Identification of Thienopyrimidine Scaffold as Inhibitor of the ABC

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Identification of Thienopyrimidine Scaffold as Inhibitor of the ABC Transport Protein ABCC1 (MRP1) and Related Transporters Using a Combined Virtual Screening Approach Katja Silbermann, Sven Stefan, Randa Elshawadfy, Vigneshwaran Namasivayam, and Michael Wiese J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01821 • Publication Date (Web): 29 Mar 2019 Downloaded from http://pubs.acs.org on March 30, 2019

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Journal of Medicinal Chemistry

Identification of Thienopyrimidine Scaffold as Inhibitor of the ABC Transport Protein ABCC1 (MRP1) and Related Transporters Using a Combined Virtual Screening Approach. Katja Silbermann,Ɨ Sven Marcel Stefan,**/Ɨ Randa Elshawadfy, Vigneshwaran Namasivayam,* Michael Wiese.*

Pharmaceutical Chemistry II, Pharmaceutical Institute, Rheinische Friedrich-WilhelmsUniversity of Bonn, An der Immenburg 4, 53121 Bonn, Germany

Key Words: ABC transporter; ABCC1 (MRP1); ABCB1 (P-gp); ABCG2 (BCRP); broad-spectrum inhibition; Multidrug Resistance (MDR).

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Abstract A virtual screening protocol with combination of similarity search and pharmacophoremodelling was applied to virtually screen a large compound library to gain new scaffolds regarding ABCC1 inhibition. Biological investigation of promising candidates revealed four compounds as ABCC1 inhibitors, three of them with scaffolds not associated with ABCC1 inhibition until now. The best hit molecule – a thienopyrimidine – was a moderately potent, competitive inhibitor of the ABCC1-mediated transport of calcein AM which also sensitized ABCC1-overexpressing cells toward daunorubicin. Further evaluation showed that it was a moderately potent, competitive inhibitor of the ABCB1-mediated transport of calcein AM, and noncompetitive inhibitor of the ABCG2mediated pheophorbide A transport. In addition, the thienopyrimidine could also sensitize ABCB1- as well as ABCG2-overexpressing cells toward daunorubicin and SN-38, respectively, in concentration ranges that qualified it as one of the ten best triple ABCC1/ABCB1/ABCG2 inhibitors in literature. Besides, three more new multitarget inhibitors were identified by this virtual screening approach.


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Introduction Multidrug resistance-associated protein 1 (MRP1/ABCC1) is an ATP-binding cassette (ABC) transport protein that utilizes the energy of ATP hydrolysis to export solutes from the cytosol into the extracellular space. Besides P-glycoprotein (P-gp/ABCB1) and Breast Cancer Resistance Protein (BCRP/ABCG2), ABCC1 represents a major target in cancer-related multidrug resistance (MDR). During antineoplastic treatment it recognizes and exports intracellularly present antineoplastic agents. This leads to a decreased

intracellular

concentration,

diminishing

the

success

of

cancer

chemotherapy. In addition, these cancer cells become cross-resistant toward other structurally diverse chemotherapeutics. The substrate range of ABCC1 includes intercalators, topoisomerase II inhibitors, mitosis inhibitors, antifolates, and antiandrogens.1,2,3,4 ABCC1 upregulation and overexpression has been found in several cancers of different origin, e.g., brain,5 lung,6 pancreas,7 colon,8 kidney,9 bladder,10 or blood.11 In some cases, malignant tissues simultaneously express ABCC1 and other ABC transporters, such as ABCB1 and ABCG2,12 e.g., certain stomach,13 liver,14 bone marrow,15 or breast cancers.16 Due to differing substrate ranges, certain ABC transporters might act in concert. In addition, due to a partially overlapping substrate range of these transporters, downregulation or inhibition of one transporter might be compensated by upregulation of another.17,18 ABC transport protein-mediated MDR in general, and ABCC1-mediated in particular, remain until today an unresolved obstacle in cancer chemotherapy. Modern approaches to gain ABCC1 inhibitors have been developed within the last approximately 20 years originating from high throughput screenings (HTS) of huge compound libraries. Hit compounds were synthetically improved until a specific desired pharmacological profile regarding potency, selectivity, and toxicity had been 3 ACS Paragon Plus Environment


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achieved.19 The beginning of this so-called ligand-based inhibitor design was marked by the reports of certain pharmaceutical companies, e.g. Eli Lilly, or Xenova Ltd., revealing several quinazolinones,20 pyrrolo-,21,22,23,24 and indolopyrimidines,24,25 or tricyclic isoxazoles26,27 (Figure 1) as the very first purely HTS- and synthesis-derived ABCC1 inhibitors.

