure 7E) has been proposed to target MmpL3
(detected by sequencing mutants that arose under drug
pressure), but the sites of these mutations, shown as blue
spheres in Figure 7F, are spread throughout the protein,
suggesting, perhaps, multisite targeting of MmpL3/11 as an
additional basis for the lack of resistant mutations with 1.
Overall, however, the effects of 1 on the PMF and respiration,
the menaquinone-reversal experiments, activity against diverse
organisms as well as the ability to make generally good
predictions of cell activity without MmpL3 inhibition data
suggests that MmpL3 may not be the primary target for 1, in M.
tuberculosis. In addition, of course, other targets may exist.
A Multitarget Model for Antiinfective Activity. We
show in Figure 8 a summary of the proposed sites of action for
1 and its analogues in M. tuberculosis and in M. smegmatis. Some
of these targets are also present in the other pathogens
investigated but not in human cells. In addition to its previously
proposed role in targeting MmpL3, 1 and its analogues also
inhibit MenA and MenG and, as described above, the inhibition
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Figure 8. Proposed sites of action of SQ109 and its analogues. MenA, MenG targeting can affect respiration/electron transfer; PMF (ΔpH, Δψ)
collapse leads to decreased ATP biosynthesis, reduction in PMF/ATP-powered transporters (e.g., MmpL3), increased TMM accumulation, and
decreased cell wall biosynthesis.
of M. tuberculosis cell growth or activity is rescued by MK-4. We
also find that the PMF is inhibited by the most active
compounds, which act as protonophores/uncouplers. This
results in a decrease in ATP synthesis and, we propose,
decreased activity of MmpL3/11, helping explain the
accumulation12 of TMM (with 1).
This multiple-targeting is perhaps best thought of as
involving network inhibition in which both series and parallel
paths are involved (Figure 1C) because at least in the
mycobacteria, MenA, MenG, electron transport, ΔpH, Δψ,
and MmpL3 (and presumably other pumps dependent on the
PMF) can all be affected. There are, of course, likely to be
differences in the mechanisms of action of different inhibitors in
different organisms (and in the same organisms under different
growth conditions), although effects on the PMF are expected
to be quite common because they are based on more “physical”
properties, rather than purely enzyme inhibition. The
uncoupling effects we observe could also help explain the
growth inhibition seen in human cell lines, as could inhibition
of the human MenA, UbiA, and MenG/UbiE orthologues:
UbiAD1, CoQ2, and CoQ3.
Also of interest are the likely differences in time scales (and
concentrations) for the different reactions involved. The effects
on the collapse in Δψ and ΔpH are very rapid-on the seconds
to minutes time scale and are observed (in vesicle experiments)
at low μM concentrations, for the most active species. The
effects on respiration as determined by methylene blue
reduction (in intact cells) are also rapid, typically observable
in minutes, and may reflect the time required for inhibitors to
enter the cell and accumulate (because they could also be
actively pumped out). Little is known about the rate of
menaquinone turnover, but it is likely that several cell divisions
are required for a large reduction in menaquinone levels, so
while MenA/MenG inhibition may be rapid, the effects on cell
growth may take many hours or (with M. tuberculosis) days to
occur likewise, because MmpL3 is thought to be involved in cell
wall biosynthesis, its inhibition would also be expected to result
in observable effects on growth inhibition on a time scale of
hours to days.
provide cationic, protonatable, and neutral species, and in
addition we varied the adamantyl headgroup. The most active
compound against M. tuberculosis was ∼5× more potent than
was 1 and was also less toxic to an MCF-7 human cell line. We
tested all compounds against a panel of bacteria, fungi, and a
protozoan parasite, and the results obtained showed that at
least one cationic (or basic) group was essential for activity.
