Rational design of an apoptosis-inducing photoreactive DNA intercalator

Article abstract of DOI:10.1002/anie.201302439

Light on DNA intercalators: Molecular modeling and mutasynthesis were employed to rationally tailor the antitumoral agent chartreusin into a vinyl-substituted derivative. Exposure with visible light dramatically improved antiproliferative activities owing to covalent binding with DNA and induction of apoptosis. The results hold promise for a more efficient chemotherapy, in particular for selectively treating tumors with light probes. Copyright

Full text of DOI:10.1002/anie.201302439

Angewandte
Chemie
DOI: 10.1002/anie.201302439
Antitumor Agents
Rational Design of an Apoptosis-Inducing Photoreactive DNA
Intercalator**
Nico Ueberschaar, Hans-Martin Dahse, Tom Bretschneider, and Christian Hertweck*
One of the greatest medicinal challenges is the treatment of
cancer, which has recently become the leading cause of death
in economically developed countries and the second leading
cause of death in emerging nations.[1] Natural products from
plant, fungi, and bacteria represent the prime source of
medicines that help to extend the life expectancy of patients.[2]
The most effective chemotherapeutics either interfere with
the tumor cell cycle and division or bind to DNA and cause
apoptosis through various downstream processes.[3] This
prominent mode of action is known for the bacterial
metabolites chartreusin (1) and elsamicin A (2; Figure 1a).[4]
Common to both of these polyketide glycosides is the
pentacyclic aglycone named chartarin (Figure 1, highlighted
in blue). This rare chromophore is capable of intercalating
into DNA, thereby inhibiting RNA synthesis and causing
radical-mediated single-strand scission of DNA.[5] Moreover,
1 and 2 efficiently inhibit topoisomerase II, which is a prime
target of chemotherapeutics that are used to treat human
malignancies.[5] Although 1 and synthetic prodrugs are highly
effective against various cancer cell lines, such as murine
leukemia, and melanoma cells,[6] a challenge associated with
such chemotherapeutics is that they typically lack selectivity
Figure 1. Structures of DNA intercalators and model of DNA binding.
a) Structures of the antitumor agents chartreusin (1) and elsamicin (2)
sharing the chartarin aglycone (highlighted in blue), b) Model of
vinylchartreusin binding to DNA. View of the major groove shows
proximity of the aglycone vinyl residue to thymine (double bonds
marked in green). c) Distances of the thymine and vinyl carbon atoms
measured in the model.
against tumor cells and thus damage healthy tissue. A
promising avenue that is currently being explored to decrease
unwanted side effects is addressing tumors with light after
systemic or local drug administration.[7] Various leads have
been developed for photodynamic therapy,[8] yet the clinical
use of these compounds is hampered by severe side effects.
For example, hypericin and porphyrin-like photosensitizers
generate reactive oxygen species, which cause unselective
oxidative damage and light hypersensitivity.[7,9] For the light-
induced release of cytotoxic agents various UV-dependent
systems have been explored, for example, doxorubicin
prodrugs with photolysis activation.[10] However, the muta-
genic effect of short-wave irradiation is well-known, and it has
been shown that the UV light employed for photoactivation
may induce secondary cancers.[11] Examples of antitumoral
agents that can be activated with visible light are scarce and
encompass mainly photocleavable Pt or Ru complexes.[12]
Herein we report the first successful tailoring of the
potent chartarin pharmacophore by merging the strengths of
molecular modeling, biosynthesis, and chemical synthesis. We
demonstrate a viable chemobiosynthetic route to a novel
vinyl-substituted chartreusin analogue, which forms covalent
links to DNAupon mild photoactivation with visible light and
which has a markedly higher antitumoral potency than the
parent compound.
To rationally design chartreusin analogues with poten-
tially improved potencies we modeled the structures of
chartreusin and ring-substituted analogues into DNA and
took into account current data on the multivalent, nonrandom
binding properties. Since chartreusin preferentially binds to
sequences containing CpG or TpG triplets,[13] we docked it
into a canonical B DNA between the T and G nucleotides of
the sequence GCGTATGATGCG by using a standardized
[*] N. Ueberschaar, Dr. H.-M. Dahse, T. Bretschneider,
Prof. Dr. C. Hertweck
Leibniz Institute for Natural Product Research and Infection
Biology, HKI
Beutenbergstr. 11a, 07745 Jena (Germany)
E-mail: Christian.Hertweck@hki-jena.de
Prof. Dr. C. Hertweck
Friedrich Schiller University, Jena (Germany)
[**] We thank A. Perner for MS analysis, H. Heinecke for NMR
measurements, T. Palenta for assistance in synthesis and E.-M.
Neumann for assistance in biological assays. This work was
supported by the Federal Ministry of Science and Technology
(BMBF (Germany)).
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201302439.
