Pyrrolopyrimidine derivatives and purine analogs as novel activators of Multidrug Resistance-associated Protein 1 (MRP1, ABCC1)

Article abstract of DOI:10.1016/j.bbamem.2016.10.017

Multidrug resistance (MDR) is the main cause of diminished success in cancer chemotherapy. ABC transport proteins are considered to be one important factor of MDR. Besides P-glycoprotein (P-gp, ABCB1) and Breast Cancer Resistance Protein (BCRP, ABCG2), Mu

Full text of DOI:10.1016/j.bbamem.2016.10.017

Pyrrolopyrimidine derivatives and purine analogs as novel activators of
Multidrug Resistance-associated Protein 1 (MRP1, ABCC1)
Sven Marcel Schmitt, Katja Stefan, Michael Wiese
PII:
DOI:
Reference:
S0005-2736(16)30350-9
doi:10.1016/j.bbamem.2016.10.017
BBAMEM 82339
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14 June 2016
26 October 2016
27 October 2016
Please cite this article as: Sven Marcel Schmitt, Katja Stefan, Michael Wiese,
Pyrrolopyrimidine derivatives and purine analogs as novel activators of Multidrug
Resistance-associated Protein 1 (MRP1, ABCC1), BBA - Biomembranes (2016),
doi:10.1016/j.bbamem.2016.10.017
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Pyrrolopyrimidine derivatives and purine analogs as novel activators of
Multidrug Resistance-associated Protein 1 (MRP1, ABCC1)
Sven Marcel Schmitt†, Katja Stefan†, Michael Wiese*
University of Bonn, Pharmaceutical Institute, An der Immenburg 4, 53121 Bonn,
Germany
*E-mail: mwiese@uni-bonn.de, phone: +49/228/73-5212
† these authors contributed equally
Key words: ABC transporter; MRP1; ABCC1; transport activation; activator; flux
ratio.
Abstract. Multidrug resistance (MDR) is the main cause of diminished success in
cancer chemotherapy. ABC transport proteins are considered to be one important
factor of MDR. Besides P-glycoprotein (P-gp, ABCB1) and Breast Cancer Resistance
Protein (BCRP, ABCG2), Multidrug Resistance-associated Protein 1 (MRP1, ABCC1)
is associated with non-response to chemotherapy in different cancers. While
considerable effort was spent in overcoming MDR during the last two decades,
almost nothing is known with regard to activators of transport proteins. In this work
we present certain pyrrolo[3,2-d]pyrimidine derivatives with variations at positions 4
and 5 and purine analogs as novel activators of MRP1-mediated transport of the
MRP1 substrate calcein AM and the anticancer drug daunorubicin in low nanomolar
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concentration range. Two different MRP1 overexpressing cell lines were used, the
doxorubicin-selected human lung cancer cell line H69 AR and the transfected Madin-
Darby Canine Kidney cell line MDCK II MRP1. No effect was observed in the
sensitive counterparts H69 and MDCK II wild type (wt). Derivatives with higher
molecular weight possessed also inhibitory properties at low micromolar
concentrations, although most compounds were rather poor MRP1 inhibitors. Purine
analogs derived from potent MRP1 inhibitors of the pyrrolopyrimidine class showed
equal activating, but no inhibiting effects at all. All tested compounds were non-toxic
and had only minor impact on P-gp or BCRP, showing no inhibition or activation.
Introduction. The overexpression of ABC transport proteins confers resistance to a
broad range of cytotoxic agents. Besides P-gp and BCRP, MRP1 is associated with
reduced drug retention in different cancers resulting in the phenomenon called MDR
[1]. MRP1-mediated resistance is related to lung cancer [2] and other cancer types
[3], affecting structurally diverse antineoplastic agents, like Vinca alkaloids,
anthracyclines, alkylating agents, intercalators, or podophyllotoxines [4,5]. The export
of drugs by MRP1 is mostly connected to co-binding and co-transportation of reduced
glutathione (GSH) using the energy of ATP hydrolysis [5,6]. Inhibition of this transport
protein has been studied extensively during the last two decades, providing a bulk of
structurally diverse inhibitors [7,8]. Especially compounds with existing
pharmacological use were found as MRP1 inhibitors, such as the calcium antagonist
verapamil, the immunosuppressant cyclosporine A [9], the organic anion transporter
inhibitor probenecid [10], or several NSAIDs [11], e.g. indomethacin [12]. Some
inhibitors were evaluated in clinical trials against cancer and cancer-related diseases,
but with limited success due to severe side effects amongst other reasons
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[13,14,15,16,17]. Even compounds specifically designed to affect ABC transporters
such as biricodar were clinically ineffective [18,19,20].
