Mitochondrial-Targeting MET Kinase Inhibitor Kills Erlotinib-Resistant Lung Cancer Cells

Article abstract of DOI:10.1021/acsmedchemlett.6b00223

Lung cancer cells harboring activating EGFR mutations acquire resistance to EGFR tyrosine kinase inhibitors (TKIs) by activating several bypass mechanisms, including MET amplification and overexpression. We show that a significant proportion of activated MET protein in EGFR TKI-resistant HCC827 lung cancer cells resides within the mitochondria. Targeting the total complement of MET in the plasma membrane and mitochondria should render these cells more susceptible to cell death and hence provide a means of circumventing drug resistance. Herein, the mitochondrial targeting triphenylphosphonium (TPP) moiety was introduced to the selective MET kinase inhibitor PHA665752. The resulting TPP analogue rapidly localized to the mitochondria of MET-overexpressing erlotinib-resistant HCC827 cells, partially suppressed the phosphorylation (Y1234/Y1235) of MET in the mitochondrial inner membrane and was as cytotoxic and apoptogenic as the parent compound. These findings provide support for the targeting of mitochondrial MET with a TPP-TKI conjugate as a means of restoring responsiveness to chemotherapy.

Full text of DOI:10.1021/acsmedchemlett.6b00223

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Letter
A Mitochondrial-targeting MET Kinase Inhibitor
Kills Erlotinib-resistant Lung Cancer Cells
Tianming Yang, Wai Har Ng, Huan Chen, Kamon
Chomchopbun, The Hung Huynh, Mei-Lin Go, and Oi Lian Kon
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.6b00223 • Publication Date (Web): 23 Jun 2016
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A Mitochondrial-targeting MET Kinase Inhibitor Kills Erlotinib-
resistant Lung Cancer Cells.
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Tianming Yang #, Wai Har Ng ^, Huan Chen #, Kamon Chomchopbun #, The Hung Huynh ǂ, Mei Lin Go
# *, Oi Lian Kon ^ §
*
# Department of Pharmacy, National University of Singapore, 18 Science Drive 4, Singapore 117543; ^ Division of Medical
Sciences, Laboratory of Applied Human Genetics, ǂ Division of Cellular & Molecular Research, Laboratory of Molecular
Endocrinology, Humphrey Oei Institute of Cancer Research, National Cancer Centre Singapore, 11 Hospital Drive, Singaꢀ
pore 169610 and § Department of Biochemistry, National University of Singapore, 8 Medical Drive, Singapore 117596.
KEYWORDS : Mitochondrial targeting, Triphenylphosphonium conjugate, MET kinase, PHA665752, Drug resistance, Non-
small cell lung cancer.
ABSTRACT: Lung cancer cells harboring activating EGFR mutations acquire resistance to EGFR tyrosine kinase inhibitors
(TKIs) by activating several bypass mechanisms, including MET amplification and overexpression. We show that a significant
proportion of activated MET protein in EGFR TKIꢀresistant HCC827 lung cancer cells resides within the mitochondria. Targeting
the total complement of MET in the plasma membrane and mitochondria should render these cells more susceptible to cell death
and hence provide a means of circumventing drug resistance. Herein, the mitochondrial targeting triphenylphosphonium (TPP)
moiety was introduced to the selective MET kinase inhibitor PHA665752. The resulting TPP analog rapidly localized to the mitoꢀ
chondria of METꢀoverexpressing erlotinibꢀresistant HCC827 cells, partially suppressed the phosphorylation (Y1234/Y1235) of
MET in the mitochondrial inner membrane and was as cytotoxic and apoptogenic as the parent compound. These findings provide
support for the targeting of mitochondrial MET with a TPPꢀTKI conjugate as a means of restoring responsiveness to chemotherapy.
