Photoswitchable Antimetabolite for Targeted Photoactivated Chemotherapy

Article abstract of DOI:10.1021/jacs.8b08249

The efficacy and tolerability of systemically administered anticancer agents are limited by their off-target effects. Precise spatiotemporal control over their cytotoxic activity would allow improving chemotherapy treatments, and light-regulated drugs are well suited to this purpose. We have developed phototrexate, the first photoswitchable inhibitor of the human dihydrofolate reductase (DHFR), as a photochromic analogue of methotrexate, a widely prescribed chemotherapeutic drug to treat cancer and psoriasis. Quantification of the light-regulated DHFR enzymatic activity, cell proliferation, and in vivo effects in zebrafish show that phototrexate behaves as a potent antifolate in its photoactivated cis configuration and that it is nearly inactive in its dark-relaxed trans form. Thus, phototrexate constitutes a proof-of-concept to design light-regulated cytotoxic small molecules and a step forward to develop targeted anticancer photochemotherapies with localized efficacy and reduced adverse effects.

Full text of DOI:10.1021/jacs.8b08249

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Article
A photoswitchable antimetabolite for targeted photoactivated chemotherapy
Carlo Matera, Alexandre Gomila-Juaneda, Núria Camarero,
Michela Libergoli, Concepció Soler, and Pau Gorostiza
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b08249 • Publication Date (Web): 10 Oct 2018
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A photoswitchable antimetabolite for targeted photoactivated
chemotherapy
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Carlo Matera†,§, Alexandre Gomila-Juaneda†,§,‡, Núria Camarero†,§,‡, Michela Libergoli†,
Concepció Soler#, and Pau Gorostiza†,§,¶,*
† Institute for Bioengineering of Catalonia (IBEC), Barcelona Institute for Science and Technology (BIST)
§ Biomedical Research Networking center in Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN)
# Departament de Patologia i Terapèutica Experimental, Facultat de Medicina i Ciències de la Salut, IDIBELL, Universitat de
Barcelona
¶ Catalan Institution for Research and Advanced Studies (ICREA)
photopharmacology, photodynamic therapy, antiproliferative, arthritis, psoriasis, nanomedicine
ABSTRACT: The efficacy and tolerability of systemically administered anticancer agents are limited by their off-target effects.
Precise spatiotemporal control over their cytotoxic activity would allow improving chemotherapy treatments, and light-regulated
drugs are well suited to this purpose. We have developed phototrexate, the first photoswitchable inhibitor of the human
dihydrofolate reductase (DHFR), as a photochromic analog of methotrexate, a widely prescribed chemotherapeutic drug to treat
cancer and psoriasis. Quantification of the light-regulated DHFR enzymatic activity, cell proliferation, and in vivo effects in
zebrafish show that phototrexate behaves as a potent antifolate in its photoactivated cis configuration, and that it is nearly inactive
in its dark-relaxed trans form. Thus, phototrexate constitutes a proof-of-concept to design light-regulated cytotoxic small
molecules, and a step forward to develop targeted anticancer photochemotherapies with localized efficacy and reduced adverse
effects.
chemotherapeutic at the tumor site. The increased availability
of versatile materials and recent advances in the field of
INTRODUCTION
nanotechnology are certainly boosting this field, but yet the
clinical translation of such drug carriers has been very limited
so far.3
Although the neoplastic process has been recognized for
centuries, the treatment and cure of cancer still represent one
of the main challenges in medicine.1 Chemotherapy —the use
of cytotoxic agents to kill the rapidly proliferating cells
present in tumors— is one of the main actors in this fight,
together with radiation therapy, hormone therapy and surgery.2
However, the efficacy, tolerability and acceptance of cancer
chemotherapy are in many cases dramatically limited by low
therapeutic indices, non-specific targeting, and off-target
toxicity of the drugs used, which is intrinsic to their
antiproliferative effects.3 Alternative strategies to overcome
these drawbacks have emerged over the last years.
A third approach to improve cancer therapies is to activate the
drugs exclusively at their desired place of action.8 In fact, in
cancer types and stages that would benefit from a highly
localized treatment, a precise spatiotemporal control over the
activity of a cytotoxic agent would allow reducing the
concentration of active compound outside the targeted
neoplasm region, and would permit the administration of
higher doses of drug. As a result, localizing drug activity
would improve the efficacy of the treatment, reduce off-target
effects, enhance the patients’ quality of life and therapeutic
compliance. Light is a powerful tool in this respect.9,10 It offers
unparalleled opportunities as a non-invasive regulatory signal
for biological and pharmacological applications because it can
be delivered with high precision regarding space, time,
intensity and wavelength, it generally interacts weakly with
tissue (except for scattering), and causes negligible toxicity for
the human body.11,12,13,14,15 Several cancer treatments relying
on the use of light have emerged during the last decades.8 One
of those, known as photodynamic therapy (PDT), is already
well established and clinically applied. The procedure involves
administration of a photosensitizer that forms reactive oxygen
One approach relies on the design of pharmacological agents
interfering with targets that are overexpressed in tumor cells,
i.e. growth factors, signaling molecules, cell-cycle proteins,
and modulators of apoptosis and angiogenesis.1,4,5 However,
despite remarkable achievements obtained recently, the
development of such targeted cancer therapies is hampered by
the identification of tumor biomarkers, the selection of patient-
personalized treatments, and high costs.6,7 Another strategy to
reduce off-target toxicity in cancer treatments relies on the
design of engineered delivery systems (e.g., nanoparticles,
liposomes, micelles) that can selectively release the
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species (ROS) upon irradiation with light at a specific independent treatments relying on the use of photoactivatable
metal complexes22 or photoactivatable prodrugs (“caged
compounds”)23,24,25 have emerged as an alternative, but in
these cases the irreversibility of activation of the cytotoxic
species and their subsequent diffusion outside the intended site
of action represent in turn major disadvantages.
