A Red-Light-Activated Ruthenium-Caged NAMPT Inhibitor Remains Phototoxic in Hypoxic Cancer Cells

Article abstract of DOI:10.1002/anie.201703890

We describe two water-soluble ruthenium complexes, [1]Cl₂ and [2]Cl₂, that photodissociate to release a cytotoxic nicotinamide phosphoribosyltransferase (NAMPT) inhibitor with a low dose (21 J cm^?2) of red light in an oxyg

Full text of DOI:10.1002/anie.201703890

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Accepted Article
Title: A red light-activated ruthenium-caged NAMPT inhibitor remains
phototoxic in hypoxic cancer cells
Authors: Lucien N Lameijer, Daniël Ernst, Samantha L Hopkins,
Michael S Meijer, Sven HC Askes, Sylvia E Le Dévédec, and
Sylvestre Bonnet
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201703890
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A red light-activated ruthenium-caged NAMPT inhibitor remains
phototoxic in hypoxic cancer cells
Lucien N. Lameijer[a], Daniël Ernst[a], Dr. Samantha L. Hopkins[a], Michael S. Meijer[a], Dr. Sven H. C.
Askes[a], Dr. Sylvia E. Le Dévédec[b], Dr. Sylvestre Bonnet *[a]
Abstract: Herein we describe two water-soluble ruthenium
complexes [1]Cl2 and [2]Cl2 that photodissociate cytotoxic
compounds [1]Cl2 and [2]Cl2 (Scheme 1). Red light is superior to
previously reported blue- or green-light activation of PACT
compounds due to deeper tissue penetration (0.5-1.0 cm),[10]
and can be used with higher doses without significant light-
induced cytotoxicity.[11] Two sterically hindered ruthenium
photocaging scaffolds were chosen based upon earlier work by
the group of Turro and Kodanko:[12] [Ru(tpy)(dmbpy)(L)]2+ (tpy =
2,2’;6’-2”-terpyridine; dmbpy = 6,6’-dimethyl-2,2’-bipyridine) and
a
nicotinamide phosphoribosyltransferase (NAMPT) inhibitor with a
low dose (21 J cm-2) of red light in an oxygen-independent manner.
Using a specific NAMPT activity assay, up to an 18-fold increase in
inhibition potency was measured upon red-light activation of [2]Cl2,
while [1]Cl2 was thermally unstable. For the first time the dark and
red light-induced cytotoxicity of these photocaged compounds could
be tested under hypoxia (1% O2). In skin (A431) and lung (A549)
cancer cells a 3- to 4-fold increase in cytotoxicity was found upon
red light irradiation for [2]Cl2, whether the cells were cultured and
irradiated with 1% or 21% O2. These results demonstrate the
potential of photoactivated chemotherapy for hypoxic cancer cells
where classical photodynamic therapy, which relies on oxygen
activation, is poorly efficient.
[Ru(tpy)(biq)(L)]2+ (biq
=
2,2’-biquinoline). Both types of
complexes have an absorption band that extends in the red
region of the spectrum,[13] and photodissociate their ligand when
the monodentate is a thioether or a pyridine moiety.[12a, 14] These
scaffolds
were
conjugated
to
4-[({[4-(2-Methyl-2-
propanyl)phenyl]sulfonyl}amino)methyl]-N-(3-
pyridinyl)benzamide (STF-31),
compound natively containing
a
a
known cytotoxic organic
pyridine moiety that can
coordinate to ruthenium. The toxicity of STF-31 was reported to
originate from inhibition of both NAMPT enzyme activity[15] and
GLUT1.[16] We synthesized the two STF-31-containing
compounds [1]Cl2 and [2]Cl2 (Scheme 1), demonstrated that red
light can uncage STF-31, and show that this leads to efficient
PACT under both normoxia (21% O2), and hypoxia (1% O2).
