Mitochondrial uncoupler MB1-47 is efficacious in treating hepatic metastasis of pancreatic cancer in murine tumor transplantation models

Article abstract of DOI:10.1038/s41388-021-01688-7

Pancreatic ductal adenocarcinoma (PDA) is aggressive cancer characterized by rapid progression, metastatic recurrence, and highly resistant to treatment. PDA cells exhibit aerobic glycolysis, or the Warburg effect, which reduces the flux of pyruvate into mitochondria. As a result, more glycolytic metabolites are shunted to pathways for the production of building blocks (e.g., ribose) and reducing agents (e.g., NADPH) for biosynthesis that are necessary for cell proliferation. In addition, PDA cells are highly addicted to glutamine for both maintaining biosynthetic pathways and achieving redox balance. Mitochondrial uncoupling facilitates proton influx across the mitochondrial inner membrane without generating ATP, leading to a futile cycle that consumes glucose metabolites and glutamine. We synthesized a new mitochondrial uncoupler MB1-47 and tested its effect on cancer cell metabolism and the anticancer activity in pancreatic cancer cell models and murine tumor transplantation models. MB1-47 uncouples mitochondria in the pancreatic cancer cells, resulting in: (1) the acceleration of pyruvate oxidation and TCA turnover; (2) increases in AMP/ATP and ADP/AMP ratios; and (3) a decrease in the synthesis rate of nucleotides and sugar nucleotides. Moreover, MB1-47 arrests cell cycle at G₀–G₁ phase, reduces clonogenicity, and inhibits cell growth of murine and human pancreatic cancer cells. In vivo studies showed that MB1-47 inhibits tumor growth in murine tumor transplantation models, and inhibits the hepatic metastasis when tumor cells were transplanted intrasplenically. Our results provide proof of concept for a potentially new strategy of treating PDA, and a novel prototype experimental drug for future studies and development.

Full text of DOI:10.1038/s41388-021-01688-7

Oncogene (2021) 40:2285–2295
https://doi.org/10.1038/s41388-021-01688-7
ARTICLE
Mitochondrial uncoupler MB1-47 is efficacious in treating hepatic
metastasis of pancreatic cancer in murine tumor transplantation
models
1
1
Amer Alasadi1,2 Bin Cao3 Jingjing Guo1 Hanlin Tao1 Juan Collantes
Victor Tan
Xiaoyang Su4
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●
●
●
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●
David Augeri3 Shengkan Jin
1
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Received: 27 May 2020 / Revised: 19 January 2021 / Accepted: 27 January 2021 / Published online: 1 March 2021
© The Author(s), under exclusive licence to Springer Nature Limited 2021
Abstract
Pancreatic ductal adenocarcinoma (PDA) is aggressive cancer characterized by rapid progression, metastatic recurrence, and
highly resistant to treatment. PDA cells exhibit aerobic glycolysis, or the Warburg effect, which reduces the flux of pyruvate
into mitochondria. As a result, more glycolytic metabolites are shunted to pathways for the production of building blocks
(e.g., ribose) and reducing agents (e.g., NADPH) for biosynthesis that are necessary for cell proliferation. In addition, PDA
cells are highly addicted to glutamine for both maintaining biosynthetic pathways and achieving redox balance.
Mitochondrial uncoupling facilitates proton influx across the mitochondrial inner membrane without generating ATP,
leading to a futile cycle that consumes glucose metabolites and glutamine. We synthesized a new mitochondrial uncoupler
MB1-47 and tested its effect on cancer cell metabolism and the anticancer activity in pancreatic cancer cell models and
murine tumor transplantation models. MB1-47 uncouples mitochondria in the pancreatic cancer cells, resulting in: (1) the
acceleration of pyruvate oxidation and TCA turnover; (2) increases in AMP/ATP and ADP/AMP ratios; and (3) a decrease in
the synthesis rate of nucleotides and sugar nucleotides. Moreover, MB1-47 arrests cell cycle at G0–G1 phase, reduces
clonogenicity, and inhibits cell growth of murine and human pancreatic cancer cells. In vivo studies showed that MB1-47
inhibits tumor growth in murine tumor transplantation models, and inhibits the hepatic metastasis when tumor cells were
transplanted intrasplenically. Our results provide proof of concept for a potentially new strategy of treating PDA, and a novel
prototype experimental drug for future studies and development.