The development of new compounds inhibiting ABC transport proteins in general,
and MRP1 in particular with high potency and selectivity with no serious side effects
is desirable. But it is as much important to get a detailed understanding of the
function of ABC transporters, as the current knowledge of the mechanistic aspects of
these targets and the drug-protein interaction is still unsatisfactory. Activators of drug
transport mediated by ABC transport proteins might help to elucidate the mode of
action with respect to drug recognition, binding, transport and release.
The first study in this field was published by Sharom et al., who found that synthetic
hydrophobic peptides with tyrosine C-terminus increased the ATP-dependent
colchicine transport of P-gp accompanied by ATPase activation [21]. Since then,
several authors reported on the activating characteristic of many structural diverse
compounds with regard to P-gp, e.g. prazosin [22], (-)epicatechin [23], aged garlic
extracts [24], the tyrosine kinase inhibitor erlotinib [25] and the imidazothiazole-
derivative QB102 [26] as well as other benzimidazole and -thiazole derivatives [27].
In contrast to P-gp, however, almost nothing is known referred to transport activation
and binding sites of MRP1 [28], although certain amino acids were found to be crucial
for the binding and transport of endo- and xenobiotics [29,30], especially tryptophan
[31,32], cysteine [33,34], and charged amino acids [35,36]. Wesolowska et al.
showed in 2005 that phenothiazine maleates stimulated the MRP1-mediated
transport of the fluorescence dyes BCPCF and BCECF [37]. The findings were
confirmed with respect to pharmacologically used phenothiazines, like
thiethylperazine or perphenazine [38], compounds which were known before as P-gp
inhibitors [39]. The flavonoid genistein was found to stimulate the transport of
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rhodamine 123 by MRP1 [40]. Analogs of GSH synthesized and evaluated by Leslie
et al. proved to accelerate MRP1-mediated transport of [3H]estrone 3-sulfate [41], a
known substrate of this transport protein.
Activation of MRP1-mediated transport is often associated with enhanced efflux of
GSH, since GSH is a co-substrate of this transporter [5,6]. The cardiovascular drug
verapamil, a known P-gp and MRP1 inhibitor as well as a P-gp ATPase activator [42],
was found to stimulate GSH transport [43,44], leading to decreased intracellular GSH
levels and cell apoptosis [45,46]. This has also been shown for certain flavonoids by
Lorendeau et al. [47]. Loe et al., who also reported on the stimulating effect of
verapamil before [48], synthesized and evaluated dithiane derivatives of this
cardiovascular drug. These compounds had a stimulating effect on GSH export by
MRP1 [49]. Furthermore, Brandmann et al. showed that the protease inhibitors
indinavir and nelfinavir stimulated MRP1-mediated GSH transport in human
astrocytes [50], whereas Perloff et al. found that ritonavir increased the activity of
MRP1 by inducing its expression in LS-180 cells [51].
An enhanced transport velocity can also be accompanied by an increase of the
ATPase activity, as has been shown for several flavonoids by Leslie et al. and others
[47,52,53]. Guggulsterone, a compound derived from the plant Commiphora mukul.
and analyzed by Nabekura et al., increased MRP1 ATPase activity in high magnitude
[54]. The same applies to the endogenous compound GSSG, as this was shown by
Mao et al. [55].
In this study we provide two new classes of MRP1 activators, pyrrolopyrimidine
derivatives and purine analogs. These compound classes were shown by us and
others before to contain potent and selective inhibitors of MRP1 and P-gp [56,57,58].
Our aim was to analyze the transport increasing effect of these compounds in detail
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with respect to MRP1 as well as to P-gp and BCRP. Using the MRP1 substrates
calcein AM and daunorubicin, determination of the quality and quantity of transport
activation of MRP1 was in focus of this study. The intrinsic toxicity of the tested
compounds was evaluated using the MTT viability test utilizing different sensitive,
selected and transfected cancer cell lines.
Results and Discussion.
Chemistry.
Scheme 1 A illustrates the preparation of compounds 6-11. Ethoxymethylene-
malononitrile was reacted by a nucleophilic substitution with different primary amines
yielding the malononitril derivatives 1a-d. These were cyclized by addition of ethyl
bromoacetate providing intermediates 2a-d. The amino side chain was derivatized by
adding dimethyl formamide dimethyl acetale, yielding compounds 3a-d. Exposure to
gaseous ammonia gave the cyclized products 4a-d. Aromatization using phosphoryl
chloride provided the precursors 5a-d. Microwave-assisted synthesis yielded
compounds 6-11 by nucleophilic substitution of the aromatic ring system at position 4
with different primary amines. Scheme 1 B shows the synthesis of the purine analogs
12-15. 6-chloropurine and 1H-piperazine derivatives were combined by nucleophilic
1
13
substitution. The identity of all intermediates was confirmed using H and C NMR
spectroscopy. The purity of the desired compounds 6-15 was determined using LC-
MS analysis.
Biological Investigations.