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method.[14] Interestingly, according to this model the glycoside
residue fits into the minor groove of the DNA helix,[13] thus
supporting aglycone intercalation. We noted that the aryl
methyl group would be placed next to the nucleobases.
Likewise, a vinyl-substituted chartarin aglycone could be
positioned in a way that the vinyl group and the double bond
of the thymine were in proximity (Figure 1b,c). Conse-
quently, such an nonnatural chartreusin derivative could in
principle be capable of forming [2+2] photo adducts in
analogy to the gilvocarcin V family of angucyclic polyketide
glycosides, which are efficiently activated in the near UVA
spectrum (398 nm).[15] Since the absorption maxima of the
chartarin chromophore are shifted to longer wavelengths
compared to the angucyclins, we envisaged the possibility of
generating an improved chartreusin analogue that can be
activated with visible light. To test this hypothesis we aimed at
preparing a chartreusin derivative substituted with a vinyl
group.
gene cluster coding for chartreusin (cha) biosynthesis from
Streptomyces chartreusis and successful expression of the
entire cha gene cluster in the heterologous host Streptomyces
albus,[17] genes coding for the type II polyketide synthase
(PKS)[18] were excised by PCR targeting.[19] HPLC–MS
monitoring showed that the cha PKS null mutant does not
produce chartreusin or related metabolites (Figure 2, trace a).
Figure 2. Mutasynthesis of the vinyl-substituted chartreusin analogue
13. HPLC profiles (l=400 nm) of extracts from cultures of DPKS
mutant supplemented with the following substrates: a) DMSO (neg-
ative control), b) synthetic vinylchartarin (12) in DMSO.
Alteration of the alkyl substitution of chartreusin is
challenging owing to the lack of protocols for the selective
chemical derivatization of the methyl moiety. Since a total
synthesis of chartreusin would be impracticable, we sought to
harness the biosynthetic potential of the producing organism
We next aimed at chemically complementing the mutant
with the synthetic chartarin analogue. For the synthesis of the
vinyl-substituted aglycone a Hauser tandem annulation
strategy was employed, which has proven successful for the
synthesis of the native aglycone[20] (Figure 1, highlighted in
blue). However, the corresponding vinyl-substituted aglycone
could not be obtained by simply employing the vinyl-
substituted coumarin 8. Thus, we introduced a bromo sub-
stituent that could be replaced by another functional group at
a later stage in the synthesis (Scheme 1). The bromo-
substituted coumarin 7 proved to be a suitable reactant in
the Hauser annulation, yielding bromochartarin (9) after
deprotection. The coupling of 9 with a vinyl synthon,
however, proved to be arduous, as reflected by the generally
unsatisfactory examples of vinyl-group transfers onto (poly)-
phenolic aryl bromides.[21] We found that a Stille-type reaction
with a bridged [Pd(dppp)Cl2] catalyst in the presence of
lithium chloride gave the best results (Scheme 1). Finally, the
vinyl-substituted aglycone (12) was added to the DPKS
mutant broth for biotransformation. Indeed, the formation
of a new compound with the molecular mass of 652 amu
pointed to the successful production of the vinyl derivative
(Figure 2, trace b). The mutasynthesis was repeated at
a preparative scale (18 mg starting material), providing
a sufficient amount of vinylchartreusin (13; 100% turnover
rate, 31% yield of isolated 13) for a full characterization and
biological evaluation. The identity of the new compound was
verified by physicochemical data (HRESI-MS, ESI-MS2, 1D-,
2D-NMR spectroscopy, UV/Vis spectroscopy).
(Scheme 1). Specifically, we aimed at
a mutasynthesis
approach[16] using a mutant that is unable to prepare the
natural aglycone. On the basis of the known sequence of the
Evaluation of the antiproliferative and cytotoxic activities
of the new chartreusin variant 13 gave a surprising result.
Compared to chartreusin, 13 exhibits only slightly lower
activity against K-562 and HeLa tumor cells, and is about four
times less active than chartreusin against the HUVEC cell
line (Figure 3a). However, the situation changed dramatically
under photoinducing conditions. For the photoactivation
assay we employed a blue gallium(III) nitride (GaN) LED,
taking advantage of its high spectral purity combined with
Scheme 1. Mutasynthesis of a chartreusin analogue. a–h) Synthesis of
vinylchartarin (12). a) Methoxymethyl chloride (MOM-Cl), diisopropyl-
ethylamine, CH2Cl2, 65%; b) tBuLi, N,N,N’,N’-tetramethylethylenedi-
amine, THF, DMF, 71%; c) trimethylsilyl cyanide, KCN, [18]crown-6,
THF; d) AcOH, 60% (2 steps); e) vinylboronic acid pinacol ester,
[Pd(PPh3)4], dioxane water, 92% f) tBuOLi, THF; 71%; g) BBr3, CH2Cl2,
100%; h) [Pd(dppp)Cl2] (dppp=l,3-bis(diphenylphosphanyl)propane),
LiCl, nBu3Sn-vinyl, DMF, 20%; i) biotransformation, 100% turnover
rate, 31% yield of isolated 13.