The most potent activity was against M. tuberculosis (MIC =
0.02−0.05 μg/mL) and the intraerythrocytic form of the
malaria parasite, P. falciparum (IC50 = 30 ng/mL). To explore
possible targets, we tested several compounds for activity
against a panel of cis- and trans-prenyl transferases (cis-FPPS,
FPPS, DPPS, GGPPS, UPPS, CrtM, and SQS) as well as
against the menaquinone biosynthesis enzymes, MenA and
MenG. Activity was seen against MenA and MenG, and we
proposed a structural model for the MenA active site, as well as
a likely binding site for 1. In addition, we found that
menaquinone (MK-4) rescued both aerobic H37Rv M.
tuberculosis cell growth and the activity of nonreplicating M.
tuberculosis (streptomycin-starved 18b). We found that 1 as well
as several analogues inhibited oxygen consumption in M.
smegmatis, and there was a correlation between oxygen
consumption and cell growth inhibition. We tested 1 and
each of the 11 analogues for their effects on the PMF (ΔpH
and Δψ) in fluorescence-based assays, as well as in some cases
in intact cells (via 31P NMR). The results obtained showed that
1 and the most potent cell growth inhibitors collapsed both Δψ
and ΔpH, and there were good correlations between
experimental and predicted cell growth inhibition results
based on MenA/ΔpH (E.coli) and respiration/Δψ (M.
smegmatis). Taken together, the results obtained suggested a
model for 1/analogue activity in mycobacteria in which the
increase in TMM levels seen on treatment with 1 have a
contribution from (indirectly) inhibiting the TMM transporter
MmpL3 by blocking the PMF/ATP biosynthesis. Overall, the
results are of general interest because they indicate that 1 (and
its analogues) can have diverse effects: on O2-consumption/
electron transport/MenA/MenG inhibition; on Δψ, ΔpH, and
ATP biosynthesis, likely helping to explain activity against non-
MmpL3-containing pathogens such as H. pylori, C. albicans,
and, here, P. falciparum. Moreover, the possibility of developing
more potent compounds that can inhibit these targets is of
general interest in the context of developing drug leads that are
“resistance resistant”, due to multitargeting.
CONCLUSIONS
■
The results we have described above are of interest for drug
discovery against tuberculosis, as well as against other bacterial,
fungal, and protozoan pathogens, for several reasons. We
synthesized a series of analogues of the antituberculosis drug 1
in which we varied the nature of the ethylenediamine linker to
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(Amersham Biosciences). IC50 values were calculated by using GraFit
Software (Version 5.0.13).
EXPERIMENTAL SECTION
■
Chemical Syntheses: General Methods. All chemicals were
reagent grade and were used as received. Moisture-sensitive reactions
were performed under an inert atmosphere (dry nitrogen) with dried
solvents. Reactions were monitored by TLC using Merck silica gel 60
F-254 thin-layer plates. Flash column chromatography was carried out
Expression and Purification of EcMenA. The gene encoding
EcMenA with a N-terminal strep tag was amplified by polymerase
chain reaction (PCR) with forward primer 5′-GACGACGACAA-
G A T G A G C G C G T G G A G C C A T C C G C A G T T T -
GAAAAAGGCGGTGGCAGCGCGGAGAATCTTTATTTT-
CAGGGCGCTGGTGC-3′ and reverse primer 5′-GAGGAGAAG
CCCGGTTATTATGCTGCCCACTGGCTTAGGAATAT-3′, and
then cloned into the pET46 Ek/LIC vector. The recombinant plasmid
was transformed to E. coli C43 (DE3) and the protein induced with 1
mM isopropyl thiogalactopyranoside (IPTG) at 37 °C for 5 h. The cell
paste was harvested by centrifugation at 7000g and resuspended in
buffer A containing 25 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 20
mM imidazole. A cell lysate was prepared with a JNBIO pressure cell
(JN-3000 PLUS), the membrane, and soluble proteins being separated
by ultracentrifugation at 150 000g for 1.5 h. The resulting pellet was
solubilized by incubation in buffer A supplemented with 1% (w/v)
DDM detergent overnight at 4 °C. The latter solution was centrifuged
(100 000g for 1 h at 4 °C in a Beckman Ti70 rotor) and the
supernatant loaded onto a Ni-NTA column and washed with buffer A
containing 0.05% DDM. The buffer and gradient for the Ni-NTA
column were 25 mM Tris, pH 7.5, 150 mM NaCl, 0.05% DDM, and
20−500 mM imidazole. The protein was then loaded onto a Strep-
Tactin (IBA) column equilibrated with washing buffer containing 100
mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, and 0.05% DDM
and washed with five column volumes of washing buffer. EcMenA was
finally eluted with eluting buffer containing 100 mM Tris-HCl, pH 8.0,
150 mM NaCl, 1 mM EDTA, 0.05% DDM, and 2.5 mM desthiobiotin.
