Small Molecules Targeting Mycobacterium tuberculosis Type II NADH Dehydrogenase Exhibit Antimycobacterial Activity

Article abstract of DOI:10.1002/anie.201800260

The generation of ATP through oxidative phosphorylation is an essential metabolic function for Mycobaterium tuberculosis (Mtb), regardless of the growth environment. The type II NADH dehydrogenase (Ndh-2) is the conduit for electrons into the pathway, and

Full text of DOI:10.1002/anie.201800260

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Accepted Article
Title: Small molecules targeting Mycobacterium tuberculosis type II
NADH dehydrogenase with antimycobacterial activity
Authors: Michael B Harbut, Renhe Liu, Baiyuan Yang, Takahiro Yano,
Catherine Vilcheze, Jonathan Lockner, Hui Guo, Scott G
Franzblau, Mike Petrassi, William R Jacobs, Harvey Rubin,
Arnab K Chatterjee, and Feng Wang
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201800260
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Small molecules targeting Mycobacterium tuberculosis type II
NADH dehydrogenase with antimycobacterial activity
Michael B. Harbut, Baiyuan Yang, Renhe Liu, Takahiro Yano, Catherine Vilchèze, Bo Cheng, Jonathan
Lockner, Hui Guo, Chenguang Yu, Scott G Franzblau, H. Mike Petrassi, William R. Jacobs Jr., Harvey
Rubin, Arnab K. Chatterjee, and Feng Wang*
Abstract: Generation of ATP via oxidative phosphorylation is an
essential metabolic function for Mycobaterium tuberculosis (Mtb)
regardless of the growth environment. The type II NADH
dehydrogenase (Ndh-2) is the conduit for electrons into the pathway,
and is absent in the mammalian genome, making it a potential drug
target. Herein, we report the identification of two types of small
molecules as selective inhibitors for Ndh-2 via a multi-component
high-throughput screen. Both compounds block ATP synthesis, lead
to effects consistent with loss of NADH turnover, and importantly,
exert bactericidal activity against Mtb. Extensive medicinal chemistry
optimization afforded the best analog with an MIC of 90 nM against
Mtb. Moreover, the two scaffolds have differential inhibitory activities
against the two homologous Ndh-2 enzymes in Mtb, which will allow
precise control over Ndh-2 function in Mtb to facilitate assessment of
this anti-TB drug target.
validates oxidative phosphorylation as a pharmacologic target in
Mtb.[3]
Electrons enter the respiratory chain from NADH oxidation,
coupled to the reduction of menaquinone. The Mtb genome
encodes both type
I
(nuoA-N) and two type II
NADH:menaquinone oxidoreductases (Ndh-2: ndh and ndhA),
though nuo appears dispensable for growth.[4] The role(s) for
each individual copy of Ndh-2 are uncertain, but genetic knockout
studies have suggested only ndh is essential for Mtb growth and
persistence.[5] Importantly, while common among bacteria and
protozoan parasites, these enzymes are absent from the
mammalian genome, which further highlights their potential as
drug targets.[ A well-validated, specific Ndh-2 inhibitor would
provide both a novel tool to study Ndh-2 function as well as enable
preclinical pharmacological validation of Ndh-2 as a target for TB
therapy.