O

N

R

R

O

O

N

R

N N

N N

R

N R

quinazolinones (Wang et al., 2002)

R

N N R

pyrrolopyrimidines (Wang et al., 2004)

Cl

R N

N

N

R

O

N O

N R

indolopyrimidines (Wang et al., 2004)

tricyclic isoxazoles (Norman et al., 2005)

Figure 1: Chemical structures of ABCC1 inhibitors derived by HTS-based synthetic approaches.

Since then, several compound classes have been identified to contain ABCC1 inhibitors,19 such as quinazolines,28 quinolines,29 tetrahydroisoquinolines,30,31,32 pyrazolopyrimidines,9 thienopyridines,33 thiophenes,34 furoxans,35 chalcogen pyrylium compounds,36 gallic acids,37 or thioureidocarboxylic acids.34 A couple of compounds


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even proved to be multi-target inhibitors of ABCC1, ABCB1, and ABCG2 (e.g. compounds 1-4; Figure 2).24,29,33,38 O O O

O O

H 2N O O

O

O S

O

N

O O 1 (benzoflavone 16; Juvale et al. 2013)

2 (thienopyridine 6r; Krauze et al., 2014)

N HN

N

S

N

N N

N

N

HN 3 (quinoline 29; Krapf et al., 2016)

4 (pyrrolopyrimidine 55; Stefan et al., 2017)

Figure 2: Chemical structures of triple inhibitors of ABCC1, ABCB1, and ABCG2 derived from HTS screenings and synthetic improvement as reported by Juvale et al. in 2013 (1),38 Krauze et al. in 2014 (2),33 Krapf et al. in 2016 (3),29 and Stefan et al. in 2017 (4).24

Although ligand-based inhibitor design yields compounds with basic inhibitory activity and selectivity, it proceeds with high financial and occupational costs resulting in a few active compounds, accumulating a huge amount of less desired structures. Diverse or 5 ACS Paragon Plus Environment


combined ligand-based virtual screening approaches have rarely been applied to search for inhibitors of ABCC1,39,40 but these methods might be of great use to gain new scaffolds as lead molecules.

Results and Discussion Virtual Screening. In order to identify new scaffolds as inhibitors of ABCC1, we selected two ligand-based screening methodologies, similarity search and pharmacophore model, for screening against the ZINC database. Initially, we have validated the two approaches with a small data set of molecules manually assembled from the ChEMBL database. The data set comprises 288 molecules which were classified based on their half-maximal inhibition concentration (IC50). Activity values ≤ 1 µM were considered as active, while IC50 values > 10 µM were classified as inactive against ABCC1 (Figure 3). The remaining molecules were considered as moderately active.

Figure 3: Total number of molecules for each class as assembled from ChEMBL database.


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As a first step, we developed a pharmacophore model based on four randomly selected molecules from the active class. The four molecules were aligned using the flexible alignment method and the consensus features from the alignment were manually selected for generating the pharmacophore model. The created pharmacophore model was validated using the active and inactive molecules from the data set assembled from ChEMBL. Based on the hit molecules, which were correctly identified as active, we further modified the selection of molecules and features to generate a new pharmacophore model. According to the manual selection of the molecules and features, we finally selected four molecules as shown in Figure 4 (compounds 5-8) for similarity search and the pharmacophore model.