NAMPT is a key enzyme in the salvage pathway of NAD+
biosynthesis that is abnormally up regulated in cancer cells.[1]
Importantly, high NAMPT expression has been associated with
poor prognosis in different types of cancer, which makes
NAMPT a potential therapeutic target.[2] It has been shown that
NAMPT inhibition leads to reduction of NAD+ intracellular levels,
which can induce apoptosis in cancer cells.[2a, 3] However, it has
also been reported that targeting of NAMPT might lead to side
effects such as blindness.[4] A strategy called PhotoActivated
ChemoTherapy (PACT) might solve selectivity issues.[5] PACT
consists in hiding the toxicity of a drug with a caging agent that
is released upon light irradiation, together with the free drug.[5b, 6]
Ruthenium polypyridyl complexes are particularly promising
photocaging groups as they can be removed using visible light,[7]
whereas most organic caging groups require UV light for
activation.[8] Unlike photodynamic therapy (PDT), a clinically
approved therapy that relies on the photocatalytic activation of
Scheme 1. STF-31 caged compounds [1]Cl2 and [2]Cl2
3O2 into 1O2 by
a
photosensitizer,[9] PACT is oxygen
Compounds [1]Cl2 and [2]Cl2 were synthesized by reacting STF-
31 with the precursors [Ru(tpy)(dmbpy)(Cl)]Cl[14] and
[Ru(tpy)(biq)(Cl)]Cl, respectively (Scheme S1). Compounds
were isolated as PF6 salts, purified over Sephadex LH-20 and
converted to their chloride salt by salt metathesis, to afford [1]Cl2
and [2]Cl2 as red or purple solids in 50% and 44% yield,
respectively.
independent[5b, 9] which makes it a promising and complementary
therapeutic strategy for addressing hypoxic tumours. However,
proof of efficacy of PACT under hypoxia is still lacking.
Herein we describe a setup using monochromatic red light on
living cells under hypoxia (1%), allowing the study of PACT
One of the challenges in PACT is to find the ideal balance
between thermal stability and photoactivation efficiency,
expressed as the photosubstitution quantum yield (Φ). Previous
research had shown that [Ru(tpy)(dmbpy)(SRR’)]2+ complexes
[a]
[b]
L. N. Lameijer, D. Ernst, Dr. S. L. Hopkins, M.S. Meijer, Dr. S.
Bonnet
Leiden Institute of Chemistry, Leiden University
Gorlaeus Laboratories, P.O Box 9502, 2300 RA Leiden (The
Netherlands)
E-mail: bonnet@chem.leidenuniv.nl
Dr. S. Le Dévédec
Leiden Academic Centre for Drug Research, Leiden University
Gorlaeus Laboratories, P.O Box 9502, 2300 RA Leiden (The
Netherlands)
are
less
stable
and
more
photoreactive
than
[Ru(tpy)(biq)(SRR’)]2+ in water,[14,17] therefore preventing their
application in PACT.[18] In contrast, Turro et al. have
demonstrated that complexes with L=pyridine are stable enough
to be isolated while retaining photosubstitution properties under
19]
low-energy visible light (λirr>590 nm).[12b,
Here the
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10.1002/anie.201703890
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photoreactivity of [1]Cl2 and [2]Cl2 in water was tested under red
light irradiation. Figure 1 shows the evolution of the electronic
absorption spectrum of [1]2+ upon activation at 625 nm under
deoxygenated conditions in H2O. The initial metal-to-ligand
charge transfer (1MLCT) band at 473 nm was gradually replaced
by a new 1MLCT band at λ = 484 nm with a clear isosbestic
point at 477 nm, to afford [Ru(tpy)(dmbpy)(H2O)]2+ (m/z found
536.1, calc m/z 536.1 for [Ru(tpy)(dmbpy)(OH)]+). According to
1H NMR (Figure S2) under white light irradiation in D2O the
doublet of [1]Cl2 at 6.89 ppm was replaced by two doublets at
the dose-response curves of treated vs. non-treated wells
(Figure 2 and Table S1).