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wavelength, leading to direct tumor cell death, damage to the
microvasculature and induction of an inflammatory reaction.16
Although PDT is on the rise as a cancer treatment modality, its
dependence on the presence of molecular oxygen17,18,19 and the
frequent occurrence of unbearable inflammation-induced pain
represent important drawbacks that can remarkably limit its
applicability and success rate.20,21 Actually, oxygen-
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Figure 1. Mechanism of action of methotrexate (MTX) and design strategy of phototrexate (PHX). (a) Chemical structure of the
DHFR inhibitor MTX. (b) Mechanism of action of DHFR inhibitors (left) and cartoon representation of cell death (right) induced by a
conventional versus a light-regulated DHFR inhibitor: the latter should be constitutively inactive and allow DHFR inhibition upon
illumination at the proper wavelength. (c) Chemical structures of phototrexate in the trans (in the dark and under blue/white light
illumination, structure in blue) and cis (under UVA illumination, structure in purple) configurations. The red dotted rectangle defines the
moiety involved in the azologization strategy. (d) Crystal structure of the human DHFR in complex with MTX (in orange) and the cofactor
NADPH (in khaki) (PDB 1U72, protein surface representation with sectioning to reveal the active site pocket). The folded cis-like
configuration adopted by MTX in the active site is shown. Docked structures of trans (in blue) and cis (in purple) phototrexate are also
represented. (e),(f) Zoomed views of trans (in blue) and cis (in purple) phototrexate (best docked poses) superimposed on the
crystallographic structure of MTX (in orange) in the active site of the human DHFR. In our docking simulations (see SI for details), unlike
its trans counterpart, cis-phototrexate shows a high degree of overlap to the crystallographic pose of MTX. Nitrogen and oxygen atoms of
the ligands are colored in blue and red, respectively.
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candidates using medical and scientific literature42,43 and
public databases, including DrugBank,44 ChEMBL,45 Protein
Data Bank,46 and Drugs.com.47 This survey ranked
methotrexate (MTX) as one of the most prominent drugs
matching the identikit to develop a light-regulated cytotoxic
agent (Figure 1a).
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Overall, current strategies to improve the safety and efficacy
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of cancer treatment are promising, but the availability of truly
tumor-selective therapies remains an unmet medical need.
Photopharmacology, which relies on the use of reversibly
photoswitching drugs, is well-suited to this task.26,27,28,29
Several light-regulated compounds have been described to
reversibly control a diversity of proteins. They are obtained by
introduction of a molecular photoswitch into the structure of
an existing drug in order to alter its pharmacological
properties upon illumination.27 Azobenzenes, and more in
general arylazo compounds, have been widely employed as
molecular photoswitches because of their favorable
characteristics, namely design flexibility, ease of synthesis,
large changes in geometry upon isomerization, relatively
stable photostationary states and high quantum yields, fast
photoisomerization rates, spontaneous relaxation to the
thermodynamically more stable configuration (T-type
photoswitch), and low photobleaching rates.15 Furthermore,
the use of compounds bearing arylazo moieties has been
proven safe in humans, including approved drugs and food
colorants.27 Examples of bioactive molecules with
photoswitchable efficacy include G protein-coupled receptor
modulators,30,31,32 ion channel modulators,33 enzyme
inhibitors,34 and inhibitors of protein-protein interactions.35
Recently, a number of photoswitchable cytotoxic agents acting
on different biological targets have also been reported, thus
demonstrating an increasing attention to this approach.9,36,37
Indeed, photopharmacology has a transformative potential, by
bringing together the advantages of targeted therapies and
light-based oxygen-independent therapies in order to improve
the safety and efficacy of anticancer treatments. Of relevance
are the results obtained with photoswitchable combretastatin
A-4 analogues, named photostatins or azo-combrestatins,
acting as tubulin polymerization inhibitors. Four research
groups have independently published their findings on the
efficacy of such derivatives as photoswitchable light-regulated
cytotoxic agents in different cell lines.38,39,40,41 Another
example was published by Szymanski and coll.10 Their
photoswitchable histone deacetylase inhibitor displayed a
remarkable cytotoxicity in vitro and emerges as a putative lead
compound for photopharmacological chemotherapy.