Calculation of Flux Ratio and Activation Ratio of MRP1.
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The following calculations were performed assuming first order kinetics for both
active transport and passive diffusion in order to relate the resistance ratio of MRP1
expressing cells to the reduced intracellular concentration [59].
The influx or efflux velocity of a substrate, e.g. fluorescence dye or cytotoxic agent, is
described as product of concentration and rate constant:
v = k ∙ c
(1)
v = transport velocity
c = substrate concentration
k = velocity constant
Under steady state conditions the influx equals the efflux:
v(p,out) = v(p,in) + v(a,in)
(2)
(p,out)= passive transport from outside to inside
(p,in) = passive transport from inside to outside
(a,in) = active transport from inside to outside the cell mediated by a
transport protein
Combination of equations 1 and 2 yields:
kp ∙ cout = (kp + ka) ∙ cin
(3)
Rearrangement gives the relative flux ratio (ka/kp) of active transport in relation to
passive diffusion:
(ka + kp)/kp = cout / cin

ka/kp = (cout / cin) - 1
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or
cin / cout = kp /(ka + kp)

cin / cout = kin /(ka + kp)
The effect of increased active transport in relation to passive diffusion on intracellular
concentration is shown in Figure 1. The depicted relationship is in agreement with
observations made for different anthracyclines with different lipophilicity and passive
diffusion velocity [59]. Here, the cells were less resistant to more lipophilic
compounds with a higher diffusion coefficient. The hyperbolic relationship makes it
evident that low active transport velocity has initially a large effect on intracellular
concentration, lowering it considerably for flux ratios of less than approximately two.
However, the active transport velocity has to be increased strongly in order to further
decrease the intracellular drug concentration. Thus, in a resistant cell a significant
increase in active transport velocity is necessary for a noticeable further decrease of
intracellular concentration. An infinite active transport rate would be needed to reach
zero intracellular concentration, which is impossible. Even in the rare case of an
active uptake of a drug, the same hyperbolic relationship between active efflux and
intracellular concentration is observed (Figure 1). In such a case the numerator would
be the sum of passive diffusion and active uptake rate.
In case of fluorescent probes, the fluorescence intensity can be used as surrogate for
the concentration, as it was done extensively to characterize inhibitors of transport
proteins in accumulation assays or inside-out membrane vesicles. The fluorescence
intensity correlates in a linear manner with the substrate concentration, if no
interfering effects like quenching occur. We have checked that our compounds do not
influence the fluorescence of daunorubicin or calcein. Additionally we found a linear
relationship between the extracellular substance concentration and measured
fluorescence, for both, the active and inhibited transporter, as it is shown in Figure 2.
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Therefore, no self-quenching occurs. The linear relationship also implies that other
effects that could change the fluorescence intensity like protein binding or binding to
DNA accompanied by fluorescence quenching in case of daunorubicin are
proportional to the used concentration and not saturated under the assay conditions.
Thus, such effects have no influence on the linear relationship between concentration
and fluorescence intensity.
Theoretically, the decreased fluorescence in case of calcein AM could be caused by
inhibition of intracellular hydrolases by the investigated compounds. However, taking
into account that the compounds are used at nanomolar concentrations, this seems
to be improbable. This conclusion is supported by the analogous results obtained for
daunorubicin, where no hydrolases are involved in the measurement process. Thus,
possible interfering processes do not change the linear relationship between
concentration and measured fluorescence. Hence, the maximal fluorescence
obtained when the transporter is completely inhibited is proportional to cout and the
fluorescence observed when the transporter is fully active corresponds to cin. These
two concentrations conform to the top and bottom values of calculated dose-
response curves. The activation ratio was defined as flux ratio in presence of an
activator divided by the flux ratio of the control in absence of any activator or inhibitor
in order to make different experiments comparable:
activation ratio = flux ratio (activator) / flux ratio (control)
The activation ratio gives an X-fold increase (or decrease) of transport velocity and
makes different data sets comparable to each other.
Calcein and Daunorubicin Accumulation Assays. We showed in our previous work
that pyrrolopyrimidines are potent and selective inhibitors of MRP1, and correlated
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their inhibitory potency with physicochemical parameters like molecular weight and
lipophilicity [57]. In further experiments we found that the small, low-weight
representatives 18-21 (Figure 3 A) enhanced MRP1-mediated transport of calcein
AM and daunorubicin in low nanomolar concentration range (Figure S1 and S2),
which led to a detailed screening of our compound library consisting of various
structural classes. In this work we present the most potent activators in low
nanomolar concentration range with none to modest inhibitory activity at low
micromolar concentrations. We established compound 12 (SC12, Figure 3 B) as
standard inhibitor of MRP1 in our previous work [57], which was also used in the
present study.