Table 1. Absorption maxima (λmax), molar absorption coefficients at λmax (ε)
and at 625 nm (ε625), photosubstitution quantum yields (Φ625) at 298 and 310
K, 1O2 generation quantum yields (ΦΔ) at 293 K, and photosubstitution
reactivity (ξ= Φ625 x ε625).
t
6.80 and 7.78 ppm, while the characteristic Bu singlet at 0.94
ppm disappeared, confirming photodissociation of STF-31. The
photosubstitution quantum yield (Φ625) 0.058 at rt and 0.080 at
37 °C, are consistent with thermal promotion of the triplet metal-
centred states (3MC) from the photochemically generated 3MLCT
states.[12b] For [2]Cl2, irradiation at 625 nm resulted in a shift of
the MLCT band at 531 nm to 549 nm, and an aqua photoproduct
[Ru(tpy)(biq)(H2O)]2+ (found m/z=607.8, calcd m/z=608.1 for
[Ru(tpy)(biq)(OH)]+). According to 1H NMR in D2O the final
spectrum (Figure S3) showed a new, distinctive quartet at 8.86
ppm, and a decrease of the doublet at 6.69 ppm and singlet at
0.90 ppm. Photosubstitution occurred with a quantum yield Φ625
of 0.013 and 0.019 at rt and 37 °C, respectively (Table 1). The
lower photoreactivity of [2]Cl2 compared to [1]Cl2 is consistent
with previous work.[12b] Both [1]Cl2 (logP=-0.63±0.04) and [2]Cl2
(logP=-0.08±0.04, see ESI) are water-soluble, but STF-31 is not
(logP=+3.92), resulting in ligand precipitation during
photosubstitution of STF-31 in the NMR tube. Hence the caging
Ru complexes significantly increase water solubility of the
inhibitor. Before testing these compounds in cancer cells the
dose of red light necessary to obtain full activation in the cell
irradiation setup was evaluated to be 20.6 J.cm-2, which
corresponds to 10 minutes irradiation under normoxia (Figure
S4).[11]
[a]
[a]
ξ[20]
λmax
in
ε625
Φ625
ΦΔ [b]
at 293 K
Complex
nm[a]
at 298 K
(at 310 K)
in M-1cm-1
(ε in M-
1cm-1)
473
(8050)
0.058
(0.080)
2.2
(3.0)
[1]Cl2
[2]Cl2
379
609
<0.005
0.036
531
(9320)
0.013
(0.019)
0.79
(1.2)
[a] In H2O. [b] In CD3OD.
Figure 1. Electronic absorption spectra of [1]Cl2 (left) and [2]Cl2 (right) in
deoxygenated H2O under red light irradiation (625 nm, photon flux 1.30×10-7
mol.s-1). tirr= 5 min (every 30 s). T = 298 K..
The cytotoxicity of STF-31 and of its caged analogues [1]Cl2 and
[2]Cl2 was first tested in normoxic conditions (21% O2) against
three human cancer cell lines (A549, MCF-7, and A431) and a
Figure 2. a) (Photo)cytotoxicity (EC50) in μM for [1]Cl2 and [2]Cl2 in human
cancer cells under normoxia and hypoxia. b) EC50 for STF-31 and cisplatin
under normoxia and hypoxia (dark). Data points are averages (n = 3) with 95%
CI (in μM). * = p ≤ 0.05, ** ≤ 0.01, *** ≤ 0.001. Also see Table S1 .
[11]
non-cancerous cell-line (MRC-5).