Drug resistance to the EGFR tyrosine kinase inhibitors
(TKIs) gefitinib and erlotinib seriously impedes the therapeuꢀ
tic usefulness of these drugs on nonꢀsmall cell lung cancers
(NSCLC) that harbor activating mutations at the catalytic doꢀ
main of EGFR, the most common of which are found at exon
21 (L858R) and exon 19 (delE746ꢀA750).1 Acquired reꢀ
sistance due to a single point mutation at the gatekeeper resiꢀ
due (T790M) on the kinase domain,2 enhances affinity for
ATP and displaces gefinitib and erlotinib which are ATPꢀ
competitive inhibitors from the kinase binding pocket.3 Aside
from acquired secondary EGFR mutations, resistance to TKIs
may arise from constitutive activation of alternative signaling
pathways that bypass or lie downstream of EGFR.4 The
hepatocyte growth factor (HGF)ꢀMET signaling pathway is
frequently cited as a bypass mechanism coꢀopted by cancer
cells with acquired or innate resistance to TKIs.5ꢀ7 MET is
Other strategies to address resistance to TKIs have been reꢀ
cently reviewed. 12
We have previously shown that MET overexpression in
cancer cells is associated with MET protein localization to the
mitochondria while also remaining on the plasma membrane.13
The presence of MET in the mitochondria shifted cellular meꢀ
tabolism to a state of heightened glycolysis, increased tricarꢀ
boxylic acid (TCA) cycle activity and oxidative phosphorylaꢀ
tion.13 Such metabolic remodeling would conceivably enꢀ
hance the fitness of cancer cells for survival by increasing
ATP production and providing abundant TCA cycle intermeꢀ
diates for biomass production. Thus we postulate that in these
and other ways yet to be uncovered, mitochondrial MET inꢀ
creases the fitness of cancer cells for survival and may counꢀ
teract antiꢀcancer therapies designed to achieve cell killing.
The corollary is that inhibition of mitochondrial MET would
serve to restore responsiveness to chemotherapy. However,
current MET kinase inhibitors are designed to inhibit MET in
plasma membranes, not MET sequestered within the mitoꢀ
chondria. Thus, if the total complement of MET that resides in
plasma and mitochondrial membranes is to be inhibited, inhibꢀ
itors should have a mitochondrialꢀtargeting motif that would
direct delivery to the organelle while not compromising the
inherent ability of the entity to inhibit MET. To test this hyꢀ
pothesis, we prepared a probe molecule (TM608) in which a
triphenylphosphonium (TPP) moiety is conjugated to a selecꢀ
amplified in approximately 20% of tumors from NSCLC paꢀ
8ꢀ10
tients with acquired resistance
and its presence correlates
with poor prognosis in surgically resected patients.10 Engelꢀ
man and coꢀworkers described the focal amplification of the
MET protoꢀoncogene in an NSCLC cell line that was resistant
to gefitinib and the modest restoration of sensitivity when
MET signaling was inhibited.11 Viewed in this context, inhibiꢀ
tion of the HGFꢀMET axis may potentially reverse resistance
to TKIs, hence restoring the curative efficacies of these drugs.
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tive MET inhibitor, PHA665752 (Scheme1A). TPP is one of
the more widely employed mitochondrial directing motifs.
ing equal numbers (105) of HCC827A or HCC827B cells and
the subcellular fractions derived from this number of cells as
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TPP conjugates of several bioactive molecules (antioxidants,
anticancer agents) have been reported to specifically accumuꢀ
they were fractionated to obtain purified mitochondria. The
signal from each lane in Figure 1 reflects phosphorylated MET
(pꢀMET) present in the same number of cells (105 cells) but
not derived from identical total protein loading because the
amounts of protein would progressively decline as purification
progressed. The subcellular fractions (heavy membranes were
obtained by centrifuging whole cell lysates at 500g for 5min
and were enriched in plasma membranes; the flowꢀthrough
fraction did not bind to the antiꢀTOM22 beads used to capture
mitochondria) were derived at each sequential fractionation
step leading to pure mitochondria. Quantification by densiꢀ
tometry confirmed that whole cell lysates of HCC827B cells
had nearly six times more pꢀMET (Y1234/1235) than
HCC827A cells (B/A ratio 5.71). Moreover, the increase in pꢀ
MET (Y1234/Y1235) was more pronounced in the mitochonꢀ
dria (B/A ratio 6.07) than in the other fractions.
late in the mitochondria, with enhancement of the desired acꢀ
15ꢀ17
tivity.