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MTX belongs to a specific class of chemotherapeutic agents
known as antimetabolites, which kill hyperproliferative cells
by targeting their metabolism. Although antimetabolites have
been known for long, they are still actively developed for
cancer chemotherapy and are widely used in the treatment of
diverse human tumors.48,49 Notably, MTX inhibits
dihydrofolate reductase (DHFR), which catalyzes the
conversion of dihydrofolate (dhf) to tetrahydrofolate (thf), a
key step in folate metabolism (Figure 1b). Interrupting folate
metabolism compromises thymidine synthesis, and interferes
with purine synthesis and other reactions involving single-
carbon transfers, leading to cell death (Figure 1b).48,50 MTX is
commonly used alone or in combination with other
antineoplastic drugs in the treatment of certain cancers
including but not limited to breast cancer, epidermoid cancers
of the head and neck, lung cancer (particularly squamous cell
and small cell types) and bladder cancer. In addition, because
of its immunosuppressive properties, it is also indicated in the
symptomatic control of severe, recalcitrant, disabling
psoriasis, and in the management of severe, active rheumatoid
arthritis.51 However, the importance and ubiquity of the MTX
target (DHFR) in all cell types is at the origin of a poor drug
safety profile that often leads to unbearable side effects
requiring dose reduction or treatment cessation. The most
frequently reported adverse reactions include ulcerative
stomatitis, leukopenia, nausea, abdominal distress, malaise,
undue fatigue, chills and fever, dizziness, and decreased
resistance to infection.52
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Besides the motivation to enhance the pharmaco-toxicological
profile of MTX, two reasons supported the feasibility of
photocontrolling MTX activity. The first one is that
azobenzene analogs of the MTX structure can be readily
designed. There are two general strategies to devise arylazo-
based light-regulated drugs: (1) by elongation of the drug
structure with an arylazo moiety (“azo-extension”),53,54,55 and
(2) by incorporation of an arylazo moiety into the
pharmacophore of the drug (“azologization”).30,56 MTX is
suitable for the latter, which is generally preferred because it
requires minimal modifications of the original structure, thus
maintaining the druglikeness of the parent compound, and
largely preserving its pharmacokinetic and pharmacodynamic
properties (Figure 1c).57 The second reason is that the activity
of MTX azologs is expected to be higher in their cis state
(“cis-on”) than in the most thermodynamically stable trans
configuration. This prediction is based on the crystal structure
of the ternary complex of MTX with the wild-type human
DHFR and the cofactor NADPH (PDB 1U72),58 in which
MTX is bound to the enzyme active site in a bent “cis-like”
configuration (Figure 1d-f). When designing bioactive
azologs, a “trans-off/cis-on” behavior is often desired because
such compounds can be safely applied (e.g., systemically) in
the inactive relaxed form and they can be photoactivated at the
required times and locations. Thus, MTX azologs fulfill all the
above-mentioned structural and (photo)pharmacological
Here, we have taken one step further towards the development
of light-regulated cancer chemotherapy by photocontrolling
the in vitro antiproliferative action and in vivo toxicity of a
target-specific, widely prescribed chemotherapeutic agent. For
that purpose, we first performed a selection of protein targets
and anticancer drugs complying with several criteria: (1)
clinically validated target, (2) wide application in high-
incidence diseases, (3) in use to treat cancer forms affecting
organs or tissues potentially accessible to light, either directly
or through natural orifices (e.g., skin, bladder, intestine), (4) a
drug safety profile including severe systemic side effects
which currently limit its use, (5) drug structure suitable for
photoregulation with minimal modifications, (6) biological
inertness in the dark-relaxed form, (7) compatibility with
phototherapy and/or used as adjunctive treatment, and (8)
synthetic accessibility. We extensively documented the
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design requirements of a cis-on drug-like antifolate candidate
for light-regulated chemotherapy.
intermediates were then combined under Mills conditions
(55% yield) to give (S,E)-diethyl 2-(4-((2,4-
diaminoquinazolin-6-yl)diazenyl)benzamido)pentanedioate
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(3), which was finally hydrolyzed in alkaline conditions to
provide phototrexate in high yield (95%). Physicochemical
characterization of each intermediates and the final compound
is provided in the SI file.
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RESULTS AND DISCUSSION
Design strategy and synthesis. MTX structural azologs
could be designed by replacing the methyleneamino bridge
between the diaminopteridine and the benzamido moieties
with an azo (–N=N–) group. However, before applying this
strategy, we reviewed the structure-activity relationships of
MTX and related analogs in order to define the role of this
bridge. We found out that a great variety of structural variants
of the bridge are tolerated, and some of them can even favor
the accumulation and persistence of the drug in responsive
tumor cells rather than in normal proliferative tissues.59,60
Moreover, it is reported that 5,8-dideaza (quinazoline) DHFR
inhibitors obtained by isosteric replacement of the nitrogen
atoms at positions N5 and N8 (–N=C) of the pteridine ring
with –CH= groups are endowed with comparable inhibitory
activity.61,62 In this regard, generating a diazo bond at position
C6 of a quinazoline is expected to be more synthetically
accessible than at a pteridine, while still providing a strong
DHFR inhibitor. Thus, we conceived a putative photochromic
DHFR inhibitor by MTX azologization and two bioisosteric
substitutions, and named it phototrexate (PHX, Figure 1c).