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O NH2

O O

N

Cl

N

N

O

O

HN

N

O

N

O

O

N

N

O

F F 5 (indolopyrimidine 66; Wang et al., 2004)

6 (tricyclic isoxazole 14b; Norman et al., 2005)

IC50 = 0.122 µM

IC50 = 0.079 µM

N

N N

N N

N

N

N

N

N

N

N

7 (pyrrolopyrimidine 23; Schmitt et al., 2016)

8 (pyrrolopyrimidine 29; Schmitt et al., 2016)

IC50 = 0.981 µM

IC50 = 1.24 µM

Figure 4: 2D representation of the four selected molecules used as query molecules for similarity search and pharmacophore model generation as reported by Wang et al. in 2004,25 Norman et al. in 2005,27 and Schmitt et al. in 2016.22


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The alignment of the four selected molecules and the pharmacophore model is shown in Figure 5A and B. The five features F1-F5 (F1: aromatic; F2 and F3: aromatic/hydrophobic; F4: hydrophobic; and F5: acceptor) classify 78 active molecules and identified 72 molecules correctly as hit molecules from the data set. As shown in table 1, the pharmacophore model yielded high sensitivity and specificity values of approximately 90%.

Table 1: Prediction results obtained for the four selected molecules using the pharmacophore model against the molecules in the data set assembled from ChEMBL.

Molecules 5-8

Hit Molecules Active Inactive (78) (129) 72 14

Prediction (%) Sensitivity

Specificity

Balance accuracy

92.31

89.15

90.73

Next, the four molecules identified from our pharmacophore model were used in a similarity search using FTrees method with a similarity threshold value of 0.8. Each of the four molecules was used as a query and searched against the molecules in the data set assembled from ChEMBL. Among the four selected molecules the pyrrolopyrimidine scaffolds 5 and 8 found an efficient number of 68 and 69 molecules, respectively, as active class. Although the tricyclic isoxazole 6 identified the inactive molecules correctly with a high specificity value of 95.35%, it didn’t yield better values for the active class of compounds. This is possibly due to the fact that the dataset contained less molecules related to the tricyclic isoxazole scaffold. The detailed number of molecules obtained as hits from the active and inactive class of compounds for the data set from ChEMBL is provided in table 2.



A)

B)

F3

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F2 F1 F4

C)

D)

E)

F)

F5

Features Distance (Å) F1-F2 2.44 F1-F3 9.74 F1-F4 4.30 F1-F5 1.65 F2-F3 9.21 F2-F4 5.03 F2-F5 3.95 F3-F4 6.10 F3-F5 9.58 F4-F5 3.69

Figure 5: (A) The overall alignment of the selected four molecules 5 (magenta), 6 (orange), 7 (pink) and 8 (cyan) with the five pharmacophore features F1-F5 (F1: aromatic; F2 and F3: aromatic/hydrophobic; F4: hydrophobic; and F5: acceptor). (B) Distances between the pharmacophore features are shown in Å as red lines. On the right side, distances between each feature are provided as table. (C-F) Each individual molecule is shown with the five identified pharmacophore features (F1–F5). Oxygen atoms are colored in red, nitrogen atoms in blue, fluorine atoms in light green, chlorine atoms in dark green, and hydrogen atoms in silver white; non-polar hydrogen atoms are omitted.


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Table 2: Prediction results obtained for the four selected molecules using the Ftreesbased similarity search against the molecules in the data set assembled from ChEMBL.

Molecules 5 6 7 8

Hit Molecules Inactive Active (78) (129) 68 17 2 6 49 20 69 24

Prediction (%) Sensitivity

Specificity

87.18 2.56 62.82 0.88

86.82 95.35 84.50 81.40

Balance accuracy 87.00 48.96 73.66 84.93

In our work, we selected a data set of molecules from ChEMBL and on the basis of four selected molecules two ligand-based virtual screening methods, similarity search and pharmacophore modeling, were validated. For exploring the selected molecules and approaches further, we applied the two screening methods based on the four selected molecules with the combination of physicochemical property filter to identify novel scaffolds as ABCC1 inhibitors from the ZINC database and verify this by biological studies. In total, 16 million compounds classified as AllClean were screened against the similarity search and the pharmacophore-based approach. The flowchart of the overall screening process is shown in Figure 6.



Figure 6. The schematic representation of the overall workflow of the virtual screening and the number of molecules screened at each stage.