The lipophilicity of the TPP cation coupled with
extensive delocalization of the positive charge favors accumuꢀ
lation in the organelle due to the substantial potential differꢀ
ence that exists across the mitochondrial membranes.18
To rationally design TM608 so that kinase inhibitory activiꢀ
ty is retained, we examined the orientation of PHA665752 in
the ATPꢀbinding pocket of MET (SI, Figure S1).19 Noting that
PHA665752 binds to the pocket with its basic side chain diꢀ
rected towards the solvent space and away from the critical
hinge region (Met1160, Pro1158) that is required for catalytic
activity, we inserted the TPP moiety in place of the basic side
chain and attached it to the core scaffold via an ethylaꢀ
minocarbonyl linker. Reꢀdocking of TM608 showed that it
was retained within the hinge region with the TPP side chain
directed towards the solvent space (SI, Figure S1). Scheme 1B
outlines the synthesis of TM608. Briefly, the pyrrole carboxꢀ
ylate 4 was formylated in a Vilsmeier reaction, hydrolysed to
yield the free acid 6 and condensed with indolinone 3 to give
intermediate 7. EDC coupling between 7 and (2ꢀaminoethyl)
triphenyl phosphonium bromide 8 gave TM608 in satisfactory
yields. Details are provided in SI.
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Scheme 1: (A) Modification of PHA665752 to give TM608.
(B) Synthesis of TM608.
Figure 1: Expression of activated MET protein (phosphoꢀ
Y1234/1235) in whole cells, heavy membranes, flowꢀthrough
fraction and pure mitochondria derived from HCC827A and
HCC827B cells. B/A ratios report the extent to which HCC827B
cells or fractions were enriched in pꢀMET (Y1234/Y1235) comꢀ
pared to the corresponding HCC827A cells/fractions.
The susceptibility of the mitochondrial MET in HCC827B
to pharmacological inhibition would depend on whether MET
is located externally at the outer membrane or internally, withꢀ
in the inner mitochondrial membrane. To determine the site(s)
at which MET was localized, we carried out controlled trypꢀ
sinization of pure mitochondria derived from HCC827B cells
and probed for the presence of αꢀ and βꢀsubunits of MET,
TOM20 (an outer mitochondrial membrane protein) and
SDHA (an inner mitochondrial membrane protein). As seen
from Figure 2, trypsinization degraded TOM20 but had little
effect on MET and SDHA. Thus we concluded that MET was
located at a similar intraꢀmitochondrial compartment as
SDHA, namely the inner mitochondrial membrane. Taken
together, these experiments affirmed the presence of stable
and high levels of activated MET sequestered within the inner
membranes of the mitochondria of HCC827B cells. Hence
HCC827B is a clinically relevant resistant phenotype of
NSCLC on which investigations on TM608 could be pursued.
Reagents and Conditions: (a) Chlorosulfonic acid; (b) 2,6ꢀ
Dichlorobenzylbromide, NaH2PO4, Na2SO3, 30ꢀ60oC, acetone; (c)
POCl3, DMF; (d) NaOH reflux; (e) Piperidine, ethanol, 60oC; (f)
PPh3, 80oC; (g) EDCI, DMAP, DMF, rt.
With the probe molecule TM608 on hand, we proceeded to
interrogate its ability to inhibit MET kinase in NSCLC cells
that are resistant to TKIs. An isogenic pair of NSCLC cells
(HCC827A and HCC827B) was used for this purpose. The
parental HCC827A cells harbor an activating mutation in exon
19 of EGFR and is erlotinibꢀsensitive.