The pyrrolopyrimidine derivatives 6-11 accelerated calcein AM and daunorubicin
efflux as can be seen in Figure 4 A and B. The compounds were compared to each
other at 10 nM, as this concentration was found to result in maximal activation for
most compounds. A representative dose-response curve of compound 9 (Figure 3 C)
in comparison to SC12 is shown in Figure 5 A. Figure 5 B depicts the concentration-
dependent curve of the MRP1 activation ratio. Calculation of the activation ratio of the
compounds yielded an increase of 68-98% in the daunorubicin and about 32-50% in
the calcein accumulation assay, respectively (table 1), in comparison to basal pump
velocity.
While H69 AR cells were used in the latter experiment, the former was conducted
with MDCK II MRP1 cells. Comparable effects were observed for both substrates and
both cell lines, showing that the effect of activation is not substrate or cell line
specific. The sensitive cell lines H69 and MDCK II wt have been used to prove that
the compounds have no nonspecific effects on the cells. As can be seen in Figure 5
A and B, daunorubicin accumulated in both sensitive cell lines to the same extent as
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it was observed for fully inhibited MRP1 overexpressing cells. These results show
that the activators do not interfere with the distribution of this fluorescence dye
caused by changes in membrane fluidity or challenge of membrane integrity.
Lineweaver-Burk analysis of enzyme kinetic experiments showed a competitive
interaction with daunorubicin (Figure 8 A). Although linearization according to
Lineweaver-Burk gives direct information on the type of interaction, this method has
been criticized as being notoriously susceptible to experimental errors due to the
double reciprocal plot. Therefore we additionally analyzed the data using the
linearization method according to Hanes-Woolf. The parallel lines seen in Figure 8 B
are indicative of a competitive behavior. As a third method the direct linear plot
according to Cornish-Bowden was used to investigate the type of interaction. This
method has been claimed to be insensitive to outliers. As can be seen in Figure 8 C,
Km decreases while Vmax remains constant. This clearly shows that the affinity for the
substrate is increased. These results are in agreement with the behavior of a non-
essential activator [60].
While small pyrrolopyrimidines with a molecular weight of less than 350 Da had no or
only poor inhibitory power toward MRP1, compounds with higher molecular weight
(9-11) showed moderate to good inhibition (table 1). This is in agreement with the
already described observation that within this substance class, high molecular weight
is preferred with respect to MRP1 inhibition [57]. Compound 9 was the most effective
inhibitor with an IC50 value of 0.790 µM in the daunorubicin accumulation assay
(Figure 5).
Screening of our compound library showed also purine analogs (Figure 3 D) to be
equally stimulating with respect to calcein AM and daunorubicin efflux (Figures 4 A,
B and 6 A, B), accelerating MRP1-mediated efflux of daunorubicin and calcein AM by
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46-94% and 32-43%, respectively (table 1). While the substituents at position 4 were
crucial partial structures of already described potent inhibitors of MRP1 (cmpds. 12-
15 and 22-25 in ref. 57), these residues seem not to have any influence on MRP1
inhibition when linked to the purine scaffold. Therefore, the purine scaffold seems to
abolish the inhibitory nature of the compounds. Figure S3 A and B show
representative dose response curves of pyrrolopyrimidine derivatives and purine
analogs in the calcein and daunorubicin accumulation assay.
Calcein Accumulation Assay to Screen for P-gp Activation and Inhibition. Due to the
known functional and structural analogy of MRP1 and P-gp [1], we analyzed the
capability of the compounds to activate or inhibit the latter transport protein (Figure 9
A). Except for compound 9, which had poor P-gp inhibiting potency with an IC50 value
of 14.0 µM, no activating or inhibiting effect of these compounds could be observed
at 10 nM and 10 µM, respectively, compared to the standard inhibitor cyclosporine A
at 10 µM (Figure 9 A, table 1).
Pheophorbide A Accumulation Assay to Screen for BCRP Activation and Inhibition.
We screened our library for activation or inhibition with regard to BCRP-mediated
transport of the chlorophyll-breakdown product and fluorescence dye pheophorbide
A. Figure 9 B shows that except for compounds 6 and 9, which had low inhibitory
power toward BCRP with IC50 values of 20.6 µM and 20.4 µM, respectively, neither
inhibition nor activation with regard to BCRP in comparison to the standard inhibitor
Ko143 at 10 µM was observed (Figure 9 B, table 1). These findings indicate that the
compounds are selectively activating MRP1.