Introduction
These authors contributed equally: Amer Alasadi, Bin Cao
Pancreatic ductal adenocarcinoma (PDA) is an incurable
and devastating cancer characterized by rapid progression,
Supplementary information The online version contains
supplementary material available at https://doi.org/10.1038/s41388-
metastatic recurrence, and high resistance to treatments
[1, 2]. PDA is the fourth leading cause of cancer-related
death in the United States with a 5-year survival rate of only
6% and a median survival rate of fewer than 6 months after
diagnosis [1, 3]. In many patients, PDA is not diagnosed
until it has reached an advanced metastatic stage [4]. Only
about 20% of patients are eligible for resection surgery, of
which most will eventually have tumor recurrence [5].
Current chemotherapeutic options for patients are not
effective [6]. Although the reason why PDA is insensitive to
chemotherapeutic agents is not fully understood, it was
proposed that the high degree of genetic heterogeneity
might be a factor [3, 7–10].
021-01688-7.
* Shengkan Jin
victor.jin@rutgers.edu
1
Department of Pharmacology, Robert Wood Johnson Medical
School, Rutgers, The State University of New Jersey,
Piscataway, NJ, USA
2
Graduate Program of Physiology and Integrative Biology, Robert
Wood Johnson Medical School, Rutgers, The State University of
New Jersey, Piscataway, NJ, USA
3
Medicinal Chemistry Department, Ernest Mario School of
Pharmacy, Rutgers, The State University of New Jersey,
Piscataway, NJ, USA
4
Department of Medicine, Robert Wood Johnson Medical School,
Regardless of the genetic heterogeneity, the PDA cells
share a potential therapeutic vulnerability lying in the
Rutgers, The State University of New Jersey, Piscataway, NJ,
USA

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A. Alasadi et al.
reliance on aerobic glycolysis, known as the Warburg effect
[11–13]. In the presence of oxygen, the PDA cancer cells
convert a majority of pyruvate derived from glycolysis to
lactate, in contrast to normal cells in which the majority of
the pyruvate enters the mitochondria for oxidative phos-
phorylation [14, 15]. It is becoming clear that the most
important function of the Warburg effect to tumorigenesis is
to reduce glycolysis flux going into mitochondria, thus
preventing the complete oxidation of glucose [14, 16]. Due
to aerobic glycolysis, more glycolytic metabolites are
shunted to pathways of production of building blocks (e.g.,
ribose) and reducing agents (NADPH), both of which are
required for macromolecule biosynthesis in cell prolifera-
tion [17–19]. In addition, PDA cells are also highly
dependent on glutamine as a nitrogen source for the bio-
synthesis necessary for PDA cell proliferation [20–23].
Mitochondrial uncoupling is a process that facilitates
protons influx across the inner mitochondrial membrane
without generating ATP [24, 25], which causes a futile
TCA cycle with mitochondrial oxidation and ultimately
promotes glucose and glutamine oxidation. For that reason,
we postulate that mitochondrial uncoupling could poten-
tially diminish the anabolic effect of aerobic glycolysis and
exhaust glutamine, thereby inhibiting cancer cell pro-
liferation. Small molecule mitochondrial uncouplers have
been used in humans before. In the 1930s, 2,4-dini-
trophenol (DNP) was a proved drug used by more than
100,000 people for weight loss in a period of 4 years [26].
However, DNP has a narrow therapeutic index, and it was
withdrawn from the market. Another mitochondrial
uncoupler used in humans is niclosamide, an anthelmintic
drug [27]. In contrast to DNP, NEN exhibits a wide ther-
apeutic index, partly due to its limited absorption and
restricted distribution, such as enriched in the liver with
limited exposure to other tissues [28]. Previously, we
demonstrated that the ethanolamine salt of niclosamide,
niclosamide ethanolamine (NEN), and oxyclozanide (a
veterinary anthelmintic drug with mitochondrial uncou-
pling activity) are efficacious for treating hepatic metastasis
of colorectal cancer in the murine models [29]. The similar
anticancer activity was observed with a slow-release form
of DNP [30].
mitochondrial uncouplers with better druggable properties.
One compound, MB1-47 (5-chloro-2-hydroxy-3-methyl-
N-(6-(trifluoromethyl)benzo[d]thiazol-2-yl)
benzamide,
2-aminoethan-1-ol salt) was identified. In this report, we
examined the metabolic effect of MB1-47, and its antic-
ancer activity in cultured pancreatic cancer cells and in
murine models of PDA hepatic metastasis.