24 h after seeding cells
were treated with STF-31, [1]Cl2 or [2]Cl2, and after 6 hours
incubation one plate was irradiated with red light (628 nm, 20.6
J∙cm−2) while the other was left in the dark. At t = 48 h medium
was replaced. Cell viability was then assayed by using
sulforhodamine B (SRB) 96 h after seeding.[21] Cell growth
inhibition effective concentrations (EC50) were calculated from
Under normoxia (Figure 2a), STF-31 was highly cytotoxic in all
cell lines, including MRC-5. [1]Cl2 also caused a great cytotoxic
effect on all cancerous cell lines, but its effect was limited on the
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non-cancerous MRC-5 cell line (EC50>20 μM). Importantly, a
negligible difference was found between the irradiated and non-
irradiated wells. This result was in great contrast to [2]Cl2, which
was less cytoxic against the non-cancerous MRC-5 cells in the
dark (EC50>40 μM) and highly toxic (EC50<10 μM) to cancerous
cells after irradiation, with a marked difference in cytotoxicity
between dark and irradiated cells for both A549 and A431.
Considering the minimal O2 production efficiency (0.036, Table
1), this effect is most likely attributed to the anti-proliferative
effect of the photoreleased STF-31.
commercial Cyclex® assay (Figure 3b). At 2 μM concentration
STF-31 showed the largest effect on NAMPT activity, confirming
that it is a NAMPT inhibitor.[15a] [2]Cl2 showed a dramatic
reduction in NAMPT activity after red light activation, whereas
the non-irradiated sample had much less inhibition. A similar
effect was observed for [1]Cl2, although the dark activity was
found much higher than for [2]Cl2. The dark inhibitory
concentrations (IC50) of 4.8 μM for [2]Cl2 was lowered by a factor
18 down to 0.26 μM after irradiation, which is similar to the value
obtained for STF-31 (0.25 μM, Table 1). The NAMPT inhibitory
effect of STF-31 was thus fully recovered upon red-light
activation of [2]Cl2.
1
To validate this hypothesis, [1]Cl2, [2]Cl2, and SFT-31, were
tested under hypoxia, in which 1O2 generation is impaired. We
therefore modified our published LED-based irradiation setup[11]
allowing irradiation on living cells while controlling O2
concentrations (1.0-21%, Figure S7). We then repeated the
cytotoxicity assay using the same protocol and light dose of 20.6
J.cm-2, but now at 1.0% O2 (see Figure S7, lower left). As shown
in Figure 2 and Table S1, the EC50 values for all compounds
were found higher than under normoxia, which is consistent with
earlier reports on the higher resistance of hypoxic cells to
chemotherapy.[22] No photocytotoxicity was observed for [1]Cl2.
However, the photoindices found for [2]Cl2 under hypoxia (3.6
and 2.4 for A431 and A549) were identical to those found under
normoxic conditions (3.3 and 2.6 for A431 and A549),
demonstrating for the first time that a lower O2 concentration
does not affect the photoindex of the PACT compound [2]Cl2.
We then investigated the enzyme inhibition properties of STF-31,
which is both a reported GLUT-1 and NAMPT inhibitor.[15a, 16a, 23]
When GLUT-1 overexpressing A549 cells[24] were starved using
glucose-free medium, incubated 2 h with a vehicle control (2%
DMSO), 50 μM STF-31, or 100 μM Phloretin, a well-known
GLUT-1 inhibitor,[25] treated with the fluorescent D-glucose
analogue NBDG,[26] and analysed by flow cytometry (Figure
S10), STF-31 showed a minimal glucose uptake inhibition
compared to phloretin.[27] Thus, the cytotoxicity of STF-31 is
most likely not related to impaired glucose uptake and GLUT-1
inhibition.
Table 1. NAMPT activity inhibitory concentration (IC50 with 95% confidence
intervals, in μM) obtained for STF-31, [2]Cl2 in the dark and [2]Cl2 after red
light irradiation. Photoindex (PI) = IC50,dark/IC50,light
.
[2]Cl2
(Dark)
[2]Cl2
Compound
STF-31
0.25
PI
(625 nm)[a]
IC50 in µM
(CI)
+0.027
-0.027
4.8
0.89
0.75
0.26
+0.079
-0.094
18
[a] Samples were irradiated for 10 minutes at 37 °C. See ESI.