The purified protein was finally concentrated to 5 mg mL−1 in a 25
mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.05% DDM buffer.
on Merck silica gel 60 (230−400 mesh). H NMR and 13C NMR
1
spectra were recorded on Varian (Palo Alto, CA) Unity spectrometers
at 400 and 500 MHz for 1H and at 100 and 125 MHz for 13C.
Coupling constants (J) are reported in hertz. High-resolution mass
spectra (HRMS) were recorded in the University of Illinois Mass
Spectrometry Laboratory. Elemental analyses were carried out in the
University of Illinois Microanalysis Laboratory. HPLC/MS was
performed using an Agilent LC/MSD Trap XCT Plus system (Agilent
Technologies, Santa Clara, CA) with an 1100 series HPLC system
including a degasser, an autosampler, a binary pump, and a multiple-
wavelength detector. All final compounds were ≥95% pure as
determined by elemental analysis, analytical HPLC/MS analysis, or
qNMR analysis. qNMR spectra were recorded using Varian (Palo Alto,
CA) 500 MHz Unity spectrometers with 1,3,5-trimethoxybenzene as
the internal total-spin-count quantitation standard; 60° pulse
excitation, 60 s recycle delay, 1.0 Hz line-broadening due to
exponential multiplication, and 16 accumulations. qNMR data were
processed using Mnova NMR software (Mestrelab, Escondido, CA).
All NMR spectra (including qNMR spectra) are provided in the
Supporting Information.
Enzyme Inhibition Assays. MenA and MenG Inhibition. MenA
and MenG inhibition assays were carried out using M. smegmatis
membrane fragments.22 Mycobacterial MenA assays were conducted
as previously reported.22 In addition, we used an expressed, purified E.
coli MenA, as described below.
EcMenA Inhibition Assay. Inhibition of EcMenA was carried out
using an HPLC-based protocol. Typically, 2.5 μg of EcMenA in 100
μL of reaction buffer (25 mM Tris-HCl, 0.1% Triton X-100, 250 μM
MgCl2, 10 mM DTT, pH = 7.5) was incubated with inhibitors for 30
min at 22 °C. 1,4-Dihydroxy-2-naphthoic acid (DHNA) and farnesyl
diphosphate (FPP) were then added to the enzyme solution to a final
concentration of 150 μM each. The reaction was incubated at 37 °C
for 3 h before quenching with 50 μL of 0.1 M acetic acid in methanol
containing 50 μM menaquinone-4 (MK-4, Sigma-Aldrich) as an
internal standard. The mixture was then extracted with 600 μL of
hexane by vortexing. After centrifugation, 500 μL of organic layer was
collected and dried under nitrogen then dissolved in 200 μL of
methanol. Twenty microliters of the methanol solution was then
subjected to HPLC analysis (0.1% formic acid in H2O to 0.1% formic
acid in CH3CN, UV: 325 nm, 250 μL/min). The amount of the MenA
reaction product demethylmenaquinone-3 (DMMK-3) was deter-
mined by comparison of integrated peak areas between DMMK and
the internal standard MK-4. IC50 values were estimated by using
Origin 6.1 software to analyze the dose−response curves.
Cell Lines. Mycobacterium tuberculosis ATCC 27294, Mycobacterium
smegmatis ATCC 700084, Bacillus subtilis subsp. subtilis ATCC 6051,
E. coli ATCC 29425, and Saccharomyces cerevisiae ATCC 208352 were
purchased from the American Type Culture Collection. The C.
albicans strain was CAI-4; the P. falciparum strain was 3D7 and the
human cell line MCF-7 (breast adenocarcinoma), obtained from the
National Cancer Institute.