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First, a Ftrees-based similarity search was applied to screen against the molecules from the ZINC database with the similarity threshold value of 0.8. From the four selected molecules, compounds 5 and 8 were used as query molecules to identify the hit molecules and the results obtained from the similarity search were combined. The similarity search results showed that 95,409 of the molecules in the database were predicted as inhibitors. These were clustered using MACCS fingerprints and certain molecules were selected from each cluster. The clustering process reduced the number of molecules, and finally, 3,029 molecules were selected. Next, the molecules were investigated for the spatial arrangement of pharmacophore sites required to be active as an ABCC1 inhibitor. Thus, the generated pharmacophore model was screened against the 3,029 molecules and among them, 1,510 molecules were identified as hit molecules. Since a certain lipophilicity was found to correlate with inhibitory activity,22,41 1,098 molecules having a log P within the range from 2 to 5 were chosen. In the following step, the diversity of the molecules was analyzed by visualizing the hit molecules. We manually selected 200 molecules to make sure that the hit molecules covered a reasonable distribution of molecular scaffolds. Finally, on the basis of availability and cost we selected 32 molecules to purchase for testing against ABCC1 and related ABC transporters. Subsequent LC-MS analysis of the purchased compounds in our laboratory revealed that 15 candidates had purities of less than 95% and were therefore excluded from biological investigation. The list of the 17 investigated compounds is given in Supplementary Material.

Biological Investigation.



Calcein AM Assay to Screen for ABCC1 Inhibition. In order to evaluate the 17 selected compounds regarding inhibition of ABCC1, the calcein AM assay was performed as described previously22,23,42 using H69AR small-cell lung cancer cells, which are known only to express ABCC1.1,6 In short, the ABCC1 substrate calcein AM accumulates intracellularly when ABCC1 function is inhibited, and becomes cleaved by unspecific esterases to the fluorescent calcein. The higher the degree of inhibition, the higher the fluorescence (effect) values. These effect values have been compared to our standard ABCC1 inhibitor, compound 26 (Figure 7B),24 which has been established in our laboratory before.41 The complete screening results can be seen in Figure 7A.

A 100 80 60 40 20 0

B N N N

N N N

O O

compound 26


25

24

23

22

21

20

19

18

17

16

15

14

13

12

11

10

-20 9

percentage inhibition

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Figure 7: (A) Results of biological screening against ABCC1 for compounds 9-25 selected by virtual screening. Data obtained in the calcein AM assay using the human small-cell lung cancer cell line H69AR. Compound 26 was taken as standard inhibitor of ABCC1,41 giving 100% (full inhibition at 10 µM). Compounds with an inhibition level [+ standard error of the mean (SEM)] of at least 20% at 10 µM were taken for further evaluation of the IC50 value (compounds 12, 14, 18, 25). Shown is mean of at least three independent experiments with duplicate measurements ± SEM. (B) 2D representation of the standard ABCC1 inhibitor 26. Table 3: Inhibitory potency (IC50) and scaffold class of compounds that reached an inhibition level (+ SEM) of at least 20% in the screening assay using ABCC1overexpressing H69AR cells as determined in the calcein AM assay. The concentration-effect curves had to be constrained to the maximum effect value of compound inhibitor 26 (100%).41 Shown is mean of at least three independent experiments with duplicate measurements ± SEM. compound

scaffold class

IC50 ± SEM [µM]

12

quinoline

39.3 ± 6.3

14

thienopyrimidine

5.04 ± 1.18

18

pyrrolotriazine

36.3 ± 6.7

25

pyrimidine/benzothiazole

22.7 ± 4.0

Four compounds (12, 14, 18, 25; at 10 µM) reached an inhibition level (+ SEM) of at least 20% in comparison to the standard ABCC1 inhibitor 26. The four hit compounds were further examined regarding their IC50 values, which are presented in Table 3. In addition, the table outlines also the basic scaffold classes of the hit compounds. In principle, three scaffolds have been found that are already known to be present in 15 ACS Paragon Plus Environment


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certain MRP1 inhibitors: pyrimidine (e.g., biricodar,43 imatinib,44 NIK-250 (bis(pyridin4-ylmethyl) 2,6-dimethyl-4-(3-methyl-5,6-dihydro-1,4-dithiin-2-yl)-1,4-dihydropyridine3,5-dicarboxylate),5 PAK-104 P (2-(4-benzhydrylpiperazin-1-yl)ethyl 5-(4,6-dimethyl-2oxido-1,3,2-dioxaphosphinan-2-yl)-2,6-dimethyl-4-(3-nitrophenyl)nicotinate),45 or V104