6]
Mycobacterium tuberculosis resides in
a
variety of
microenvironments, depending on the stage of infection, and to
survive they are aided by a remarkable metabolic plasticity, which
allows remodeling of energy utilization and biomass producing
pathways, dependent on the environmental conditions and
nutrient availability.[1] Recent work suggests that survival of Mtb,
under a variety of conditions, is completely dependent on the
existence of an energized plasma membrane, which couples
nutrient oxidation to ATP synthesis via the production of an
electrochemical gradient.[2] The discovery that TMC207
We performed a high-throughput screen for compounds that
reduced the production of ATP (via oxidative phosphorylation)
through the inhibition of Ndh-2. We utilized membrane vesicles
derived from mycobacteria and measured ATP production upon
the addition of NADH. The NADH is then oxidized through a
cascade of redox reactions, resulting in the formation of a proton
gradient across the vesicle membrane. This proton gradient
drives the synthesis of ATP from ADP and inorganic phosphate
by the F F ATP synthase. In this assay bedaquiline inhibits ATP
1
0
synthesis with an IC50 of 6.9 nM (Figure S1).[7] In a miniaturized
536-well plate format of this assay we screened over 800,000
(
bedaquiline) exerts its antimycobacterial activity via shutdown of
ATP production through inhibition of the F-type ATP synthase
1
compounds and identified over 7,000 primary hits (Figure S2). Hit
compounds were progressed through a variety of triage assays to
eliminate false positives, exclude cytotoxic compounds, and to
determine the mode of inhibition in the pathway.
[*]
Dr. H. Guo, Prof. F. Wang
Institute of Biophysics, Chinese Academy of Sciences
Beijing 100101, China
Compounds CBR-1825 and CBR-4032 each inhibited
electron transport-dependent ATP production initiated by NADH,
as well as NADH oxidation assayed in mycobacterium
membranes with IC50 < 0.5 M (Figure 1 and S3). Furthermore,
they were inactive when assayed for succinate-initiated oxidative
phosphorylation in membranes (Figure S4). This activity profile
suggests that both small molecules block the electron transport
chain at Ndh-2. Multiple active structural analogs were identified
in the primary screen for both series. The major structural motif
for CBR-1825 and the related compounds was the thioquinazoline
E-mail: wangfeng@ibp.ac.cn
Drs. M. B. Harbut, B. Yang, R. Liu, B. Chen, J. Lockner, H. C. Yu,
M. Petrassi, A.K. Chatterjee, F. Wang
California Institute for Biomedical Research
La Jolla, California 92037, USA
T. Yano, H. Rubin
Department of Medicine
University of Pennsylvania
Philadelphia, PA 19104, USA
C. Vilchèze, W. R. Jacobs Jr.
Howard Hughes Medical Institute
Department of Microbiology and Immunology
Albert Einstein College of Medicine
Bronx, NY 10461, USA
(
TQZ) core; CBR-4032 and like molecules possessed a
tetrahydroindazole (THI) core. Importantly, both scaffolds showed
Scott G. Franzblau
growth inhibition of Mtb in liquid medium (MIC50 = 0.43 M and
Institute for Tuberculosis Research
University of Illinois at Chicag
Chicago, IL 60612, USA
6
.6 M for CBR-1825 and CBR-4032, respectively) while
displaying no gross toxicity towards mammalian cells (Figure 1 A
B).
&
Supporting information for this article is given via a link at the end of
the document.
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the loss of function of Ndh-2. Moreover, treatment of Mtb with
complex I inhibitors, such as rotenone, has little effect on
mycobacterial growth or NADH oxidation in purified membranes,
suggesting that Ndh-2 is the dominant NADH dehydrogenase.[10]
Though our preliminary triage data suggested that both
scaffolds block the electron transport chain (ETC) at Ndh-2, we
initiated further target identification studies for confirmation. We
attempted to identify the target(s) of the TQZ scaffold by isolating
spontaneous arising mutants resistant to it. Single-step selection
-8
produced resistant mutants at a frequency of 3x10 . Sequencing
of 3 resistant mutants revealed a C-to-G mutation in the promoter
region of ndhA (Rv0392c), 44 nucleotides upstream of the start
codon. We analyzed gene expression levels of ndh, ndhA, and
nuoG, in wild-type and one of the resistant strains and found that
ndhA was expressed at a level approximately 50-fold greater than
the wildtype strain (Figure 3A). Determination of MIC50 values of
the resistant strains showed a greater than 10-fold decrease in
the susceptibility to either scaffold suggesting resistance is
conferred by compensatory upregulated expression of ndhA.