Results
MB1-47 is a mitochondrial uncoupler with favorable
pharmacokinetic properties
The mechanism by which chemical uncouplers uncouple
oxidative phosphorylation is illustrated in Fig. 1A [32]. In
essence, a chemical uncoupler is often a lipophilic weak
acid distributed in the mitochondrial inner membrane
(MIM), where it exists in equilibrium between the proto-
nated form (UH) and deprotonated form (U-). When a
protonated uncoupler (UH) moves to the inner side of the
MIM (matrix side), the protonated uncoupler (UH) tends to
release the proton to the mitochondrial matrix and become
deprotonated. The protonation–deprotonation of an uncou-
pler within the MIM thus catalyzes a passive translocation
of protons across the MIM.
The synthesis route and chemical structure of MB1-47 are
shown in (Fig. 1B) with detailed synthesis conditions descri-
bed in the “Material and Method” section. The hallmark of
mitochondrial uncoupling is the induction of oxygen con-
sumption in the presence of ATP synthase inhibitor oligo-
mycin (i.e., increasing the electron transport chain activity
independent of ATP synthesis). We tested the uncoupling
activity of MB1-47 with oxygen consumption rate (OCR)
assay with the Seahorse XF24 instrument in murine pancreatic
adenocarcinoma cells, Panc02. MB1-47 increases the OCR in
the presence of oligomycin at 2.5 μM (Fig. 2A), indicating
MB1-47 is a mitochondrial uncoupler. The mitochondrial
uncoupling process is usually correlated with a reduction in
mitochondrial membrane potential (MMP). To confirm the
uncoupling effect of MB1-47, Panc02 cells were treated with
various concentrations of MB1-47, and MMP was measured
in these cells with the TMRE staining method. Our data show
that MB1-47 begins to reduce the MMP at 0.5 µM (data not
shown), reduces roughly 30–50% of staining at 1.0 µM, and
nearly completely dissipates the MMP at 2.5 µM (Fig. 2B).
Similar results were obtained with another pancreatic cancer
cell line, Panc1 (Supplementary Fig. S1a, b). As mitochondrial
uncouplers usually have high protein binding capacity, we
determined the effect of MB1-47 on MMP in the medium
containing no fetal bovine serum (FBS). As summarized in
Fig. S1c, MB1-47 exhibits robust activity in reducing MMP at
10 nM in the medium containing no FBS, as observed with
It remains to be tested if pancreatic cancers would
respond to the mitochondrial uncoupling therapeutic strat-
egy as colon cancers [29]. Our initial in vivo efficacy
testing of NEN in murine models did not produce con-
sistent results (data not shown). NEN has multiple intrinsic
limitations as a cancer drug candidate. For example, NEN
contains a moiety of nitrobenzene structure, which is often
mutagenic and Ames positive [31]. Moreover, the absorp-
tion of NEN is low and varies between individuals. To
overcome these shortcomings, we have implemented a
medicinal chemistry program to synthesize and screen new

Mitochondrial uncoupler MB1-47 is efficacious in treating hepatic metastasis of pancreatic cancer in. . .
2287
+
H
Intermembrane Space
Heat
+
H
A
+
H
+
H
+
H
+
H
+
H
+
H
+
H
+
H
+
H
H+
-
-
e
UH
UH
U
-
IV
Mitochondrial
Inner Membrane
eII
I
ATP Synthase
-
III
U
-
2
e
FAD+
FADH2
+
H
2O
2H +1/2 O2
NAD+
NADH
H+
+
H
+
ADP
Pi
ATP
Mitochondrial
Matrix
TCA Cycle
Acetyl CoA
β oxidation
Fatty acyl
CoA
O
O
CF3
O
O
N
O
Cl
O
Cl
Cl
OH
B
OH
S
N
H
Cl
OH
O
CH3
O
Br
CH3
3-Br-BA
2)
4)
3)
1) BA
3-Me-BA
3-Me-benzamide
CF3
CF3
N
N
O
O
Cl
Cl
S
S
N
H
N
H
OH
OH
OH
CH3
CH3
H2N
5)
6) MB1-47
2-OH-3-Me-benzamide
Fig. 1 The schematic of the mitochondrial uncoupling mechanism
and the synthetic route of MB1-47. A Mechanistic illustration of a
chemical uncoupler (UH) showing a schematic of oxidative phosphor-
ylation and chemical uncoupling. The ETC (electron transport chain)
complexes pump protons out of the mitochondrial inner membrane
(MIM), resulting in a proton gradient driving ATP synthesis. A chemical
uncoupler (U−) catalyzes a protonation/deprotonation cycle that facilitates
a proton influx which does not generate ATP, leading to a “futile” oxi-
dation of acetyl-CoA. B The synthetic route and chemical structure for
MB1-47 (detailed synthetic methods are described in “Materials and
Methods”): 1 BA (5-chloro-2-methoxybenzoic acid), in sulfuric acid,
TFA, and NBS to yield 2 Br-BA (3-bromo-5-chloro-2-methoxybenzoic
acid). Next the reaction was acidified using HCl and extracted using ethyl
acetate to eventually yield 3 3-Me-BA (5-chloro-2-methoxy-3-methyl-
benzoic acid). It was then further catalyzed using DMF and purified to
yield
4
3-Me-benzamide (5-chloro-2-methoxy-3-methyl-N-(6-(tri-
2-OH-3-Me-bena-
fluoromethyl)benzo[d]thiazol-2-yl)benzamide).