M. tuberculosis Growth Inhibition Assay. All 12 compounds (1,
3−13) were assayed for inhibition of M. tuberculosis cell growth as
described previously.42
MenG Assay. Vitamins K1 and K2 and kanamycin were purchased
from Sigma-Aldrich (St. Louis, MO). Authentic MK9 was purchased
from Toronto Research Chemicals (TRC, Canada). S-Adenosyl-L-
[methyl-14C]methionine (14C-SAM) obtained from Perkin-Elmer (47
mCi/mmol). DMK8 was prepared from an E. coli ΔubiE mutant
(CGSC #11636), which accumulates DMK8, and was purchased from
the E.coli Genetic Stock Center, Yale University (http://cgsc.biology.
yale.edu).
MenG assays were conducted using the membrane fractions
prepared from M. smegmatis grown in 7H9 medium (supplemented
with oleic acid, albumin, dextrose, and 0.05% Tween 80). Washed cells
were resuspended in buffer A (50 mM MOPS pH 7.9, 5 mM MgCl2, 5
mM DL-dithiothreitol (DTT), 10% glycerol (V/V)) and disrupted by
probe sonication on ice with a Sanyo Soniprep 150 (10 cycles of 60 s
on and 90 s off). The whole cell lysate was centrifuged at 27 000g for
20 min at 4 °C. The supernatant was further centrifuged at 100 000g
(for 2 h at 4 °C) in an Optima TLX Ultracentrifuge (Beckman). The
membrane-enriched pellet was washed with buffer A followed by
ultracentrifugation at 100 000 g. The washed pellet was resuspended in
buffer A, divided into aliquots, and frozen at −80 °C. The membrane
protein concentration was estimated by using a BCA protein assay kit
(Pierce).
Assay mixtures (100 μL) contained 100 mM Tris-HCl pH 8.0, 1
mM DTT, 5 mM MgCl2, 0.1% CHAPS, 600 ng of DMK8, 40 μM
radiolabeled SAM, and varying concentrations of inhibitor 1 (0 to 25.0
μg/mL). Reactions were initiated by the addition of 50−100 μg of M.
smegmatis membrane protein and incubated at 37 °C for 1 h. Reactions
were stopped by the addition of 0.1 M acetic acid in methanol (0.5
mL), and radiolabeled products were extracted with hexane (2 × 3
mL). Pooled extracts were washed with 1 mL of water, evaporated to
dryness under a N2 stream, and dissolved in CHCl3/CH3OH (2:1 v/
v). An aliquot was subjected to liquid scintillation counting (LS 6500,
Beckman Coulter); a second aliquot and authentic standards (DMK8
and MK9) were subjected to reverse-phase TLC (Whatman KC 18F
Silica gel 60 A) developed in acetone/water (97:3). Standards were
visualized under UV light, and distribution of radioactivity was
detected by phosphorimaging (Typhoon TRIO, Amersham Bio-
sciences) and quantified with ImageQuant TL v2005 software
Menaquinone Rescue Experiments with M. tuberculosis
Treated with 1. We measured the activity of 1 against both actively
growing M. tuberculosis (H37Rv) and nonreplicating M. tuberculosis
(streptomycin-starved 18b28) using a resazurin microplate reduction
assay. The effects of menaquinone supplementation on the dose−
response curves were investigated using medium that was
supplemented with 0, 10, 100, and 1000 μM menaquinone (MK-4,
Sigma-Aldrich) in the presence of between 10 ng/mL and 10 μg/mL
1. The activity of 1 against nonreplicating 18b was determined after 7
days of drug exposure by plating the culture followed 28 days later by
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CFU counting after plating serial dilutions on 7H10 agar plates
(Difco) .
Candida albicans Growth Inhibition Assay. C. albicans growth
inhibition was carried out according to a reported protocol43 except
that YPD media was used instead of RPMI 1640.
cyanine (DisC3(5)). M. smegmatis were grown for 8 h in Middlebrook
7H9-ADC-Tween 80 medium and diluted to an OD600 of 0.3 in the
same medium plus 10 mM glucose and 1 μM nigericin. Different
concentrations of 1 and its analogues were added to the bacterial
suspension, and changes in fluorescence due to the disruption of Δψ
were continuously monitored with a fluorescence spectrophotometer
(FLUOstar Omega, BMG LABTECH) employing an excitation
wavelength of 643 nm and an emission wavelength of 666 nm, at
30 °C.