Figure 1. Representative hit compounds from the primary screen and their
activities against Mtb H37Ra and oxidative phosphorylation assessed in
mycobacterial membranes. A) CBR-1825,
thioquinazoline scaffold. B) CBR-4032,
tetrahydroindazole series.
a
representative of the
representative of the
a
We prioritized medicinal chemistry optimization for the TQZ
series due to its high cellular potency. A few key structure-activity-
relationships were observed during this process (Figure 2 and SI).
Replacement of the thioether with oxygen, nitrogen, or methylene
(
Figure 3B and S6).
Figure 2. Selected SAR results of TQZ scaffold
significantly reduced activity, as did oxidation of the sulfide.
Compared to saturated fused rings, a fused phenyl ring was
preferred. CBR-5992, a quinazoline compound was identified with
MIC of 0.67 M, two-fold more active than the original hit CBR-
The biochemical and genetic data suggested the activity of
both scaffolds is mediated through one or both of the type II
NADH:quinone oxidoreductases, yet those data do not
specifically distinguish the protein target(s) of these inhibitors.
The Ndh-2 enzymes Ndh and NdhA in Mtb share high homology
(67% identity), are both expressed under aerobic culture
1
825. Further modifications of phenyl ring (various substitutions
and heterocyclic rings) afford 5-fluoro substituted analog CBR-
465 with further improved activity (MIC = 0.16 M). Next SAR
[11]
3
conditions, and retain oxidoreductase activity. Little is known
though about their respective roles; in organisms with multiple
copies of Ndh-2, their roles are often non-redundant.[12] To further
examine target engagement, we utilized a temperature sensitive
(TS) Mycobacterium smegmatis mutant to probe the activity of
study was conducted around cyclohexane amide side chain in the
context of the optimized 5-fluoroquinazoline. While a lot of
analogs are not active, fluoride, chloride, and methyl substituents
are relatively tolerated. Among them, introduction of difluorines
into to cyclohexane ring affords the most potent analog CBR-1922
against virulent Mtb with an MIC of 0.09 M in growth inhibition
assays. This compound compares favorably with the most
advanced inhibitors from two recently described medicinal
chemistry efforts targeting Ndh-2, the quinolinyl pyrimidines and
a quinolone series.[8] The MIC50 of the most potent compounds
from either of these two series ranged from 0.14-1.0 M. Likewise,
a suite of phenothiazine-derived compounds were unable to
achieve an MIC50 below 10 M against aerobic Mtb.[9]
Next, we assessed whether either Ndh-2 inhibitor series
was bactericidal. Indeed, compounds from each scaffold
produced over 1-log killing within the first three-day treatment, and
greater than 4-log killing against cultures over a period of 32 days
when tested at 10x the MIC (Figure S5). These data suggest that
any assumed activity by the type I Ndh is unable to complement
Figure 3. Upregulation of ndhA expression confers resistance to TQZ. A)
Gene expression in the H37Ra ndhA -44 G>C background. B) Activity of TQZ
against wild type and ndhA -44 G>C H37Ra strains.
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each compound against Ndh and NdhA individually. When grown
above the permissive temperature, this TS strain downregulates
expression of ndh due to a base substitution of G250 to T of the
ndh gene that alters amino acid Gly84 (GGC) to Cys (TGC) of
Ndh (Yano and Rubin, unpublished data). When grown at 30°C
this strain shows low susceptibility to either inhibitor series (similar
to the WT M.smegmatis), but as the incubation temperature
increases to 40°C, ndh expression correspondingly decreases,
which results in an >8-fold shift in the MIC of the two compounds
relative to their potency at the permissive temperature (Table S1
and Figure S7). In addition, the TS mutant can be complemented
with Mtb malate dehydrogenase (mdh), which restores growth at
to INH, consistent with the previous observations in type II NADH
dehydrogenase mutants isolated during selection for INH
resistance.[
13b]
Some strains with Ndh mutations are also
auxotrophic for serine, possibly due to inhibition of the initial step
in serine biosynthesis by elevated levels of NADH.[15] In
corroboration of this, we found that addition of serine to Ndh-2
inhibitor-treated Mtb cultures could rescue bacterial growth
(Figure 4B).