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In order to achieve the desired biological properties (Figure
1b), phototrexate should be constitutively inactive in its most
thermodynamically stable configuration (trans isomer, dark-
adapted state), and it should mimic the pharmacological
effects of the parent drug (MTX) upon illumination at the
proper wavelength (cis isomer). To test the feasibility of our
design, we performed molecular docking simulations of cis-
and trans-phototrexate in the atomic structure of the human
DHFR (PDB 1U72).58 The results (see also SI for details)
show that cis-phototrexate binds to the active site of the target
enzyme in a mode that strictly mimics the orientation and
conformation adopted by MTX in its crystallographic pose,
with a root mean square deviation (RMSD) of 0.5 Å between
the best-scoring pose of cis-phototrexate and the
crystallographic pose of MTX, and a predicted binding energy
of −10.8 kcal/mol (Figure 1f). In contrast, trans-phototrexate
displayed a set of binding poses that barely overlapped with
MTX, with a RMSD of 4.8 Å between its best docked pose
and the crystallographic pose of MTX, and a predicted binding
energy of −9.3 kcal/mol (Figure 1d-f). Overall, these
computational results lend support to the assumption of a “cis-
on” antifolate behavior of phototrexate and encouraged
pursuing the synthesis of the target compound.
Scheme 1. The chemical synthesis of phototrexate.
Photochemical
characterization.
An
essential
requirement for using phototrexate as a light-dependent DHFR
inhibitor is that it efficiently responds to light, which means
that it can be quickly photo-isomerized between two different
configurations with
a
relatively high degree of
photoconversion (trans/cis ratio). UV/Vis absorption studies
showed a robust photochromic behavior in aqueous solution.
Phototrexate can be effectively isomerized from trans to cis
with UVA light (375 nm), and back-isomerized from cis to
trans with blue (460 nm) or white light (Figure S2). This
transition is reversible and can be repeated several times
without any noticeable loss of absorption (Figure S3). We then
Phototrexate was prepared via a convergent 4-step synthetic
route starting from commercially available compounds
(Scheme 1). To generate the diazo bond of phototrexate, we
planned to perform
a coupling reaction between its
quinazoline and benzamido building blocks. The required
amine and nitroso intermediates were therefore prepared for
this purpose. A catalytic hydrogenation of 6-nitroquinazoline-
2,4-diamine afforded the desired quinazoline-2,4,6-triamine
(1) in high yield (98%), while (S)-diethyl 2-(4-
nitrosobenzamido)pentanedioate (2) was obtained by
oxidation of the corresponding amine with Oxone® in a
biphasic system of dichloromethane and water. These
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quantified by H-NMR analysis the ratio between the two
isomers at the photostationary state (PSS). The amount of the
thermodynamically less stable cis isomer shifted from 0%
(dark-adapted state) to 76% upon illumination with 375 nm
light for 30 minutes (4.6 J/cm2, Figure 2a). In addition, as
phototrexate is a T-type photoswitch, we also studied its
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spontaneous thermal relaxation (from cis to trans) in dark photoconversion is achieved with a fluence greater than 0.6
J/cm2. Overall, the photochromic properties of phototrexate
show that it can be efficiently and reversibly photoisomerized
using UVA and white light, and that it has a stability of
several hours in the dark at the normal human body
temperature.
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conditions. The measured half-life was approximately 690 min
at 25 °C and 236 min at 37 °C in aqueous solution (Figure 2b).
Finally, we investigated the time dependence (Figure 2c) and
the light fluence dependence (Figure 2d) of the
photoisomerization (from trans to cis) under UVA (375 nm)
illumination.
These
results
show
that
maximal
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Figure 2. Photochemical characterization of phototrexate (PHX). (a) Composition of the photostationary state achieved after
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illumination with 375ꢀnm light for 30 min (4.61 J/cm2) determined by H NMR (5 mM in methanol-d4). (b) Thermal relaxation of
phototrexate (from trans to cis) in the dark at 25 °C (50 μM) and 37 °C (100 μM), measured by absorbance at 350 nm. Half-lives were
estimated using the one-phase decay function in GraphPad Prism 6 (nonlinear regression curve fitting). (c) Time dependence of
photoisomerization of phototrexate (from trans to cis) under 375 nm light measured by absorbance of a 200 μM solution at 350 nm. Data
were fitted to the one-phase decay function in GraphPad Prism 6 (nonlinear regression curve fitting). (d) Light fluence dependence of
photoisomerization of phototrexate (from trans to cis) under 375 nm light measured by absorbance of a 200 μM solution at 350 nm. Data
were fitted to the four-parameter dose-response curve in GraphPad Prism 6 (nonlinear regression curve fitting).
strongly (70%) than the trans isomer at the same concentration
In vitro photopharmacological characterization. After
(21%) (Figure 3a). The stronger inhibition capacity exhibited
obtaining the target chemical compound with the desired
by cis-phototrexate thus matched with our molecular docking
simulations and fulfilled the design requirements of a cis-on
antifolate (Figure 1).