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even illumination of the cell culture well plate. Furthermore,
the emission wavelength (420 nm) perfectly matches with the
absorption maximum of 13, which is a prerequisite for
efficient activations of the molecule (Figure 3b).
purity similar to the LED combined with a reproducible high
power density. Electrophoretic movement of the accurately
defined DNA fragment was not altered in samples containing
1 or 13 without irradiation. In stark contrast, we observed
a clear band shift to a higher molecular mass for DNA
irradiated in the presence of 13 (Figure 4a, lane 7). Since this
result strongly suggests that a covalent DNA adduct was
formed, we explored the structural details of adduct forma-
tion. As a prerequisite, we optimized the irradiation proce-
dure using a GaN laser diode as blue light source, which
allowed us to photoactivate the chartreusin derivative under
near physiological conditions. Buffered aqueous herring
sperm DNA solution was treated with 13 and irradiated for
3 min with blue light. Through hydrolysis with hydrochloric
acid the glycosides (DNA and chartreusin) were cleaved and
the mixture was subjected to HPLC–HRMS analyses (Fig-
ure 4b). Our earlier modeling studies revealed that the vinyl
residue is in proximity to the double bond of a thymine, and
the plausible formation of two possible regioisomers (Fig-
ure 1c) was experimentally confirmed. According to UV
absorption, HRMS data and HPLC retention times, two
isomeric vinylchartarin thymine adducts (m/z 471.0838
[MÀH]À, 12.6 min, T#1, and m/z 471.0835 [MÀH]À,
12.3 min, T#2) were identified. Two additional vinylchartarin
adducts were detected: adducts of vinylchartarin with guanine
(9.2 min, m/z 456.0836 [MÀH]À, G#) and cytosine (9.7 min,
To validate the photoinduced activity of vinylchartreusin
(13) in an antiproliferation assay we chose a colon adeno-
carcinoma cell line (HT-29). We compared standardized
conditions in darkness with incubations in the presence of the
blue LED cluster. Notably, the activity of 13 in conjunction
with the LED proved to be 12-fold higher (GI50 0.6 mm) than
without light (GI50 7.1 mm) (Figure 3d,e). In contrast, the
activity of native chartreusin (1) was not affected by treat-
ment with light. To investigate the fate of colon adenocarci-
noma cells (HT-29) we visually monitored the microscopic
appearance of the cancer cells after light exposure. Specifi-
cally, we exposed defined rows of the test plate after
application of 13 for 2, 4, 6, 8, and 16 h with blue light and
incubated them for a total duration of 72 h in darkness. Cell
blebbing, a characteristic sign of apoptosis, was observed after
irradiation for 2 h. After 4 h irradiation further signs of
apoptosis, including chromatin condensation were detectable
(Figure 3g). Longer irradiation did not have any further effect
on the cells. To elucidate mechanistic details of DNA binding
by 13, an electrophoretic mobility assay (EMSA) was
performed. For the irradiation of single samples in standard
glassware we employed a laser, because it has a high spectral
Figure 3. Evaluation of antiproliferative effects and induction of apoptosis. a) GI50 (HUVEC, K-562) and CC50 (HeLa) values for chartreusin and its
vinyl-substituted analogue. GI50 =concentration required to cause 50% reduction in proliferation; CC50 =cytostatic concentration required to
reduce cell growth by 50%. b) Overlay of absorption spectrum of 13 in presence of DNA and emission spectrum of the blue LED light source.
c) Image of the LED light source and a vial containing chartreusin showing its fluorescence. d) Light-dependent antiproliferative effect on human
colon adenocarcinoma cell line HT-29. Dose–response curve of 13 with and without light. e) Corresponding growth-inhibition values with and
without light. f) Time-resolved effect of the inhibition of proliferation by vinylchartreusin (13) on HT-29 cells at a constant concentration
(c=1.25 mgmLÀ1). g) Microscope images of the corresponding HT-29 cells show chromatin condensation (black/white arrowhead) and cell
blebbing (black arrowhead) indicative of apoptosis.
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tumor agents,[22] but also reveals a lead that holds promise for
curing skin cancers and tumors that are accessible with light
probes.
Received: March 23, 2013
Published online: May 6, 2013
Keywords: antitumor agents · glycosylation · photoreaction ·
.
polyketides · streptomyces chartreusis
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m/z 496.0903 [MÀH]À, C#). The relative occurrence of the
adducts (T#1/T#2/G#/C# = 1:0.08:0.36:0.62) indicates a strong
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