Shutdown of electron transport to the proton-pumping
terminal electron acceptors will disrupt the proton motive force,
1 0
which is utilized by the F F -ATP synthase to catalyze ATP
synthesis.[16] Consistent with this notion, the inhibition of Ndh-2
also decreased intracellular ATP levels in a dose-dependent
fashion (Figure 4C).
4
2°C by combining the M. smegmatis malate:menaquinone
[13]
oxidoreductase (Mqo) activity with Mtb Mdh NADH recycling.
When grown at 42°C, a temperature where Ndh-2 activity is
minimal and complemented by Mdh/Mqo activity, neither
compound inhibited cell-growth at 100 M, strongly suggesting
their target is Ndh-2.
This strain also provides a biochemical means to assess
inhibitor activity against either Ndh or NdhA utilizing isolated
membrane vesicles. We used the mutant strain complimented
with either Mtb ndh or ndhA and prepared membrane vesicles
from cultures grown at the non-permissive temperature. In these
membrane preparations, the complemented genes are the
majority type II NADH dehydrogenases expressed. The TQZ
analog CBR-1825 showed strong potency against both Msmeg
and Mtb Ndh (IC50 = 10.7 nM and 64.7 nM, respectively) when
expressed in temperature-sensitive background and grown under
non-permissive conditions (Table 1). Its activity against Mtb NdhA
was similarly potent. Unlike the TQZ scaffold, the THI inhibitor
CBR-4032 appeared specific for Ndh, as its IC50 against NdhA
was over 300-fold increased relative to Ndh.
Table 1: NADH oxidation in M. smegmatis membranes with complemented Mtb
ndh or ndhA inhibited by TQZ and THI scaffolds.
Mtb Ndh
IC50 (nM)
Msmeg Ndh
IC50 (nM)
Mtb NdhA
IC50 (nM)
Compound
TQZ
THI
64.7
25.0
10.7
44.3
42.5
15300
Taken these results together we felt confident that both scaffold
series were potent and specific Ndh-2 inhibitors, and we next
endeavored to characterize the phenotypic response to loss of
function of Ndh-2 in mycobacteria.
The NADH:quinone oxidoreductases are the major
enzymes that oxidize NADH to NAD+, and we therefore
Figure 4. Metabolic and physiologic effects in Mtb produced by Ndh-2
inhibition. A) Treatment with sub-inhibitory concentrations of TQZ blocks
growth inhibition by INH at a concentration (250 nM) that normally completely
prevents replication in the absence of TQZ. Percent growth was normalized to
untreated controls. B) Addition of exogenous serine rescues Mtb treated with an
Ndh-2 inhibitor. C) Quantitation of ATP levels in Mtb treated with 10x, 5x, and
hypothesized the immediate consequence of blocking Ndh-2
function would be the prevention of NADH oxidation, resulting in
+
an increase in the ratio of NADH:NAD . It has previously been
shown that increased levels of NADH cofactor can protect InhA
from inhibition by isoniazid (INH), likely due to increased
competition with the active form of INH, the INH-NAD adduct.[
Indeed, CBR-1825 abrogated the anti-mycobacterial activity of
INH when the two were tested in combination. Treating Mtb with
a sub-lethal concentration of the Ndh-2 inhibitor completely
reversed growth inhibition of isoniazid at concentration that
normally prevents proliferation (Figure 4A). These results
1
x the MIC50 of the given inhibitor after 6 hr of treatment time.