photochromic properties, we characterized its biological
activity using in vitro assays. For convenience, in the text we
will refer to the all-trans form of phototrexate in the dark-
adapted state as trans-phototrexate, and to the cis-enriched
form (obtained at the PSS upon illumination at 375 nm) as cis-
phototrexate. We first assessed the ability of the two
photoisomers of phototrexate to inhibit the purified target
enzyme. To this end, we quantified DHFR activity using a
colorimetric assay (see SI for details) and observed significant
differences between the two isomers at 10 nM and 100 nM,
with 10 nM cis-phototrexate inhibiting DHFR activity more
We next examined the cytotoxicity of phototrexate in cells
using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-
a
diphenyltetrazolium bromide] assay for cell viability. HeLa
cervical cancer cells were incubated with trans- and cis-
phototrexate for 24 hours at 37 °C, and cell viability was
subsequently measured. Methotrexate was tested in the same
conditions as the positive control. Cells were also incubated in
vehicle solution (negative control wells) and used for
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normalization of cell viability. The cis isomer caused conditions (~236 minutes at 37 °C, for an incubation time of
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significantly lower viability to HeLa cells than the trans
isomer (Figure 3b), in line with the results obtained in the
enzyme inhibition assay. Notably, 1 μM cis-phototrexate
reduced viability to 27%, whereas the trans isomer barely
altered it (88%). At the maximum concentration tested (100
μM), only 23% of cells survived to cis-phototrexate, against
78% for trans-phototrexate. A remarkable difference in IC50
was estimated for the two isomers, with a value of about 6 nM
for the cis and 34 μM for the trans compound. Interestingly,
cis-phototrexate was as effective as the reference drug
methotrexate, which reduced cell viability to 40% at 1 μM and
33% at 100 μM. Overall, these results indicate that MTX and
cis-phototrexate have a similar cytotoxicity profile in HeLa
cancer cells, whereas trans-phototrexate is nearly inactive at
the same concentrations. Considering that cis-phototrexate has
a relatively short half-life of thermal relaxation in the test
24 hours), the difference observed between cis and trans in
terms of cytotoxicity is remarkable. The effect of cis-
phototrexate can be realistically ascribed to the disruption of
the folate pathway, since this process plays an essential role in
the synthesis of DNA and RNA, in the metabolism of amino
acids, and ultimately in the process of cell division.
Nevertheless, a diversity of effects of the two photoisomers on
other biochemical pathways beside the folate pathway (e.g.,
inhibition of thymidylate synthase and glycinamide
ribonucleotide transformylase),63 dissimilarities in cellular
uptake and polyglutamation,63 or differences in off-target
binding to intracellular structures,64 as reported for MTX,
should not be ruled out and could account for the differences
in drug sensitivity between assays with purified target protein
and assays on more complex systems, such as cells and living
organisms.
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Figure 3. In vitro pharmacological characterization of phototrexate (PHX). (a) Inhibition of DHFR activity at 1 nM, 10 nM and 100
nM of trans (black) and cis (violet) phototrexate. Data are means of at least three independent experiments in triplicate or quadruplicate ±
SEM. Results were analyzed by two-way ANOVA with Bonferroni's multiple comparisons test for statistical significance (GraphPad Prism
6; adjusted p-value (*) ≤ 0.05). (b) HeLa cell viability assay at different concentrations of MTX, cis-phototrexate and trans-phototrexate.
The relatively low vertical span and Hill coefficient of the sigmoidal curves of MTX and cis-phototrexate might be ascribed to short
exposure time of the cells to the drugs,65 saturation of the carrier-mediated uptake, and partitioning of the inhibitors into an inaccessible
form at the higher concentrations.66 Data are means of at least three independent experiments in triplicate or quadruplicate ± SEM. Results
were analyzed by two-way ANOVA with Bonferroni's multiple comparisons test for statistical significance (GraphPad Prism 6; adjusted p-
value (****) ≤ 0.0001 for trans-phototrexate versus cis-phototrexate).
°C. MTX was also tested as the positive control (200 μM).
Each embryo (normally until 48-72 hpf) and hatched larva
Disruption of folate metabolism in zebrafish larvae.
In view of the efficacy and light-regulation of phototrexate
(from 48-72 hpf) was observed daily to monitor changes in
antiproliferative properties in vitro, we evaluated the
their anatomy and behavior. The four groups (vehicle, MTX,
performance of our compound in a small animal model. Since
DHFR inhibitors disrupt folate metabolism, they have a high
impact at early stages of animal development. Zebrafish
cis-phototrexate, trans-phototrexate) were compared at 72 and
96 hpf, when certain anatomical and behavioral traits become
easier to observe (Figure 4a-d). In particular, healthy hatched
(Danio rerio) embryos have been used to characterize the
larvae reach the stage of protruding-mouth at 72 hpf,68 with a
effects of methotrexate67 and thus constitute an excellent
length of about 3.5 mm. Normal anatomical traits include
iridiophores in the yolk and half of the eye, with yellow
pigmentation on the head and in the dorsal part and black
pigmented lateral and ventral stripes from the head to the tail,
model system to investigate the effects of trans- and cis-
phototrexate in vivo. Zebrafish fertilized eggs were incubated
within 5 hours post fertilization (hpf) in UV purified water
containing trans- or cis-phototrexate (200 μM) for 5 days at 28
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as well as observable heart beat and blood flow (see SI for
details).68
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Figure 4. Effects of phototrexate (PHX) on zebrafish development, viability and mortality. Experiment 1, panels a-d: cis-phototrexate