The almost identical EC50 found for [1]Cl2 in the dark and after
light irradiation, and its high NAMPT inhibition in the dark,
suggested that [1]Cl is thermally unstable: When followed for 48
hours at 37 °C in the dark in OptiMEM® (Figure S8), [1]2+ slowly
decomposed to [Ru(tpy)(dmbpy)(OH2)]2+ while [2]2+ remained
stable (Figure S8 and Figure S9). This result is in contrast to
Kodanko et al. who used [Ru(tpy)(dmbpy)]2+ to cage a steroidal
CYP17A1
inhibitor.[12a]
According
to
our
results,
[Ru(tpy)(NN)(L)]2+ complexes are only stable enough for PACT
when NN=biq, while NN=dmbpy are more photoreactive
complexes that are also unstable in the dark.[14]
Figure 4. Stability of [1]Cl2 (left) and [2]Cl2 (right) in OMEM media + 2.5%
FCS at 37 °C over 48 hours in the dark. Spectra measured each hour, the
blue spectra were measured first, the red ones last. Arrow (left) indicates
a shift of the MLCT absorption maximum due to formation of the chlorido
species.
Figure 3. a) Normalized mean fluorescence intensity (MFI) of NBDG in A549
cells treated with control, STF-31, or phloretin. Data points are averages (n =
3) with ± SD. * = p ≤ 0.05, ** ≤ 0.01, *** ≤ 0.001. b) % of NAMPT activity
observed for compounds vs. control (2% DMSO) after 1 h. Data points are
averages (n = 2) with ± SEM. • = Vehicle control, ¨ = [2]Cl2 dark (2 µM), ▲ =
[1]Cl2 dark (2 µM), ¨ = [2]Cl2 light (2 µM), ▲ = [1]Cl2 light (2 µM), ■ = STF-31
(2 µM), ▼ = FK866 (20 µM).
In conclusion, we have demonstrated for the first time the
potential of PACT in hypoxic cancer cells using a photocaged
NAMPT inhibitor. Whereas under hypoxic conditions classical
PDT type II would not be effective because of the absence of
oxygen, [2]Cl2 represents a promising form of photocaged drug,
with a similar photoindex under hypoxia (1% O2) compared to
normoxia (21% O2). Also, this compound is soluble in water and
The enzyme activity inhibition of STF-31, [1]Cl2, and [2]Cl2,
towards recombinant NAMPT was then measured using the
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10.1002/anie.201703890
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This work was supported by the Dutch Organization for Scientific
Research (NWO-CW) via a VIDI grant to S.B. The European
Research Council is kindly acknowledged for financial support
via a Starting Grant to S.B. Prof. Elisabeth Bouwman is kindly
acknowledged for scientific support and input. Dr. Bart Limburg
is kindly acknowledged for setting up the irradiation setup for
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Keywords: photopharmacology • red light • hypoxia• ruthenium • cancer
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10.1002/anie.201703890
Angewandte Chemie International Edition
COMMUNICATION
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COMMUNICATION
Under red light irradiation two water-
soluble ruthenium complexes
Lucien N. Lameijer[a], Daniël Ernst[a], Dr.
Samantha L. Hopkins[a],[b], Michael S.
Meijer[a], Dr. Sven H. C. Askes[a], Dr.
Sylvia E. Le Dévédec[c], Dr. Sylvestre
Bonnet*
photorelease STF-31, a cytotoxic
NAMPT inhibitor, thus inhibiting
NAMPT enzyme activity up to 18
times better than in the dark. In cancer
cells photouncaging increased the
cytotoxicity of [2]Cl2 by a factor 3-4,
whether the cells were cultured and
irradiated with 1.0% or 21% O2. These
results demonstrate the efficiency of
photoactivated chemotherapy for
hypoxic cancer cells.
Page No. – Page No.
A red light-activated ruthenium-caged
NAMPT inhibitor remains phototoxic
in hypoxic cancer cells
This article is protected by copyright. All rights reserved.