Its derivative
20
HCC827B is erlotinibꢀresistant and METꢀenriched but the
location of MET – at its usual site at the cell membrane or
additionally partitioned to other organelles ꢀ is not known. We
were curious if the “extra” MET is localized to the mitochonꢀ
dria as this has been observed in other METꢀamplified maligꢀ
13
nant cells. Hence, we carried out an immunoblotting analyꢀ
sis of activated MET (MET phosphorylated at the catalytic
tyrosine residues Y1234/1235) in whole cell lysates comprisꢀ
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for 3 h, followed by treatment with the mitochondrionꢀspecific
dye MitoTracker Red (MTR) and visualization by confocal
microscopy. Both TM608 and PHA665752 emitted green fluoꢀ
rescence but only TM608 coꢀlocalized with MTR in the mitoꢀ
chondria (Figure 4).
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Figure 2: Detection of MET and mitochondrial membrane proꢀ
teins (TOM20, SDHA) in intact whole cells WC (Lane1), intact
pure mitochondria PM (Lane 2) and timeꢀcontrolled trypsinized
mitochondrial fractions from HCC827B cells (Lanes 3ꢀ5).
We then proceeded to determine the growth inhibitory activꢀ
ities (IC50) of PHA665752 and TM608 on HCC827A and
HCC827B cells by the colorimetric tetrazolium assay. The
sensitivity of the HCC827A cells to erlotinib was confirmed.
In our hands, % viability at 1 µM erlotinib was 22.4 % ± 2.8
(n=3), versus 72.4% ± 4.4 (n=3) on HCC827B cells. PHAꢀ
665752 and TM608 were equipotent on the erlotinib sensitive
HCC827A cells with IC50 values (72 h) of 15.7 ± 0.4 µM
(PHA665752) and 16.7 ± 1.0 µM (TM608) (p = 0.2156, unꢀ
paired Student’s tꢀtest). On the erlotinibꢀresistant high MET
expressing HCC827B cells, TM608 was found to be modestly
more potent, with IC50 of 5.3 ± 0.4 µM compared to 8.9 ± 0.6
µM for PHA665752 (p = 0.0013, unpaired Student’s tꢀtest).
Interestingly, TM608 was less cytotoxic than PHA665752
when evaluated on TAMH (TGFα murine hepatocytes), a
stable, nonꢀtumorigenic and metabolically competent cell line
that is widely used for toxicological evaluation. 21 The
24h IC50 of TM608 was 15.1µM (±1.6) as compared to 8.9 µM
(±1.2) for PHA665752.
Figure 4: Confocal microscopy images of HeLa cells treated with
4 µM TM608 (1st row) and 4 µM PHA665752 (2nd row) for 3h
and stained with MitoTracker Red and Hoechst 33342 for visuꢀ
alization of mitochondria and nuclei, respectively. PHA665752
and TM608 are fluorescent (Ex 460nm, Em 520nm). Overlap of
the fluorescence of TM608 (green) and MTR (red) is evident from
the Merged Panel.

The localization of TM608 in the mitochondria was further
investigated by a flow cytometric analysis of purified mitoꢀ
chondria isolated from HCC827B cells that were treated with
PHA665752 or TM608 (4 µM, 1h) in the absence or presence
of MTR for mitochondrial staining (Figure 5).
Both TM608 and PHA665752 induced apoptosis in
HCC827B cells as seen from the elevated levels of apoptotic
marker proteins (cleaved PARP, cleaved caspases 3 and 7) in
cells treated with subꢀlethal concentrations (4 µM) of either
compound (Figure 3). The onset of apoptosis was delayed in
TM608ꢀtreated cells but once triggered, persisted up to the 60
h time point.
Figure 5: Representative flow cytometry dot plots from double
staining experiments using MTR and test compounds
(TM608/PHA665752) in purified mitochondria of HCC827B
cells. (A) Selection of mitochondria based on forward scatter (xꢀ
axis) and side scatter (yꢀaxis) plot. Events within the gate R1 were
selected for analysis. (B) Unstained control sample (DMSO);
(C)ꢀ(G) Fluorescence of samples stained with MTR, TM608,
PHA665752, MTR+TM608 and MTR+PHA665752 for events
within R1.