was obtained by external pre-illumination with 375 nm light (see SI for details). (a), (c) Anatomical profiles of zebrafish at 72 hpf after
treatment with vehicle (DMSO 2%), MTX (200 μM), cis-phototrexate (200 μM), and trans-phototrexate (200 μM). The graph shows the
percentage of zebrafish treated versus the treatment group, superimposing the number of abnormal larvae (black and yellow checkered
sections) to the number of viable embryos (checkered sections) and to the total number of fertilized embryos (whole columns). (b)
Illustrative pictures of individual larvae from each treatment group at 72 hpf, showing the observable aberrant developmental traits in the
MTX and cis-phototrexate groups. (d) Mortality of zebrafish at 96 hpf after treatment with vehicle (DMSO 2%), MTX (200 μM), cis-
phototrexate (200 μM), and trans-phototrexate (200 μM). The graph shows the percentage of zebrafish treated versus the treatment group,
superimposing the number of dead zebrafish (black and red checkered sections) to the number of viable embryos (checkered sections) and
to the total number of fertilized embryos (whole columns). Experiment 2, panels e-f: cis-phototrexate was obtained by in situ illumination
with 375 nm light in the presence of zebrafish. (e) Anatomical profiles of zebrafish at 96 hpf after treatment with vehicle/dark (DMSO 2%,
no illumination), vehicle/375 nm light (DMSO 2%, 0.61 J/cm2, 4 min illumination twice a day), trans-phototrexate (200 μM, dark), and
cis-phototrexate (200 μM, 0.61 J/cm2, 4 min illumination twice a day). The graph shows the percentage of zebrafish treated versus the
treatment group, superimposing the number of abnormal larvae (black and yellow checkered sections) to the number of viable embryos
(checkered sections) and to the total number of fertilized embryos (whole columns). (f) Mortality of zebrafish at 96 hpf after treatment with
vehicle/dark (DMSO 2%, no illumination), vehicle/375 nm light (DMSO 2%, 0.61 J/cm2, 4 min illumination twice a day), trans-
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phototrexate (200 μM, dark), and cis-phototrexate (200 μM, 0.61 J/cm2, 4 min illumination twice a day). The graph shows the percentage
of zebrafish treated versus the treatment group, superimposing the number of dead zebrafish (black and red checkered sections) to the
number of viable embryos (checkered sections) and to the total number of fertilized embryos (whole columns).
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in which phototrexate was switched on with light was
Embryos treated with MTX presented a low viability at 24
remarkably higher than those maintained in the dark (two- and
hpf, with six out of seventeen total embryos viable (35%,
eight-fold, respectively). These results (Figure 4e-f) are in
Figure 4a). At 72 hpf, all six individuals presented three
good agreement with those obtained in the previous
experiment (Figure 4a-d) and demonstrate that the cytotoxic
effects of phototrexate can be turned on also by direct
illumination of living animals treated with its trans “inactive”
form.
abnormalities: a deficient iridiophore ocular pigmentation,
with complete black colored eyes and a deficient black body
stripe patterns; an abnormal volume of the cardiac cavity; and
tail angle deviations, with prominent bent or curved tails
(Figure 4b). Viability in the vehicle and cis-phototrexate
groups remained within normal batch levels (only two and one
non-viable embryos, respectively), and perfect in the trans-
phototrexate group (Figure 4b-c). In the vehicle group, only
one larva presented malformation, a non-hatched individual
with unformed tail. Cis-phototrexate produced a toxicity
profile similar to that of the reference compound MTX,
generating comparable developmental abnormalities at 72 hpf
together with a high rate of mortality at 96 hpf (Figure 4d). In
stark contrast, trans-phototrexate showed a lower general
toxicity, as it did not produce any observable abnormality at
72 hpf and no mortality at 96 hpf.
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Overall, our experiments in vivo, together with the in vitro
assays (Figure 3), indicate that photoactivated cis-phototrexate
has a pharmacological profile of a potent antifolate drug,
whereas it is almost inert in its trans configuration. Slight
discrepancies observed between MTX and cis-phototrexate
activities on cellular and organism assays might be related in
part to non-identical off-target activities.
CONCLUSIONS
In summary, we have rationally designed, synthesized, and
characterized phototrexate, the first light-regulated inhibitor of
the human DHFR, and we have demonstrated its antifolate and
antiproliferative properties using in vitro and in vivo assays.
Phototrexate has been derived by azologization and
bioisosteric substitutions of methotrexate, a DHFR inhibitor
that is extensively used to treat many forms of cancer, severe
psoriasis and rheumatoid arthritis. In this way, the activity,
drug-likeness and other determinants of the pharmacokinetic
profile of the parent compound should be retained. Our results
show that phototrexate can be switched on with UVA light to
become a potent antifolate drug comparable to methotrexate,
and it can be switched off with blue or white light and by
thermal relaxation in the dark. This profile offers the
opportunity of inhibiting cellular proliferation in a target-
specific manner and at confined locations, while reducing
these effects in other regions. In addition, light-activated
phototrexate self-deactivates (thermally relaxes) upon
diffusing away from the treated region. Thus, phototrexate
constitutes a proof-of-concept to develop photoswitchable
antimetabolite drugs, and makes it possible to explore
photopharmacological chemotherapies with reduced systemic
side effects and with enhanced tolerability and antineoplastic
efficacy. Major advantages of such light-based approach over
the existing ones are that the effects of photoswitchable
chemotherapeutics like phototrexate are target specific (cf.