MTT Assay to Determine Intrinsic Cytotoxicity of Selected Compounds. We also
analyzed all compounds regarding their intrinsic cytotoxicity. Except for compound 9,
which possessed a GI50 value of 31.8 µM in MDCK II MRP1 cells, the compounds
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proved to be non-toxic using MDCK II MRP1 cells and other cell lines (Figures S4-
S8). Table 2 summarizes determined GI50 values of all compounds using different
sensitive, selected and transfected cell lines. Figure 7 gives an overview of the
viability data. At relevant concentrations of 1 µM and 10 µM, none of the compounds
had a negative influence on cell growth or viability. Only compounds 6 and 9 reduced
cell viability significantly at a concentration of 100 µM, a concentration ten times
higher than used in the assays. Contradictory, compounds 8, 10, 11, 12 and 15 had a
tendency to enhance cell viability with increased concentration. This effect was
significant for compounds 10 and 11 at 100 µM. This data points to no influence on
the membrane integrity, since this would have a destructive effect on the cell viability
within the assay time of 72 hours.
Conclusion.
In this work we provide further information on a rarely explored field of ABC transport
proteins, namely compounds that activate the transporter-mediated efflux of
xenobiotics. Since this area of transmembrane efflux systems is hardly known and
the pool of already known activators of ABC transporters in general is very limited,
the presented results might help to complement the knowledge about efflux proteins
and contribute to elucidate the mode of action and function in general of these
targets. Referring to MRP1, almost nothing is known about stimulators of drug
transport [37,40].
While pyrrolopyrimidines with high lipophilicity and molecular weight have been
presented before as potent and selective inhibitors of MRP1 [56,57], we showed that
low-weight derivatives are able to stimulate MRP1-mediated transport of two known
MRP1 substrates, calcein AM and the anticancer drug daunorubicin. The purine
analogs 12-15 were equally effective with respect to MRP1 activation, increasing the
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transport velocity to the same magnitude as the pyrrolopyrimidines 6-11. We showed
that the change in distribution of the MRP1 substrates calcein AM and daunorubicin
is solely caused by the enhanced transport velocity of MRP1, since the compounds
had no influence on sensitive cell lines H69 and MDCK II wild type, respectively. The
substitution pattern at positions 4 and 5 of the pyrrolopyrimidines 6-11 had no
influence on their activating effect. Molecular weight as a property played only a role
for MRP1 inhibition, as has been shown previously [57]. While pyrrolopyrimidines
with molecular weights of less than 350 Da (6-8) had no or only poor MRP1 inhibitory
potency, compounds 9-11 showed moderate to good MRP1 inhibition. Compound 9
has a special nature, being not only a good activator of MRP1-mediated efflux in low
nanomolar concentrations, but also inhibits the three major transport proteins
involved in MDR, MRP1, P-gp and BCRP, at low micromolar concentrations, which
makes it a rare representative of a triple inhibitor. This could be a starting point for
future investigations to generate more potent compounds affecting the three major
efflux pumps connected to MDR in cancer. The purine analogs 12-15 did not have
any inhibitory power with regard to any tested transport protein.
Except for compound 9, which showed slight intrinsic toxicity, all compounds were
nontoxic in different cell lines, which also gives evidence that the observed
distribution change of the used fluorescence probes by activation of MRP1 is not
caused by cell barrier-related effect or other nonspecific factors. This assumption is
supported by the viability data. Since none of the compounds showed destructive
effects at relevant assay concentrations and most compounds had even a viability
increasing effect on the cells, a distribution change caused by disturbing the
membrane integrity is highly unlikely, as this would lead to the opposite effect.
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The compounds are a useful tool in transporter science and might help to elucidate
the mechanisms involved in the transport process. Since biochemistry and molecular
modeling techniques are still up to elucidate the exact process of drug recognition,
binding, transport and release as well as the relation between ATP hydrolysis and
conformational changes required for transport, MRP1 activators can help to get an
insight into the function of this transport protein. Finally, elucidation of its mechanism
might help in future cancer therapy.
Experimental Section.
Chemistry. Materials. Chemicals for synthesis were purchased from Acros Organics
(Geel, Belgium), Alfa Aesar (Karlsruhe, Germany), Applichem GmbH (Darmstadt,
Germany), Fisher Scientific GmbH (Waltham, MA, USA), Merck Millipore (Billerica,
MA, USA), Sigma-Aldrich (St. Louis, MO, USA) and VWR International GmbH
(Darmstadt, Germany) and used without further purification. The reaction progress
was followed by analytical thin layer chromatography with silica gel F254 coated
aluminum plates (Merck Millipore). Methylene chloride/acetone (18:1 and 9:1,
respectively) or methylene chloride/acetone/methanol (9:1:1) were used as eluent
and an UV lamp at 254 nm was utilized for compound detection. Purification of all
compounds was performed with column chromatography using silica gel 60 (43-60
µm, Merck Millipore) and gradient elution (petroleum ether/methylene chloride 1:1,
methylene chloride, methylene chloride/acetone 18:1, methylene chloride/acetone
9:1,
methylene
chloride/acetone/methanol
18:1:1,
methylene
chloride/acetone/methanol 9:1:1, in each case 200 mL). The identity of the
1
1
intermediates was determined by H spectroscopy. Additionally, H and 13C
spectroscopy was used to characterize the desired compounds 6-15, while the purity
was determined by LC-MS analysis. The NMR spectra were recorded in DMSO-d6 on
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a Bruker Advance 500 MHz (500/126 MHz) and chemical shifts (δ) are expressed as
13
13
ppm calibrated to the solvent signal (1H NMR δ 2.50; C NMR δ 39.5). C signals
were assigned employing distortion less enhancement by polarization transfer
(DEPT) and attached proton test (APT) techniques. Spin multiplicities are depicted as
singlet (s), doublet (d), doublet of doublets (dd), doublet of triplets (dt), triplet of
doublets (td), triplet (t), quartet (q) and quintet (quint). LC-MS analysis was performed
using an Agilent 1100 series with photo diode array (DAD) detector (Agilent
Technologies, Santa Clara, CA, USA) and a Nucleodur column 100-5 C18
(Macherey-Nagel, Düren, Germany) followed by ESI mass spectrometry using an API
2000 Triple Quadrupole mass spectrometer (Applied Biosystems, Waltham, MA,
USA) and Sciex Analyst Software version 1.5.1. The purity of the investigated
compounds in biological testing was determined as ≥ 95%.