ATP/ADP Determination. M. smegmatis were grown for 8 h in
Middlebrook 7H9-ADC-Tween 80 and diluted to an OD600 of 2.
Different concentrations of 1 and its analogues were added and ATP/
ADP ratios determined (Abcam; ADP/ATP Ratio Assay Kit, catalog
number: ab65313) after 10 and 60 min of incubation at 37 °C, 200
rpm. ATP and ADP were extracted from 50 μL of cell suspension by
adding trichloroacetic acid (TCA) to a final concentration of 0.5%.
After 5 min, TAE (Tris−acetic acid−EDTA) buffer was added to
neutralize the system by diluting the sample 5-fold. The ATP and ADP
cell concentrations were measured according to the manufacturer’s
protocol.
Inverted Membrane Vesicles (IMVs). E. coli IMVs were prepared
by three passages through a precooled French pressure cell at 20 000
psi. The lysate was centrifuged at 14 000g at 4 °C for 20 min to
remove unbroken cells. The supernatant was centrifuged at 370 000g
at 4 °C for 1 h, and the pellet, consisting of the IMVs, was washed with
50 mM MOPS−KOH (pH 7.5), 2 mM MgCl2. After the second
centrifugation step, membranes were resuspended in 50 mM MOPS−
KOH (pH 7.5), 2 mM MgCl2, 10% glycerol, and stored at −80 °C.
Assay for ATP or Succinate-Driven Proton Translocation.
Proton translocation into IMVs was measured by the decrease of
ACMA fluorescence. The excitation and emission wavelengths were
410 and 480 nm, respectively. IMVs (0.1 mg/mL membrane protein)
were preincubated at 37 °C in 10 mM HEPES−KOH (pH 7.5), 100
mM KCl, 5 mM MgCl2 containing 2 μM ACMA, and the baseline was
monitored for 5 min. The reaction was then initiated by adding 1 mM
ATP or 5 mM succinate. When the signal had stabilized, 1 or its
analogues were added and proton translocation was measured,
fluorimetrically.
Determination of Δψ Collapse in IMVs. The Δψ-sensitive
fluorophore Oxonol VI (1,5-bis(5-oxo-3-propylisoxazol-4-yl)-
pentamethine oxonol) was used to determine if 1 and its analogues
were able to dissipate the membrane potential in IMVs. IMVs (0.1
mg/mL membrane protein) were added to assay buffer: 10 mM
MOPS−KOH pH 7.5, 2 mM MgCl2, 2 μM Oxonol VI. After a few
seconds, 0.5 mM NADH was added to initiate respiration-dependent
generation of Δψ (positive inside), and the resultant quenching of
Oxonol VI fluorescence was monitored at 37 °C. The emission and
excitation wavelengths were 599 and 634 nm, respectively. Uncoupling
by inhibitors was estimated based on their ability to dissipate the
established Δψ, measured as the dequenching of the fluorescence
signal.
E. coli Growth Inhibition Assay. IC50 values for E. coli growth
inhibition were determined by using a broth microdilution method. An
overnight culture of E. coli was diluted 50-fold into fresh Luria−Bertani
(LB) broth and incubated to an OD600 of ∼0.4. The culture was then
diluted 500-fold into fresh LB medium and 100 μL inoculated into
each well of a 96-well flat-bottom culture plate (Corning Inc., Corning,
NY). The starting concentration of each compound was 0.3 mM, and
this was 2× serially diluted to 292 nM. Plates were incubated for 3 h at
37 °C to midexponential phase. An MTT ((3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide) cell proliferation assay (ATCC)
was then carried out to obtain bacterial viability dose−response curves.
Briefly, 10 μL of MTT reagent was added into each well, followed by
incubation for 2−4 h until a purple precipitate was visible. Then, 100
μL of detergent reagent was added, and the plates were incubated in
the dark at 22 °C for 2 h. Absorbance was measured at 570 nm and a
nonlinear regression analysis carried out using Origin 6.1 software.