PDT), oxygen-independent (cf. PDT), and reversible (cf. PDT,
photoactivatable metal complexes, and photocleavable
prodrugs). Small molecules like phototrexate could be
administered topically in their active form, or systemically in
their inactive form followed by precise photoactivation only at
the lesion area. Target regions might be tissues that can be
exposed to illumination, like the skin and the digestive,
respiratory and reproductive tracts. Notably, the envisaged
therapy may also benefit of medical devices and technologies
currently in use for clinically approved light-based therapies
(e.g., PDT, PUVA), thus facilitating its medical and regulatory
acceptance. Furthermore, phototrexate could also be used as a
powerful tool to study developmental processes with high
spatiotemporal precision. The wavelength needed to switch
Next, we tested the performance of phototrexate by
photoswitching its pharmacological effects in situ. We used
again zebrafish as animal model. Four groups of zebrafish
fertilized eggs (n = 48 in each group) were incubated within 5
hours post fertilization (hpf) in UV purified water containing
the drug vehicle (DMSO 2%, groups 1 and 2) or phototrexate
(200 μM, groups 3 and 4) for 5 days at 28 °C (Figure 4e-f).
Groups 1 and 3 (vehicle and trans-phototrexate) were
constantly kept in the dark, whereas group 4 was illuminated
twice a day with 375 nm light (0.61 J/cm2, 4 min illumination)
to generate a significant amount of cis-phototrexate directly in
the fish medium. Group 2 (vehicle) underwent a parallel and
identical protocol of illumination to determine whether the
direct exposure to UVA light could intrinsically affect fish
development and survival. The viability of individual embryos
and hatched larvae was monitored daily. The four groups were
finally examined at 96 hpf to assess the abnormality and
mortality profiles (Figure 4e-f). All groups presented normal
development and a good viability at 24 hpf (greater than 80%).
When comparing the number of larvae with anatomical
malformations and abnormalities to the total number of
embryos, both vehicle groups (dark and UVA) presented 12%
of abnormal individuals (with anomalous volume of the
cardiac cavity and sidewise position) at 96 hpf. In the trans-
phototrexate group, 30% of the larvae were affected by
abnormalities, including cardiac cavity and curved tails, while
60% of the individuals were found with a similar profile of
abnormalities and lower pigmentation in the group where
phototrexate was switched on (cis isomer) with UVA light
(Figure 4e). Analyzing the mortality profile at 96 hpf, the
vehicle/dark group did not show any loss of individuals, and
only 2% were found dead in the vehicle/UVA group.
Therefore, the light intensity, light wavelength and
illumination protocol adopted for the experiment were well
tolerated by the fish. Importantly, we found out that only 6%
of the total embryos (7% of the viable ones) were dead in the
trans-phototrexate group against the 46% of the total embryos
(51% of the viable ones) in the cis-phototrexate group (Figure
4f). Thus, the abnormality and mortality rate in the fish wells
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65.
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Chabner, B. A.; Roberts, T. G. Nat Rev Cancer 2005, 5 (1),
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infrared region).70,71
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ASSOCIATED CONTENT
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Corresponding Author
* pau@icrea.cat
Author Contributions
All authors have given approval to the final version of the
manuscript. / ‡ AGJ and NC contributed equally.
Funding Sources
This project has received funding from the European Union’s
Horizon 2020 Framework Programme for Research and
Innovation under the Specific Grant Agreement No. 720270
(Human Brain Project SGA1), AGAUR/Generalitat de Catalunya
(CERCA Programme and 2017-SGR-1442), FEDER funds,
ERANET SynBio MODULIGHTOR, Fundaluce and Ramón
Areces foundations, and MINECO projects CTQ2016-80066R to
PG and SAF2017-83815R to CS. CM was supported by the
Ermenegildo Zegna Founder’s Scholarship.
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Notes
The authors have filed a patent application for photoisomerizable
derivatives of dihydrofolate reductase inhibitors.
ACKNOWLEDGMENT
Authors are grateful to J. M. Vela, S. Fonquerna, A. Riera, and A.
Fernández-García for useful discussions. Molecular graphics and
analyses were performed with the UCSF Chimera package.
Chimera is developed by the Resource for Biocomputing,
Visualization, and Informatics at the University of California, San
Francisco (supported by NIGMS P41-GM103311). Mass
spectrometry was performed at the IRB Barcelona Mass
Spectrometry Core Facility, which actively participates in the
BMBS European COST Action BM 1403 and is a member of
Proteored, PRB2-ISCIII, supported by grant PRB2 (IPT13/0001 -
ISCIII-SGEFI / FEDER).
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Woolley, G. A. Acc. Chem. Res. 2015, 48 (10), 2662.
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Figure 1. Mechanism of action of methotrexate (MTX) and design strategy of phototrexate (PHX). (a)
Chemical structure of the DHFR inhibitor MTX. (b) Mechanism of action of DHFR inhibitors (left) and cartoon
representation of cell death (right) induced by a conventional versus a light-regulated DHFR inhibitor: the
latter should be constitutively inactive and allow DHFR inhibition upon illumination at the proper wavelength.
(c) Chemical structures of phototrexate in the trans (in the dark and under blue/white light illumination,
structure in blue) and cis (under UVA illumination, structure in purple) configurations. The red dotted
rectangle defines the moiety involved in the azologization strategy. (d) Crystal structure of the human DHFR
in complex with MTX (in orange) and the cofactor NADPH (in khaki) (PDB 1U72, protein surface
representation with sectioning to reveal the active site pocket). The folded cis-like configuration adopted by
MTX in the active site is shown. Docked structures of trans (in blue) and cis (in purple) phototrexate are also
represented. (e),(f) Zoomed views of trans (in blue) and cis (in purple) phototrexate (best docked poses)
superimposed on the crystallographic structure of MTX (in orange) in the active site of the human DHFR. In
our docking simulations (see SI for details), unlike its trans counterpart, cis-phototrexate shows a high
degree of overlap to the crystallographic pose of MTX. Nitrogen and oxygen atoms of the ligands are colored
in blue and red, respectively.