5
mide (5-chloro-2-methoxy-3-methyl-N-(6-(trifluoromethyl)benzo[d]
thiazol-2-yl)benzamide) was combined with pyridinium chloride to
yield 6 MB1-47 (5-chloro-2-hydroxy-3-methyl-N-(6 (trifluoromethyl)
benzo[d]thiazol-2-yl)benzamide).
NEN [32]. The effect of MB1-47 on OCR correlates with its
effect on the MMP, for example, in a medium containing 10%
FBS, the indication of OCR is observable at a concentration of
0.5 μM (data not shown), becomes robust at 1.0 μM, and
reaches maximal at 2.5 μM (Fig. S1d, e).
Next, we performed pharmacokinetic studies of MB1-47.
Our data show that MB1-47 has significantly improved
pharmacokinetic properties over NEN, including higher
exposure and prolonged half-life (Fig. 2C) [28]. Moreover,
consistent with high plasma protein binding property (Fig. 2C)
[28], MB1-47 has a low volume of distribution (Fig. 2C) and
has enriched distribution in the liver (Fig. 2C). For further
testing MB1-47 in mouse models, we titrated the dosage of
MB1-47 in a chow diet and measured the blood concentration
of MB1-47. We found that feeding the mice with chow
containing 600 ppm MB1-47 mice exhibit a fluctuation MB1-
47 between 0.5 and 1.4 μM in plasma (Fig. 2D), sufficient to
induce mitochondrial uncoupling in vivo.

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Fig. 2 MB1-47 uncouples mitochondria in pancreatic cancer cells.
A Oxygen consumption rate (OCR) of Panc02 cells measured by
Seahorse X24. This was carried out with sequential addition of oli-
gomycin (final concentration of 5 μM), MB1-47 (final concentration
2.5 μM), and rotenone (final concentration of 2.5 μM), at indicated
time points. B Effective concentrations of MB1-47 for mitochondrial
uncoupling in Panc02 cells measured with TMRE staining method.
Panc02 cells were treated with various concentrations of MB1-47 or
vehicle (DMSO) for 2 h, followed by staining with TMRE for 10 min.
C Pharmacokinetic and tissue distribution properties of oral MB1-47
(10 mg/kg). PPB plasma protein binding, Vss volume of distribution,
Liver/Plasma distribution ratio between liver (mg/g) and plasma (μg/
ml). D Blood concentration analyses (μM) in mice measured at 9 a.m.
(peak) and 5 p.m. (trough) in mice fed the diet containing MB1-47
(600 ppm) for 2 weeks. B, C are representative results from three
independent experiments. D, E are representative results from two
independent experiments. Results in (B) are indicated as means SD
values.
The effect of MB1-47 on pancreatic cancer cell
metabolism
demonstrated by elevated levels of two-carbon 13C incorpo-
rated into TCA cycle metabolites. For example, [U-13C6]-
glucose generates 13C3-pyruvate and 13C2-acetyl-CoA, and in
turn generates 13C2-succinate (Fig. 3B), malate (Fig. 3C), both
of which increased in the MB1-47-treated cells. Consistent
with our hypothesis, as a result of futile oxidation, the pool
size (the absolute level) of glutamate is decreased (Fig. 3D). In
addition, the pool sizes of most amino acids (exception for
methionine) either decreased significantly after 1-h treatment
or exhibited a trend of decreasing (Supplementary Fig. S2).
Consistent with a futile cycle, our results showed that
MB1-47 treatment increased the ratios of AMP/ATP (Fig. 3E)
and ADP/ATP (Fig. 3F), as well as a decrease in UTP
(Fig. 3G) and elevation of the UMP/UTP ratio (Fig. 3H).