B. subtilis Growth Inhibition Assay. IC50 values for B. subtilis
growth inhibition were determined by using a microbroth dilution
method. A stationary starter culture of B. subtilis was diluted 50-fold
into fresh LB broth and grown to an OD600 of ∼0.4. The culture was
then diluted 500-fold into fresh LB medium to give a working solution,
and then 100 μL of working solution was transferred to each well of a
96-well flat-bottom culture plate (Corning Inc., Corning, NY).
Inhibitors were then added at 0.5 mM and 2× serial diluted to 500
nM, the volume and solvent composition constant. Plates were
incubated for 12−16 h at 37 °C, and the absorbance at 600 nm was
determined. A nonlinear regression analysis was carried out using
Origin 6.1 to obtain the IC50 values.
S. cerevisiae Growth Inhibition Assay. The protocol was the
same as for B. subtilis except that YPD instead of LB was used as the
culture medium, and the 96-well plates were incubated for 48 h instead
of 12−16 h.
Plasmodium falciparum Growth Inhibition Assay. We
determined IC50 values for P. falciparum growth inhibition using the
intraerythrocytic assay described previously.44
Human Cell Growth Inhibition Assay. The MCF-7 cell growth
inhibition assay was carried out as described previously.45 A broth
microdilution method was used to determine the growth inhibition
IC50 values. Compounds were half-log serial diluted using cell culture
media into 96-well TC-treated round-bottom plates (Corning Inc.,
Corning, NY). Cells were plated at a density of 5000 cells/well and
then incubated under the same culture conditions for 2 days at which
time an MTT ((3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide) cell proliferation assay (ATCC, Manassas, VA) was
performed to obtain dose−response curves.
Dehydrogenase Activities. Dehydrogenase activity in M.
smegmatis membranes was measured by using the MTT ((3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reduction
assay in the presence of 5 mM KCN. MTT reduction was followed
at 570 nm, after addition of the different substrates (NADH, succinate,
malate, or lactate).
Determination of ΔpH by 31P NMR Spectroscopy. M.
smegmatis was grown to a cell density of 108 cells/mL in a total
volume of 500 mL in a 4 L Erlenmeyer flask with constant shaking at
37 °C in Difco Middlebrook 7H9 media supplemented with oleic
acid/albumin/dextrose and 0.05% Tween 80. Cells were harvested by
centrifugation, and the pellet was washed twice with 5 mM phosphate
buffer, pH 6.8. The cell pellet was then resuspended in 200 μL of the
same buffer and 500 μL of the resulting cell slurry transferred to a 5
mm NMR tube. Chemical shifts were referenced with respect to 85%
phosphoric acid in D2O in a coaxial capillary. 31P NMR spectra were
obtained using a Varian INOVA 300 (at 121.5 MHz) using 60° pulse
excitation, proton decoupling, and a 1 s recycle time. A total of 1024
scans were accumulated corresponding to approximately a 60 min total
data acquisition time (without aeration). Spectra were analyzed as
described elsewhere.46 The peak corresponding to the α-phosphate of
ATP (at ∼−10.5 ppm) and the inorganic phosphate peaks of interest
(in the region of 0−1.5 ppm) were used to calculate the internal and
external pH using the following equation, where d is the distance
Oxygen Consumption. Oxygen concentration was monitored at
37 °C using a YSI model 53 oxygen electrode (Yellow Springs
Instrument Co., Yellow Springs, OH) equipped with a temperature-
controlled 1.8 mL electrode chamber. The reaction mixture consisted
of sodium phosphate buffer, pH 7.5, 50 mM NaCl, and 200−400 μg/
mL membranes. The concentration of oxygen in the air-saturated
buffer was taken to be 250 μM, and the reaction was initiated by
injecting 200 μM NADH. The electron transport rates are expressed as
mol of NADH oxidized or mol of O2 (mol enzyme)−1 s−1. Membranes
were incubated with different concentrations of inhibitors for 5 min
prior to NADH addition.
Membrane Potential Measurements in Intact Cells. The
effects of inhibitors on Δψ were determined by fluorescence
quenching of the potential-sensitive probe 3,3′-dipropylthiodicarbo-
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between the α-phosphate of ATP and the inorganic phosphate peak, in
ppm.