General procedure for the preparation of compounds 6-15. The preparation of the
herein presented intermediates 1a-d - 5a-d and their spectroscopic data has already
been published by us before [57]. The desired title compounds were prepared as
described in the literature [56,57] with minor modifications. Complete spectroscopic
data are provided in the supplementary material.
Biological Investigation. Chemicals. The standard inhibitor SC12 was synthesized,
purified and characterized as described before [56,57]. The reference compounds
cyclosporine A and Ko143 ((3S,6S,12aS)-1,2,3,4,6,7,12,12a-Octahydro-9-methoxy-6-
(2-methylpropyl)-1,4-dioxopyrazino[1′,2′:1,6]pyrido[3,4-b]indole-3-propanoic acid 1,1-
dimethylethyl ester) were purchased from Tocris bioscience (Bristol, IO, USA).
Calcein AM and pheophorbide A were supplied by Calbiochem (EMD Chemicals
(San Diego, CA, USA), supply by Merck KGaA (Damstadt, Germany)) and Frontier
Scientific Inc. (Logan, UT, USA), respectively. Daunorubicin was purchased from
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Sigma (Oakville, ON, USA). All other chemicals were provided by Carl Roth GmbH
(Karlsruhe, Germany), Merck KgaA (Darmstadt, Germany), Th. Greyer GmbH Co KG
(Renningen, Germany) and Sigma-Aldrich Chemie GmbH (Steinheim, Germany).
The reference compounds were stored as 10 mM stock solution in DMSO at -20 °C.
All dilutions were prepared in Krebs-HEPES buffer (KHB, consisting of 1.3 mM
CaCl2, 11.7 mM D-Glucose monohydrate, 10.0 mM HEPES (N-2-
hydroxyethylpiperazin-N'-2-ethansulfonic acid), 4.7 mM KCl, 1.2 mM KH2PO3, 1.2
mM MgSO4, 118.6 mM NaCl, and 4.2 mM NaHCO3, adjusted to pH 7.4 with sodium
hydroxide solution and sterilized by filtration with membrane filters (Whatman FP
30/0.2 µM CA-S filter units, GE Healthcare UK limited, Buckinghamshire, UK) with
Braun Injekt 29 mL syringe (ALMO-Erzeugnisse, Erwin Busch GmbH, Bad Arolsen,
Germany), stored in cellstar 50 mL tubes (Greiner bio one, Frickenhausen,
Germany).