To study the metabolic consequences of MB1-47 treatment,
we incubated Panc02 cells with [U-13C6]-glucose, extracted
and analyzed the metabolites using LC-MS approach. Our
focus revolves around the metabolism of the central carbon in
glycolysis and the TCA cycle (Fig. 3A), and the [U-13C6]-
glucose allows us to measure metabolic flux rates. The cells
were treated with MB1-47 at 1 μM, an intermediate con-
centration in the range between 0.5 and 2.5 μM that activates
mitochondrial uncoupling. Consistent with increased mito-
chondrial oxidation rate, our LC-MS results show higher
glucose to TCA cycle flux in the MB1-47-treated cells, as

Mitochondrial uncoupler MB1-47 is efficacious in treating hepatic metastasis of pancreatic cancer in. . .
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Fig. 3 Effect of MB1-47 on pancreatic cancer cell metabolism. A
Schematic representation of metabolic molecules measured in the LC-
MS experiments. Each oval shape represents a carbon atom of a
molecule, with 13C represented by an oval in blue ( ), and 12C by an
oval in white ( ). In (B–N), relative levels or ratios of metabolites, as
indicated in each panel, in control (DMSO treated) Panc02 cells or
cells treated with MB1-47 (1.0 μM). From B to N, Panc02 cells were
grown in a medium containing 100% U-13C glucose, treating with
1.0 μM MB1-47 or vehicle DMSO for 1 h. The metabolites were
extracted using cold 40:40:20 methanol:acetonitrile:water w/ 0.5%
formic acid (ice cold) mixture. The levels of both pooled (12C and 13
C
metabolites) and 13C labeled metabolite were measured by LC/MS (Q
Exactive PLUS hybrid quadrupole-orbitrap mass spectrometer
(Thermo Scientific) coupled to hydrophilic interaction chromato-
graphy (HILIC)). G6P glucose-6-phosphate, F6P fructose-6-phos-
phate, R-5-P ribose 5-phosphate, Glu Glutamate, αKG alpha-
ketoglutarate. The data are representative results from two indepen-
dent experiments; each has sample treatment in triplicate. Results are
showed as means SD values and statistical significance (P) was
determined by Student t test: *P < 0.05; **P < 0.01; ***P < 0.001.

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A. Alasadi et al.
The synthesis of sugar nucleotides is downregulated, which
uses UTP as a substrate, as shown in the decreased levels of
UDP-glucose (Fig. 3I) and UDP-N-acetyl glucosamine
(Fig. 3J). Both nucleotide de novo synthesis and salvage use
5-phosphoribosyl-pyrophosphate as a key intermediate,
which requires ATP to synthesize. After MB1-47 treatment,
ATP M + 5 (Fig. 2K) and UTP M + 5 (Fig. 3L) decreased,
as well as UDP-D-glucose M + 11 (Fig. 3M) and UDP-N-
acetyl glucosamine M + 11 (Fig. 3N) (six from glucose and
five from ribosyl); all are consistent with decreased
nucleotide synthesis.
MB1-47 inhibits hepatic metastasis of pancreatic
cancer
Hepatic metastasis is a major cause of death in patients with
pancreatic tumors, and the liver is the most common site of
pancreatic cancer metastasis [8, 34]. To examine the effect
of MB1-47 on the hepatic metastasis of pancreatic cancer in
a well-established mouse model by injecting Panc02 cells
intrasplenically into immune-deficient NSG mice (34). Two
days after the recovery from the surgery, MB1-47 treatment
was initiated. As shown in Fig. 6A–D, MB1-47-treated
mice have less metastatic tumor nodules in the liver and
smaller tumor volume compared to the control group.
Collectively, these results demonstrate that MB1-47
accelerates pyruvate oxidation and TCA turnover, increa-
ses AMP/ATP ratio, and decreases both nucleotide and
sugar production.
Toxicology study of MB1-47
MB1-47 arrests cell cycle progression at G0/G1 and
reduces the colony formation of pancreatic cancer
cells
To investigate the potential toxicity effect of MB1-47 at the
therapeutic dosages, a 5-day, two-dosage comprehensive
toxicology study was performed in rats, the most commonly
used rodent model for a preclinical toxicology study.
Pharmacokinetic studies were first performed to determine
the two toxicology dosages, with the goal of generating
equal or higher plasma levels of the drug observed in the
efficacy study (Fig. 2D), which was further confirmed by
toxicokinetic results (Supplementary Results).
The toxicology data set is included in the Supplementary
Results with a summary presented in Supplementary Figs. S3
and S4. As shown in Fig. S3, animal body weight continued
to grow in the treated period, food intake was slightly
higher in the high dosage group comparing to the low
dosage group (consistent with the mechanism of action of
the drug), and no elevation in body temperature (a major
concern of the mechanism) was observed. Moreover, no
clinical adverse effects were observed and no mortality
occurred in the treatment period (Supplementary Results).