Figure 3: Immunoblots of apoptotic markers (cleaved PARP,
cleaved caspases 3 and 7) in whole cell lysates of HCC827B cells
treated with 4 µM TM608 or 4 µM PHA665752 at 12h, 24h, 36h,
48h and 60h time points. Controls (C) were untreated cells at 0h
under similar conditions. GADPH was the loading control.
Mitochondria were distinguished from the background by
their light scattering properties deduced from side scatter verꢀ
sus forward scatter plots. The selected gated population (R1,
Figure 5A) was analyzed for MTR fluorescence, test comꢀ
pound (TM608 or PHA665752) fluorescence and combined
MTRꢀtest compound fluorescence. More than 98% of the
events within R1 were MTR positive (Figure 5C), confirming
To determine if the presence of the TPP moiety would prefꢀ
erentially direct TM608 to the mitochondria, we treated HeLa
cells to subꢀlethal conditions of 4µM TM608 or PHA665752
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that mitochondria were being examined. Almost the same time would have allowed more of it to accumulate within the
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proportion of R1 events (98%) were TM608 positive (Figure
5D), an indication that the mitochondria were stained with
TM608. The proportion of R1 events that were “doubleꢀ
stained” with MTR and TM608 was exceedingly high (Q2
95%, Figure 5E), confirming the coꢀlocalization of MTR and
TM608 in the mitochondria. Interestingly, a small proportion
(Q2 35%) of the R1 events depicted double staining of MTR
and PHA665752 (Figure 5G) which implied that some
PHA665752 had gained access into the mitochondria, albeit at
levels that were greatly exceeded (2.5x) by TM608. Thus,
collaboratory evidence from two different approaches point to
the mitochondrial localization of TM608.
mitochondria and potently inhibit MET in that organelle, as
observed here. In the case of TM608, the mitochondrial targetꢀ
ing motif accelerated its entry into the mitochondria but hamꢀ
pered inhibition. Consequently, a longer time was necessary to
augment its MET inhibitory activity, which was likewise reꢀ
flected in other downstream events, as evidenced from the
slower onset of TM608ꢀinduced apoptosis that nonetheless
went to completion (Figure 3). Rapid localization to the mitoꢀ
chondria would advantageously blunt extraꢀmitochondrial offꢀ
target effects and this could have contributed to the cytotoxiciꢀ
ty advantage displayed by TM608 on TAMH cells.
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We then proceeded to determine if the localization of
TM608 in the mitochondria would suppress the activation of
mitochondrial MET in HCC827B cells. Whole cells and puriꢀ
fied mitochondria were separately treated with TM608 or
PHA665752 at 4 µM for 1h and 1.5h. Immunoblotting of
whole cell lysates and mitochondrial fractions showed that
MET protein expression was not altered by TM608 or PHAꢀ
665752 under these conditions (Figure 6A). On the other hand,
PHA665752 and TM608 suppressed MET phosphorylation at
Y1234/1235 in whole cell lysates. The suppression was preꢀ
dictably complete in lysates treated with PHA665752 but parꢀ
tial in the case of TM608. It would seem that the presence of
TPP had compromised the fit of TM608 at the kinase site, thus
partially attenuating its MET inhibitory activity.
When we probed mitochondrial fractions that were initially
isolated from HCC827B cells and then treated with either
TM608 or PHA665752 over the same time period, no eviꢀ
dence of phosphoꢀMET inhibition was detected for either
compound (Figure 6A). Such an outcome was anticipated for
PHA665752 in view of its limited presence in the mitochonꢀ
dria but was unexpected for TM608. We reasoned that
TM608 would require a longer treatment time to manifest its
suppressive effects on MET because it is a weaker MET inhibꢀ
itor than PHA665752. Hence HCC827B cells were exposed
to TM608 or PHA665752 (4 µM) for 24h, after which mitoꢀ
chondria were isolated from the treated cells and probed for pꢀ
MET Y1234/1235 (Figure 6B). The longer treatment time
necessitated a change in the experimental sequence (treatment
followed by isolation of mitochondria) as the structural integꢀ
rity of isolated mitochondria would be lost if the previous
method (isolation followed by treatment) was employed.