Cell Culture. The doxorubicin-selected small cell lung cancer cell line H69 AR
overexpressing MRP1 (ATCC CRL-11351) and its sensitive counterpart H69 (NCL-
H69, ATCC HTB-119) were provided by American Type Culture Collection and
cultivated in RPMI-1640 medium (PAN Biotech GmbH, Aidenbach, Germany)
complemented with 20% fetal bovine serum (FBS, Sigma Life Science, Steinheim,
Germany), 2 mM L-Glutamine and 50 U/mL penicillin G and 50 µg/mL streptomycin
(PAN Biotech GmbH, Aidenbach, Germany). The doxorubicin-selected human
ovarian carcinoma cell line A2780 adr overexpressing P-gp was supplied by the
European Collection of Animal Cell Culture (ECACC, No 931123120). Cultivation was
performed in RPMI-1640 medium supplemented with 10% FBS, 2 mM L-glutamine
and 50 U/mL penicillin G and 50 µg/mL streptomycin. The MRP1 overexpressing cell
line Madin-Darby Canine Kidney (MDCK II MRP1), the BCRP overexpressing cell line
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MDCK II BCRP, both transfected with human wildtype cDNA C-terminally linked to
the cDNA of green fluorescent protein (GFP), and their sensitive counterpart MDCK II
wild type (wt) were a generous gift by Dr. A. Schinkel (The Netherlands Cancer
Institute, Amsterdam, The Netherlands). These cell lines were cultivated in
Dulbecco’s modified Eagles medium (DMEM, Sigma Life Science, Steinheim,
Germany) complemented with 10% FBS, 2 mM L-glutamine, 50 U/mL penicillin G
and 50 µg/mL streptomycin. The cell lines were stored under liquid nitrogen
supplemented with 10% DMSO. The cultivation of cells was maintained at 37 °C
under 5% CO2-humidified atmosphere in a cell incubator (Münchener Medizin
Mechanik GmbH, Planegg, Germany) using T75 or T175 cell culture flasks (Greiner
bio one, Frickenhausen, Germany) until a confluence of ≥ 90% was reached,
followed by subculturing or biological testing. The cell layer was rinsed three times
with 5 mL of DBPS buffer. Harvesting was performed by exposure to a solution of
0.05% trypsin and 0.02% EDTA (PAN Biotech GmbH, Aidenbach, Germany),
followed by an incubation period of 5-10 minutes. The detached cells were
transferred into a 50 mL tube for centrifugation at 266 x g and 4 °C for 5 minutes. The
supernatant was replaced by fresh medium to prepare the cell pellet for cell density
counting using a CASY1 model TT (Schärfe System GmbH, Reutlingen, Germany)
equipped with a 150 µm capillary. The necessary amount of cells was taken to
perform subculturing or biological testing.
Calcein Accumulation Assay. The assay was conducted as described earlier
[57,61,62,63,64,65]. Twenty µL of the test compound in KHB at concentrations
between 0.1 pM and 100 µM were added into clear F bottom 96-well microplates
(Greiner bio one, Frickenhausen, Germany). After cell harvesting and counting as
described above, 160 µL containing approximately 60,000 cells was seeded into
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each well, followed by an incubation time of 20 minutes at 37 °C under 5% CO2-
humidified atmosphere. Twenty µL of a 3.125 µM solution of calcein AM (protected
from light) was added. The 96 well plate was instantly measured with respect to
fluorescence increase in constant time intervals of 60 seconds with a Fluostar Optima
or Fluostar Polarstar microplate reader (BMG-Labtech, Software versions 2.00R2,
2.20 and 4.11-0, respectively) tempered at 37 °C using an excitation wave length of
485 nm and an emission wave length of 520 nm. The standard inhibitor SC12 was
used as reference as it was established before [57]. The linear part of the
fluorescence-time curve was taken for the calculation of the slope between 0 and 30
minutes. Concentration-response curves were obtained by plotting the calculated
slope values against the concentrations of the tested compound using the four-
parameter logistic equation with variable Hill slope or the three-parameter logistic
equation with fixed Hill slope, whichever was statistically preferred with GraphPad
Prism version 5.03 for Windows (San Diego, CA, USA).
Calcein Accumulation Assay to Screen for P-gp Activation or Inhibition. The test
compounds were screened for P-gp activation and inhibition as described earlier
[57,61,62,63]. Dilution series, cell harvesting and counting was performed as stated
above. 160 µL of a cell suspension containing approximately 30,000 cells per well
was added to 20 µL of the test compound solution at concentrations between 1 nM
and 100 µM followed by an incubation time of 30 minutes at 37 °C and under 5%
CO2-humidified atmosphere. 20 µL of a 3.125 µM solution of calcein AM (protected
from light) was added. The 96 well plate was measured instantly with regard to
fluorescence increase in constant time intervals of 60 seconds with microplate
readers as stated above. The effect of 10 nM and 10 µM of the test compound was
compared to the effect of 10 nM and 10 µM cyclosporine A and expressed as
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percentage activation and inhibition in comparison to the standard compound, using
GraphPad Prism as analyzation tool.
Daunorubicin Accumulation Assay. The daunorubicin accumulation assay was
performed as described before [57,66]. Cell harvesting and preparation was
conducted as stated above. The cell pellet was resuspended in fresh culture medium
and 160 µL per well was seeded into colourless 96 well plates with approximately
60,000 cells per well. The dilution series of the test compounds between 1 pM and
100 µM were prepared in culture medium. The cells were preincubated with the
compounds for 15 min, followed by addition of 20 µL of a daunorubicin solution (30
µM). After that the 96 well plate was incubated for 180 minutes at 37 °C and under
5% CO2-humidified atmosphere to reach steady state conditions. The cells were
resuspended before measurement to remove adherent cells from the bottom and to
obtain a homogenous suspension. Fluorescence was measured by flow cytometry
(FACSCalibur, Becton Dickinson Biosciences, Heidelberg, Germany). An argon laser
with an excitation wavelength of 488 nm excited daunorubicin which could be
detected in the FL3 channel (≥ 670 nm). Concentration response curves were
generated by nonlinear regression based on the 4-parameter logistic equation with
variable Hill slope or the three-parameter logistic equation with fixed Hill slope
(GraphPad Prism) as stated above.