As shown in Fig. S4, hematology did not show clinical
toxicology signs; however, statistically significant changes
were observed, including neutrophil and monocytes counts,
red blood cell size distribution (RDW-CV or RDW-SD), and
mean corpuscular hemoglobin concentration, platelet dis-
tribution width, suggesting that MB1-47 affects hematogen-
esis. The serum chemistry showed no sign of liver toxicity.
Moreover, beneficial decreases in plasma total cholesterol and
AST levels were observed in the high dosage group. A sig-
nificant increase in creatine levels was observed in the high
dosage group. The elevated creatine levels are still in normal
range, suggesting mild impact in kidney function.
Next, we studied the effect of MB1-47 on murine and
human pancreatic cancer cell proliferation. First, we studied
the effect of MB1-47 on the cell cycle profile of pancreatic
cancer cells, Panc02 and Panc1. MB1-47 inhibits cell pro-
liferation by arresting cells in G0/G1 (Fig. 4A, B). The
anticancer action of MB1-47 was further investigated with
colony formation assay. Continuous exposure to MB1-47
led to over 90% (1.0 μM) to near 100% (2.5 μM) reduction
in colony formation (Fig. 4C, D). Next, we sought to study
the MB1-47 effect on the growth and proliferation of other
types of pancreatic cancer cells. Seven pancreatic cell lines
were treated with MB1-47 for 48 h, and cell growth and
proliferation were evaluated by sulforhodamine B staining
assay [33]. The results revealed that MB1-47 inhibits pan-
creatic cancer cell growth and proliferation in a dose-
dependent manner, with IC50 between 0.5 and 1 μM
(Fig. 4E).
MB1-47 inhibits pancreatic cancer growth in the
murine models
We further tested the anticancer effect of MB1-47 in
tumor transplantation mouse models. First, intrahepatic
transplantation of Panc02 was performed in NSG/6 mice
followed by immediate MB1-47 treatment. MB1-47-
treated group exhibited a smaller tumor size compared to
the control untreated group (Fig. 5A–C). Next, we tested
the efficacy of MB1-47 on tumor growth after the tumor
has been established, by starting MB1-47 treatment
10 days after cancer transplantation. Again, the
MB1-47-treated group showed a much smaller tumor
volume compared to the control untreated group
(Fig. 5D–F).
MB1-47 induces AMPK activation and
downregulates mTOR in the pancreatic cancer
AMPK is the master regulator for cell energy hemostasis and
plays a crucial role in cell metabolism [35]. A high AMP/ATP

Mitochondrial uncoupler MB1-47 is efficacious in treating hepatic metastasis of pancreatic cancer in. . .
2291
Fig. 4 Effect of MB1-47 on cell cycle progression, clonogenicity,
and cell proliferation of pancreatic cancer cells. In (A, B), the flow
cytometry analysis charts have the Y-axis as the cell count correlated
with the fluorescence intensity, while the X-axis represents the DNA
content stained with the propidium iodide dye. MB1-47 arrests pan-
creatic cancer cells at the G0–G1 phase for both Panc02 (A), Panc1 (B)
cells that were treated either with vehicle (DMSO) (left panel) or
2.5 μM of MB1-47 (right panel) for 48 h, followed by cell cycle
progression analysis via flow cytometry. C, D are bar graphs repre-
senting results of clonogenicity assay with % of surviving colony
indicated by the Y-axis, while MB1-47 concentrations, 0, 0.5, 1, and
2.5 μM, are indicated by the X-axis. MB1-47 reduces the clonogenicity
of pancreatic cancer cells Panc02 (C) and Panc1 (D), respectively.
E MB1-47 inhibits the growth of eight pancreatic cancer cell lines:
MIA PACA-2, BxPC3, Panc1, CF-Pac1, Capan2, Pan02, and AsPC1.
Pancreatic cancer cells were treated with various concentrations of
MB1-47: 0, 0.2, 0.5, 1, 2.5, or 5 μM for 48 h (X-axis), and cell growth
was measured and quantified using the sulforhodamine B (SRB)
staining (described in the “Materials and Methods”) and calculated in
% of cell growth normalized using MB1-47 treated against untreated
samples (Y-axis). The data shown in (A–E) are representative results
from three independent experiments. Results from (C–E) are shown as
means SD. Statistical significance (P) between the control and trea-
ted cells was determined by Student t test: ***, P < 0.001.

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A. Alasadi et al.
Fig. 5 MB1-47 inhibits the growth of intrahepatically transplanted
Panc02 cancer cells. Showing the tumors from the mouse liver after
intrahepatic injection of Panc02 cells into male NSG mice, in the
absence (A) or presence (B) of 750 ppm MB1-47 diets for 3 weeks.