Whole cells treated with either compound for the extended
time were also probed for pꢀMET Y1234/1235. The results on
the whole cell lysates were essentially similar to those obꢀ
tained at the shorter time intervals, thus confirming that
TM608 partially inhibited the activation of MET in HCC827B
cells.
Figure 6: Immunoblots of MET and phosphoꢀMET
(Y1234/Y1235) in whole cell lysates and purified mitochonꢀ
drial proteins of METꢀoverexpressing HCC827B cells. (A)
Cells and pure mitochondria isolated from cells were separateꢀ
ly treated with PHA665752 or TM608 (4 µM) for 1h and 1.5h.
(B) Cells were treated with PHA665752 or TM608 (4 µM) for
24h, after which whole cell lysates and mitochondria were
isolated from treated cells and probed for phosphoꢀMET. In
both (A) and (B), 10 µg protein was loaded in each lane.
SDHA and actin were loading controls for mitochondria and
whole cells, respectively.
MET activates multiple signaling pathways and is intrinsiꢀ
cally networked for crossꢀtalk with other receptor tyrosine
kinases (RTKs).22 We have observed a hitherto unrecognized
facet of MET kinase activity, namely its localization to the
mitochondria of cancer cells which are high MET expressors
13 and the ensuing alteration of cellular functions that promote
survival and hence resistance to TKIs. Our finding resonates
with that of Ding et al. who showed that ERBB2 translocated
to mitochondria of cancer cells and that localization was assoꢀ
ciated with greater resistance to cell killing by trastuzumab, a
monoclonal antibody that blocks plasma membrane ERBB2
When we examined the treated mitochondrial fractions,
TM608 was now found to significantly suppress MET phosꢀ
phorylation but to our surprise, PHA665752 also diminished
the phosphorylation of mitochondrial MET. To explain these
unusual findings, we recalled that the preceding mitochondrial
coꢀlocalization investigations by flow cytometry detected
some PHA665752 in the mitochondria (Figure 5G) which
would imply that even without a mitochondrial targeting entiꢀ
ty, PHA665752 gains access to the mitochondria, possibly due
to its lipophilicity and charged state. Prolonging the exposure
23
kinase activation. The similarity of observations from these
independent studies on two different RTKs (MET and
ERBB2) hint at an expanded view of how these kinases affect
cellular functions beyond the canonical model of signaling
from the plasma membrane and the potential of targeting miꢀ
tochondrial RTKs as a means of exploiting a distinct vulneraꢀ
bility of cancer cells.
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4. Engelman, J.A.; Settleman, J. Acquired resistance to tyrosine kiꢀ
nase inhibitors during cancer therapy. Curr. Opin. Genet Dev. 2008,
18, 73ꢀ79.
In conclusion, our findings provide proof of concept that
TM608, a first example of a TPPꢀTKI conjugate, rapidly acꢀ
cumulates in mitochondria and suppresses the activation of
MET in high MET expressing, erlotinibꢀresistant HCC827B
cells (total cell lysates and mitochondrial fractions). That inhiꢀ
bition of MET activation by TM608 was time dependent but
incomplete points to the direction future synthetic efforts
should take, namely to reconcile targeted delivery with robust
potency in final analogs. It is however telling that in spite of
its weaker inhibitory activity, TM608 was as potent and apopꢀ
togenic as PHA665752 in abrogating NSCLC cell viability.
Directed accessibility to the mitochondria as afforded by a
targeting moiety like TPP could conceivably compensate for
deficient activity if uptake is rapid, cumulative and selective.