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d − 10.85
pH = 6.75 + log
13.25 − d
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ASSOCIATED CONTENT
* Supporting Information
■
S
Additional table and figures illustrating SCORECONS analysis
of MtMenA, MenA/MenG inhibition, methylene blue reduc-
tion assay, Δψ dissipation, NMR measurement of ΔpH, ACMA
fluorescence results, synthesis schemes and protocols, com-
pound characterization and purity data, and NMR (including
qNMR) spectra. This material is available free of charge via the
Internet at http://pubs.acs.org.
(8) Sacksteder, K. A.; Protopopova, M.; Barry, C. E.; Andries, K.;
Nacy, C. A. Discovery and development of SQ109: a new
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E. Nitrogen-containing bisphosphonates as carbocation transition state
analogs for isoprenoid biosynthesis. Biochem. Biophys. Res. Commun.
1999, 263, 754−758.
AUTHOR INFORMATION
Corresponding Author
*Tel: 217-333-3374. Fax: 217-244-3186. E-mail: eoldfiel@
illinois.edu.
■
Author Contributions
&These authors contributed equally.
Notes
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Van Beek, E.; Papapoulos, S.; Wang, A. H.; Kubo, T.; Ochi, M.;
Mukkamala, D.; Oldfield, E. Lipophilic bisphosphonates as dual
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Chen, P.; Gearhart, J.; Einck, L.; Nacy, C. A. Identification of a new
antitubercular drug candidate, SQ109, from a combinatorial library of
1,2-ethylenediamines. J. Antimicrob. Chemother. 2005, 56, 968−974.
(12) Tahlan, K.; Wilson, R.; Kastrinsky, D. B.; Arora, K.; Nair, V.;
Fischer, E.; Barnes, S. W.; Walker, J. R.; Alland, D.; Barry, C. E.;
Boshoff, H. I. SQ109 targets Mmpl3, a membrane transporter of
trehalose monomycolate involved in mycolic acid donation to the cell
wall core of Mycobacterium tuberculosis. Antimicrob. Agents Chemother.
2012, 56, 1797−1809.
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Javid, B.; Sorrentino, F.; Ioerger, T. R.; Sacchettini, J. C.; Manetti, F.;
Botta, M.; De Logu, A.; Rubin, E. J.; De Rossi, E. MmpL3 Is the
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The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the United States Public Health
Service (National Institutes of Health grants GM065307,
CA158191, HL016101, and AI049151), the National Basic
Research Program of China (grants 2011CB710800 and
2011CBA00805), the National Research Foundation of Korea
(NRF) funded by the Korean government (MSIP, no. 2007-
00559), Gyeonggi-do (no. K204EA000001-09E0100- 00110),
and KISTI, and by a grant from the NIH Director’s New
Innovator Award Program (DP2 OD008463 to D.A.M.), the
European Community’s Seventh Framework Programme
(Grant 260872, S.T.C.), and the Fondation Jacqueline Beytout
(B.L.) X.F. was an American Heart Association, Midwest
Affiliate, Predoctoral Fellow (grant 13PRE14510056).
ABBREVIATIONS USED
■
UPPS, undecaprenyl diphosphate synthase; FPPS, farnesyl
diphosphate synthase; GGPPS, geranylgeranyl diphosphate
synthase; CrtM, S. aureus dehydrosqualene synthase; MK,
menaquinone; MenA, 1,4-dihydroxy-2-naphthoate polyprenyl
transferase; MenG, 2-polyprenyl-1,4-naphthoquinone methyl-
transferase; TMM, trehalose monomycolate; IC50, half maximal
inhibitory concentration; MIC, minimum inhibitory concen-
tration required to inhibit the growth of 90% of organisms;
PMF, proton motive force; Mt, M. tuberculosis; Ms, M.
smegmatis; Sa, S. aureus; Bs, B. subtilis; Ec, E. coli; Sc, S.
cerevisiae; Ca, C. albicans; Pf, P. falciparum; MTT, 3-(4,5-
dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide;
IMV, inverted membrane vesicles; TI, therapeutic index;
CCCP, m-chlorophenylcarbonyl cyanide phenylhydrazone;
ACMA, 9-amino-6-chloro-2-methoxyacridine; TLC, thin layer
chromatography; THF, tetrahydrofuran
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