Pheophorbide A Accumulation Assay to Screen for BCRP Activation and Inhibition.
The BCRP overexpressing cell line MDCK II BCRP was used to conduct this assay
as described earlier [57,67,68,69]. Cells were harvested and prepared as described
above. 20 µL of the test compounds in different concentrations between 1 nM and
100 µM were added to an U-shaped clear 96 well plate (Greiner, Frickenhausen,
Germany). 160 µL of the cell suspension containing approximately 45,000 cells was
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added to each well followed by an incubation time of 20 min at 37 °C under 5% CO2-
huminidified atmosphere. 20 µL of a pheophorbide A solution (5 µM, protected from
light) were added to each well followed by an incubation of 120 min at 37 °C and
under 5% CO2-humidified atmosphere to reach steady state conditions. The cells
were resuspended before starting measurement to detach adherent cells from the
bottom of the plates and reach homogenous conditions. Fluorescence was measured
by flow cytometry. An argon laser with an excitation wavelength of 488 nm excited
pheophorbide A which could be detected in the FL3 channel (≥ 670 nm). BCRP
expression was measured with GFP detection in the FL1 channel (530/15). The
effect of the 10 nM and 10 µM concentration of the standard compound Ko143 was
compared to the effect of 10 nM and 10 µM of the test compound, using GraphPad
Prism as analyzation tool.
MTT Assay to Determine Intrinsic Cytotoxicity of Selected Compounds. The intrinsic
toxicity of selected compounds was determined using a MTT viability assay was
conducted as described in the literature [57,63] with minor modifications. 20 µL of
different concentrations of the test compounds at 1 µM, 10 µM and 100 µM,
respectively, were pipetted into 96-well tissue-culture treated plates (Starlab GmbH,
Hamburg, Germany) followed by addition of 180 µL of a cell suspension (MDCK II
MRP1, MDCK II BCRP or MDCK II wt: 3,000 cells per well; H69 AR: 20,000 cells per
well; 2008/MRP1: 8,000 cells per well; A2780 adr: 8,000 cells per well). Culture
medium without further supplements was used as negative control, while 10% DMSO
was used as positive control, defining 100% and 0% viability, respectively. For an
incubation time of 72 hours the microplate was kept at 37 °C and under CO2-
humidified atmosphere. Twenty µL of a 5 mg/mL solution of MTT (3-(4,5-dimethyl-2-
thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) was added to the plate and incubated
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for 1 hour. After removal of the supernatant 100 µL of DMSO was added to each
well. Spectrophotometric measurement of the absorbance at 570 nm was performed
using an Ex Multiscan microplate photometer (Thermo Fisher Scientific, Waltham,
MA, USA) with a background correction at 690 nm. GraphpadPrism was used as
analyzation tool.
Author Information.
Corresponding Author. * E-mail: mwiese@uni-bonn.de Phone: (+49) 228 735213.
Fax: (+49) 228 737929.
Abbreviations used.
ABC, ATP-binding cassette; ABCB1 synonymous for P-gp; ABCC1, synonymous for
MRP1; ABCG2, synonymous for BCRP; APT, attached proton test; ATP, adenosine
5’-triphosphate;
BCECF,
2’.7’-bis(2-carboxyethyl)-5-(and-6)-carboxyfluorescein;
BCPCF, BCRP, 3-carboxy-propyl)-5-(and-6-)-carboxyfluorescein; Breast Cancer
Resistance Protein; calcein AM, calcein acetoxymethyl ester; δ chemical shift in ppm;
DAD, diode array detector; DEPT, distortion less enhancement by polarization
transfer; DMS, dimethyl sulfate; FACS, fluorescence activated cell sorting, GI50, half-
maximal growth inhibitory concentration; GSH, glutathione, reduced; GSSG,
glutathione, oxidized; HEPES, 2-(4-(2-Hydroxyethyl)-1-piperazinyl)-ethansulfonic
acid; log P calc., predicted partition coefficient of the neutral molecule; LTC4,
leukotriene C4; LTD4, leukotriene D4; [M+H]+, ionization from protonation; MDR,
multidrug resistance; MRP1, Multidrug Resistance-associated Protein 1; MTT, 3-(4,5-
dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide; NSCLC, non-small cell
lung cancer; P-gp, permeability (P) glycoprotein; SC12, standard MRP1 inhibitor
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compound 12; SCLC, small cell lung cancer; TKI, tyrosine kinase inhibitors; wt, wild
type; (Z), cis.
Supplementary Material.
Molecular formula strings, complete spectroscopic data and additional biological data
are provided.
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