Arrows in each picture are pointing to the presence of tumors. C A bar
graph showing quantification of liver tumor in size (mm3). The
representative pictures of mouse liver samples from mice after intra-
hepatic injection of Panc02 cells to the NSG mice, fed with normal diet
for 1 week (for mouse tumor to establish), then fed the normal diet (D)
or the diet containing 750 ppm MB1-47 (E) for 2 additional weeks.
Arrows point to the locations of tumors. In (F), quantification of liver
tumor size (mm3) in the mice with or without 750 ppm of MB1-47
treatment as indicated. Data are shown in (C, F) as means SD.
Statistical significance (P) was determined by Student t test between
the control and MB1-47 fed mice. N = 6 in each group. *P < 0.05. The
data are representative results from two independent experiments.
ratio activates AMPK, which, in turn, can inhibit the anabolic
signaling pathways and stimulate the catabolic signaling
pathways [36]. In light of the effect of MB1-47 on increasing
AMP/ATP ratio, we tested AMPK activity in Panc02 and
Panc1 cells upon treatment with MB1-47, to confirm this
signaling event. Additionally, we studied the effect of MB1-
47 on AMPK’s key downstream effectors, including ACC
(acetyl-CoA carboxylase), the key regulator of lipid metabo-
lism whose activity promotes lipid synthesis and inhibits lipid
oxidation at the same time, mTOR [37], the key regulator for
protein synthesis, as well as ERK, which is involved in a pro-
proliferation pathway important for PDA cells.
Our results indicated that MB1-47 activates AMPK in a
dose-dependent manner at concentrations that induce mito-
chondrial uncoupling (Fig. 7A–C, with quantification in
Fig. S5). Moreover, MB1-47 led to an increase in ACC
phosphorylation, which is expected to diminish ACC activity,
and consequently inhibiting lipid synthesis and promoting
lipid oxidation (Figs. 7A–C and S5). As shown in (Figs. 7D–E
and S5), MB1-47 treatment decreases the phosphorylation of
P70S6K and 4E-BP1, indicative of the downregulation of
mTOR activity. The results also showed that MB1-47
decreases the activity of ERK1/2 signaling pathways in can-
cer pancreatic cancer cells (Figs. 7D–E and S5).
Discussion
In this study, we explored the strategy of treating PDA by
changing cell metabolism. Using both in vitro and in vivo
models, we demonstrated that a mitochondrial uncoupler,
MB1-47, inhibits cancer cell growth and proliferation,
decreases clonogenic activity, inhibits both tumor growth
in vivo and metastasis to the liver in mice.
Mitochondrial uncoupling leads to a futile cycle that
accelerates the rate of mitochondrial oxidation (Fig. 1A).
The anticancer mechanism is proposed in the working
shown in Fig. 7F. Primarily, the futile oxidation diminishes
the metabolites feeding into pathways for the production of
building blocks and NADPH required for the biosynthesis
of macromolecules such as nucleic acids, lipids, and pro-
teins. In parallel, the reduction of ATP synthesis activates
nutrient deprivation signal pathways mediated by AMPK,
leading to inhibition of ACC, mTOR, and ERK pathways. It
is possible that these energetic, biomaterial, and signaling
events reinforce each other, effectively shut down cell
growth and proliferation programs.
Previously, we demonstrated that mitochondrial uncouplers
NEN and oxyclozanide are efficacious for treating hepatic
metastasis of colorectal cancer in murine models [29]. When

Mitochondrial uncoupler MB1-47 is efficacious in treating hepatic metastasis of pancreatic cancer in. . .
2293
A Untreated
C
7
6
5
4
***
MB1-47
B
3
2
1
0
Untreated
MB1-47
D
14000
12000
10000
8000
6000
4000
2000
0
**
MB1-47
Untreated
Fig. 6 MB1-47 inhibits liver metastasis of the intrasplenically
injected Panc02 cells. Representative pictures of the liver from the
NSG mice after intrasplenic injection of Panc02 cells, either fed with
normal chow diet (A) or with the diet containing 750 ppm MB1-47 (B)
for 2 weeks. Arrows pointing to the tumor nodules. The quantification
of visible hepatic tumor nodules resulted from metastasis with or
without MB1-47 treatment is as described in the Experimental
Methods and plotted in (C) as a bar chart. The Y-axis represents the
liver metastasis occurrence, while the X-axis describes the type of diet,
untreated versus 750 ppm MB1-47. D Quantification of the average
tumor size measured on the mice with liver metastasis, untreated
versus 750 ppm MB1-47. Data are shown as means SD. Statistical
significance (P) was determined by Student t test between the control
and MB1-47 fed mice. N = 7 in each group. **P < 0.01 and ***P <
0.001. The data are representative results from two independent
experiments.