14]
The mycobacterium ETC and the ATP synthetic machinery-
driven by it represent a hub of vulnerability for the pathogen.[
17]
Furthermore, due to the druggability of targets in this pathway, as
evidenced here and elsewhere, it thus remains an opportunity for
anti-TB drug discovery.[
3, 18]
Using a pathway-based screen in
mycobacterial membranes we identified inhibitors that block
oxidative phosphorylation and prevent Mtb growth via disruption
+
demonstrate that increase of NADH:NAD ratio causes resistance
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of Ndh-2 activity. Inhibition of Ndh-2 not only prevents ATP
synthesis through blockade of the ETC, but also disturbs the
intracellular redox balance which leads to an array of metabolic
consequences. The identification of inhibitor from a biochemical
screen that have cellular activity is especially encouraging, given
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[8]
past low success rates in these screens in the antimicrobial
field.[
19]
The scaffolds identified here act on Ndh and NdhA
differently even though their protein sequences are highly
conserved. We believe it is reasonable to suggest that the TQZ
and THI scaffolds, occupy the quinone and NADH binding sites,
respectively, given their structural resemblance to the quinone
and adenine molecules that normally reside in those positions.[
Structural analysis coupled with kinetic enzymatic analysis of the
inhibitors would be the best way to gain more insight into their
20]
[9]
inhibitory mechanism.[
21]
Future studies will also include utilizing
[10]
our Ndh-2 inhibitors to explore the roles of Ndh-2 and Nuo in vivo,
and will allow us to shutdown both or one copies of Ndh-2,
respectively.
[11]
1
02, 4548-4553.
S. S. Lin, U. Gross, W. Bohne, Mol Microbiol 2011, 82,
09-221.
[
12]
2
Acknowledgements
[13]
aD. Molenaar, M. E. van der Rest, A. Drysch, R. Yucel,
J Bacteriol 2000, 182, 6884-6891; bC. Vilcheze, T. R.
Weisbrod, B. Chen, L. Kremer, M. H. Hazbon, F.
Wang, D. Alland, J. C. Sacchettini, W. R. Jacobs, Jr.,
Antimicrob Agents Chemother 2005, 49, 708-720.
M. Nguyen, A. Quemard, S. Broussy, J. Bernadou, B.
Meunier, Antimicrob Agents Chemother 2002, 46,
2137-2144.
The authors thank Drs. Cliff Barry and Helena Boshoff for
technical support or helpful discussion. This work was supported,
in part, by National Key R&D Program of China 2017YFA0505400，
Strategic Priority Research Program of CAS，XDPB0304, CAS
Pioneer Hundred Talents Program, the Bill and Melinda Gates
Foundation, the Global Alliance for TB Drug Development, and
NIH Grants AI026170 (WRJ) and AI051519 (WRJ). We also
ackowlwdge Ms. Jennifer Yano for her technical assistance.
[14]
[15]
16]
E. Sugimoto, L. I. Pizer, J Biol Chem 1968, 243, 2081-
2089.
[
K. Hards, J. R. Robson, M. Berney, L. Shaw, D. Bald,
A. Koul, K. Andries, G. M. Cook, J Antimicrob
Chemother 2015, 70, 2028-2037.
Keywords: Ndh-2 • Oxidative phosphorylation • ATP production
•
Tuberculosis • Drug discovery
[17]
G. M. Cook, K. Hards, E. Dunn, A. Heikal, Y. Nakatani,
C. Greening, D. C. Crick, F. L. Fontes, K. Pethe, E.
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K. Pethe, P. Bifani, J. Jang, S. Kang, S. Park, S. Ahn,
J. Jiricek, J. Jung, H. K. Jeon, J. Cechetto, T.
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A high-throughput screen against the
M. tuberculosis oxidative
phosphorylation pathway identified two
inhibitors of the type II NADH
Michael B Harbut, Feng Wang*
Page No. – Page No.
Small molecules targeting
dehydrogenase. The inhibitors are
differentiated in their activity against
one or both of the Ndh-2 isozymes.
Treatment Mtb with the compounds
leads to shutdown of ATP synthesis
and bacterial killing.
Mycobacterium tuberculosis type II
NADH dehydrogenase with anti-
mycobacterial activity
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