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Scheme 1. The chemical synthesis of phototrexate.
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Figure 2. Photochemical characterization of phototrexate (PHX). (a) Composition of the photostationary state
achieved after illumination with 375ꢀnm light for 30 min (4.61 J/cm2) determined by 1H NMR (5 mM in
methanol-d4). (b) Thermal relaxation of phototrexate (from trans to cis) in the dark at 25 °C (50 μM) and
37 °C (100 μM), measured by absorbance at 350 nm. Half-lives were estimated using the one-phase decay
function in GraphPad Prism 6 (nonlinear regression curve fitting). (c) Time dependence of
photoisomerization of phototrexate (from trans to cis) under 375 nm light measured by absorbance of a 200
μM solution at 350 nm. Data were fitted to the one-phase decay function in GraphPad Prism 6 (nonlinear
regression curve fitting). (d) Light fluence dependence of photoisomerization of phototrexate (from trans to
cis) under 375 nm light measured by absorbance of a 200 μM solution at 350 nm. Data were fitted to the
four-parameter dose-response curve in GraphPad Prism 6 (nonlinear regression curve fitting).
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Figure 3. In vitro pharmacological characterization of phototrexate (PHX). (a) Inhibition of DHFR activity at 1
nM, 10 nM and 100 nM of trans (black) and cis (violet) phototrexate. Data are means of at least three
independent experiments in triplicate or quadruplicate ± SEM. Results were analyzed by two-way ANOVA
with Bonferroni's multiple comparisons test for statistical significance (GraphPad Prism 6; adjusted p-value
(*) ≤ 0.05). (b) HeLa cell viability assay at different concentrations of MTX, cis-phototrexate and trans-
phototrexate. The relatively low vertical span and Hill coefficient of the sigmoidal curves of MTX and cis-
phototrexate might be ascribed to short exposure time of the cells to the drugs,65 saturation of the carrier-
mediated uptake, and partitioning of the inhibitors into an inaccessible form at the higher concentrations.66
Data are means of at least three independent experiments in triplicate or quadruplicate ± SEM. Results were
analyzed by two-way ANOVA with Bonferroni's multiple comparisons test for statistical significance
(GraphPad Prism 6; adjusted p-value (****) ≤ 0.0001 for trans-phototrexate versus cis-phototrexate).
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Figure 4. Effects of phototrexate (PHX) on zebrafish development, viability and mortality. Experiment 1,
panels a-d: cis-phototrexate was obtained by external pre-illumination with 375 nm light (see SI for details).
(a), (c) Anatomical profiles of zebrafish at 72 hpf after treatment with vehicle (DMSO 2%), MTX (200 μM),
cis-phototrexate (200 μM), and trans-phototrexate (200 μM). The graph shows the percentage of zebrafish
treated versus the treatment group, superimposing the number of abnormal larvae (black and yellow
checkered sections) to the number of viable embryos (checkered sections) and to the total number of
fertilized embryos (whole columns). (b) Illustrative pictures of individual larvae from each treatment group
at 72 hpf, showing the observable aberrant developmental traits in the MTX and cis-phototrexate groups.
(d) Mortality of zebrafish at 96 hpf after treatment with vehicle (DMSO 2%), MTX (200 μM), cis-phototrexate
(200 μM), and trans-phototrexate (200 μM). The graph shows the percentage of zebrafish treated versus
the treatment group, superimposing the number of dead zebrafish (black and red checkered sections) to the
number of viable embryos (checkered sections) and to the total number of fertilized embryos (whole
columns). Experiment 2, panels e-f: cis-phototrexate was obtained by in situ illumination with 375 nm light
in the presence of zebrafish. (e) Anatomical profiles of zebrafish at 96 hpf after treatment with vehicle/dark
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(DMSO 2%, no illumination), vehicle/375 nm light (DMSO 2%, 0.61 J/cm2, 4 min illumination twice a day),
trans-phototrexate (200 μM, dark), and cis-phototrexate (200 μM, 0.61 J/cm2, 4 min illumination twice a
day). The graph shows the percentage of zebrafish treated versus the treatment group, superimposing the
number of abnormal larvae (black and yellow checkered sections) to the number of viable embryos
(checkered sections) and to the total number of fertilized embryos (whole columns). (f) Mortality of
zebrafish at 96 hpf after treatment with vehicle/dark (DMSO 2%, no illumination), vehicle/375 nm light
(DMSO 2%, 0.61 J/cm2, 4 min illumination twice a day), trans-phototrexate (200 μM, dark), and cis-
phototrexate (200 μM, 0.61 J/cm2, 4 min illumination twice a day). The graph shows the percentage of
zebrafish treated versus the treatment group, superimposing the number of dead zebrafish (black and red
checkered sections) to the number of viable embryos (checkered sections) and to the total number of
fertilized embryos (whole columns).
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