testing the efficacy of NEN in treating pancreatic cancer in
animal models, we failed to obtain consistent and robust
results (data not shown). We suspected that the lack of con-
sistent efficacy is due to low systemic exposure as results of
poor absorption and poor target organ distribution. For that
reason, MB1-47 was designed and screened for retaining the
mitochondrial uncoupling potency with liver-enriched dis-
tribution as NEN, meanwhile overcoming some unfavorable
features. MB1-47 has a higher absorption after oral adminis-
tration, a higher systemic exposure, and absence of the
oncogenic potential (Ames test negative). With those
improved properties, in this study, we showed that MB1-47
exhibits robust anticancer activity in vivo in pancreatic cancer
animal models, both inhibiting growth of the transplanted
tumor as well as inhibiting the hepatic metastasis.
by body weight increase, food consumption, body tem-
perature, clinical observation, hematology, serum chem-
istry, and histology analysis. It is particularly encouraging
that no impact on body temperature and no drug accumu-
lation were observed, as those issues were previously
associated with the toxicology presentation of DNP in
humans. However, the study did show that MB1-47 may
mildly affect hematogenesis and kidney function. Long-
term toxicology studies with escalating dosages are war-
ranted to further address the potential toxicity issues.
In conclusion, we demonstrated that a new mitochondrial
uncoupler, MB1-47, has anticancer activity in vitro and is
efficacious against hepatic metastatic PDA in murine models.
The results support that the anticancer mechanism is related to
the alteration of cancer cell metabolism. The unique
mechanism of targeting mitochondria and cancer cell meta-
bolism may provide a new therapeutic strategy for PDA, the
deadly cancer essentially refractory to all current therapeutic
approaches. While further research is necessary, in particular
In addition to efficacy, potential toxicity is one of the
main hurdles for therapeutics development. Our preliminary
toxicology showed that at therapeutically relevant levels,
MB1-47 does not seem to cause overt toxicity as examined

2294
A. Alasadi et al.
Fig. 7 MB1-47 increases phosphorylation of AMPK and ACC and
downregulates mTOR activities in the pancreatic cancer cells. A
Immunoblot analyses showing that NEN and MB1-47 increase phos-
phorylation of AMPK in Panc02 cells. Immunoblot analyses showing
that MB1-47 increases phosphorylation of AMPK and ACC in Panc02
(B) and Panc1 cells (C), respectively. Immunoblot analyses of Panc02
(D) and Panc1 (E) cells, respectively, showing that MB1-47 reduces
the mTOR activity and ERK phosphorylation levels under insulin
stimulation. F The working model on the anticancer mechanism of
action of MB1-47. Immunoblotting analyses (A–E) are representative
results from three independent experiments. Quantification of the
immunoblotting analyses is shown in Supplementary Fig. S5.
in toxicology, MB1-47 represents a promising prototype for
the therapeutic strategy.
Cancer Commission Research (NJCCR) Fellowship, and Mito Bio-
Pharma, LLC. AA is supported by HCED Iraq Scholarship program. HT
was supported in part by NJCCR. AA, HT, JG, and SJ are partially
supported by NIH (1R21CA216604 and 1R21 AA027050).
Materials and methods
Author contributions Conception and design: SJ and AA. Development
of methodology: AA, SJ, BC, and DA. Acquisition of data: BC syn-
thesized the compound, AA conducted most of the biological experi-
ments, AA and HT conducted OCR Seahorse experiment, AA, JG, and
XS conducted the LC-MS experiment. Analysis and interpretation of
data (e.g., statistical analysis, LC-MS): AA, XS, and SJ. Writing, review,
and/or revision of the paper: AA and SJ. Study supervision: SJ.
All materials and methods are in the supplementary file.
Acknowledgements The authors want to thank Frank Leu for reviewing
and editing the paper. The project was partially funded by Rutgers Busch
Biomedical Award (2014), HCED Iraq Scholarship program, New Jersey

Mitochondrial uncoupler MB1-47 is efficacious in treating hepatic metastasis of pancreatic cancer in. . .
2295
Compliance with ethical standards
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Conflict of interest SJ is a founder of Mito BioPharma, which has a
license right from Rutgers University for developing safe mitochon-
drial uncouplers for treating cancer and metabolic diseases. BC, HT,
and DA are co-inventors of the patents covering the rights of MB1-47
and its derivatives. The rest of the authors declare no competing
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