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L.; Roncalli, M.; Alloisio, M.; Santoro, A.; VarellaꢀGarcia, M. Inꢀ
creased MET gene copy number negatively affects survival of surgiꢀ
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11. Engelman, J.A.; Zejnullahu, K.; Mitsudomi, T.; Song, Y.; Hyꢀ
land, C.; Park, J.O.; Lindeman, N.; Gale, CꢀM.; Zhao, X.; Christenꢀ
sen, J.; Kosaka, T.; Holmes, A.J.; Rogers, A.M.; Cappuzzo, F.; Mok,
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12. Maina, F. Strategies to overcome drug resistance of Receptor
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1607ꢀ1617.
13.Guo, T.; Zhu, Y.; Gan, C.S.; Lee, S.S.; Zhu, J.; Wang, H.; Li,
X.; Christensen, J.; Huang, S.; Kon, O.L.; Sze, S.K. Quantitative
proteomics discloses MET expression in mitochondria as a direct
target of MET kinase inhibitor in cancer cells. Mol. Cell. Proteomics
2010, 9, 2629ꢀ2641.
14. T. Madak, J.; Neamati, N. Membrane permeable lipophilic catꢀ
ions as mitochondrial directing groups. Curr. Top. Med. Chem. 2015,
15, 745ꢀ766.
15.Millard, M.; Gallagher, J.D.; Olenyuk, B.Z.; Neamati, N. A seꢀ
lective mitochondrialꢀtargeted chlorambucil with remarkable cytotoxꢀ
icity in breast and pancreatic cancers. J. Med. Chem. 2013, 56, 9170ꢀ
9179.
16.Lee, C.W.; Park, H.K.; Jeong, H.; Lim, J.; Lee, A.; Cheon,
K.Y.; Kim, C.; Thomas, A.P.; Bae, B.; Kim, N.D.; Kim, S.H.; Suh, P.;
Ryu, J.; Kang, B.H. Development of a mitochondriaꢀtargeted Hsp90
inhibitor based on the crystal structures of human TRAP1. J. Amer.
Chem. Soc. 2015, 137, 4358ꢀ4367.
Docking of PHA665752 and TM608 in the Met Kinase binding
pocket (2WKM); Synthetic protocols, spectroscopic data and
purity determination of TM608; Protocols for cell culture, evaluaꢀ
tion of cell viability, cytotoxicity determinations, protein imꢀ
munoblotting, live cell imaging, mitochondrial isolation, flow
cytometry, determination of activated MET, localization of mitoꢀ
chondrial MET. The Supporting Information is available free of
charge on the ACS Publications website (http://pubs.acs.org).
Corresponding Authors
* Oi Lian Kon (dmskol@nccs.com.sg) and Mei Lin Go
(meilin.go@nus.edu.sg)
Author Contributions
The manuscript was written through contributions of all authors.
ACKNOWLEDGMENTS
This work was supported by the National Cancer Centre to OL
Kon and Biomedical Research Council (R148000706733) to ML
Go. The authors gratefully acknowledge Professor Tetsuya
Mitsudomi and Professor Kenichi Suda (Kinki University, Japan)
for their gift of the isogenic pair of nonꢀsmall cell lung cancer
HCC827 cells and the Drug Development Unit, National Univerꢀ
sity of Singapore for carrying out the cytotoxicity determinations.
ABBREVIATIONS
EGFR: Epidermal Growth Factor Receptor; NSCLC: Nonꢀsmall
cell lung cancer; HGF: Hepatocyte growth factor; TKI: Tyrosine
kinase inhibitors; TCA: tricarboxylic acid; TPP: triꢀ
phenylphosphonium; EDC: 1ꢀEthylꢀ3ꢀ(3ꢀdimethylaminopropyl)ꢀ
carbodiimide; TOM: Translocase of outer membrane; SDHA :
Succinate dehydrogenase complex subunit A; PARP : Poly ADP
ribose polymerase;
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For Table of Contents Use Only
A Mitochondrial-targeting MET Kinase In-
hibitor Kills Erlotinib-resistant Lung Cancer
Cells
Tianming Yang, Wai Har Ng, Huan Chen, Kaꢀ
mon Chomchopbun, The Hung Huynh, Mei Lin
Go, Oi Lian Kon.
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