(1-phenyl-7-(pyridin-3-yl)heptan-4-yl

(2S)-1-(2-oxo-2-(3,4,5-

trimethoxyphenyl)acetyl)piperidine-2-carboxylate)46), quinoline (e.g., mefloquine,47 MK571

(3-(((3-(2-(7-chloroquinolin-2-yl)vinyl)phenyl)((3-(dimethylamino)-3-

oxopropyl)thio)methyl)thio)propanoic

acid),48

montelukast,49

MS-209

(1-(4-(2-

hydroxy-3-(quinolin-5-yloxy)propyl)piperazin-1-yl)-2,2-diphenylethan-1-one),50

or

quinine51), and pyrazole (e.g. AG-1393 ((E)-3-amino-5-(1-cyano-2-(3,5-di-tert-butyl-4hydroxyphenyl)vinyl)-1H-pyrazole-4-carbonitrile),44 or rimonabant52). In addition, compounds 12 and 14 contain a piperazine moiety, which was already found to be very important for ABCC1 inhibition,20,21,22,24 and is also present in many ABCC1 inhibitors.50,45,53 Compound 25 contains a piperidine linker, which can be seen as an piperazine equivalent and as spacer between two aromatic ring systems which is also present in some ABCC1 inhibitors (e.g., biricodar, or V-104 (1-phenyl-7-(pyridin-3yl)heptan-4-yl

(2S)-1-(2-oxo-2-(3,4,5-trimethoxyphenyl)acetyl)piperidine-2-

carboxylate).43,46 Other than acting as a linker, piperidine is also introduced as a peripheral substituent in known ABCC1 inhibitors, e.g., AZD1208 ((R,Z)-5-((2-(3aminopiperidin-1-yl)-[1,1'-biphenyl]-3-yl)methylene)thiazolidine-2,4-dione),51 dipyridamole,54 flavopiridol,55 GSK1904529A (N-(2,6-difluorophenyl)-5-(3-(2-((5-ethyl2-methoxy-4-(4-(4-(methylsulfonyl)piperazin-1-yl)piperidin-1yl)phenyl)amino)pyrimidin-4-yl)imidazo[1,2-a]pyridin-2-yl)-2-methoxybenzamide),53 ibrutinib,56 lonafarnib,57 mefloquine,47 piperine,58 quinine,51 or vandetanib.59 Most strikingly, compounds 14, 18, and 25 belong to the classes of thienopyrimidines, pyrrolotriazines, and benzothiazoles, respectively. None of these compound classes 16 ACS Paragon Plus Environment

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were reported to contain inhibitors of ABCC1 before.19 The concentration-effect curve of the only single-digit micromolar active compound 14 is given in Figure 8A. The corresponding 2D molecular structure of compound 14 is depicted in Figure 8B.

A 120

response [% of control]

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


100 80 60 40 20 0

-

-8

-7

-6

-5

-4

log [M]

B N

N

S

N N N O NH2

compound 14

Figure 8: (A) Concentration-effect curve of compound 14 (closed squares; IC50 = 5.04 ± 1.18 µM; Hill slope: 2.1) in comparison to the ABCC1 standard inhibitor 26 (closed circles; IC50 = 0.322 ± 0.019 µM; Hill slope: 1.2). Data obtained in the calcein AM assay using the human small-cell lung cancer cell line H69AR overexpressing ABCC1. Shown is mean of at least three independent experiments with duplicate measurements ± SEM. (B) Molecular formula of the hit compound 14.



Reversal of ABCC1-mediated MDR. The next step was to evaluate whether the inhibitory activity of the most promising candidate, compound 14, reflects in a reversal of MDR in ABCC1-overexpressing cancer cells. For this purpose, the MDR reversal assay was applied as previously described in detail.22,24 In short, ABCC1overexpressing cells are resistant toward the anticancer drug and ABCC1 substrate daunorubicin due to its extrusion out of the cell mediated by this transport protein. Inhibition of ABCC1 leads to an increased persistence of daunorubicin and mid-term cancer cell death. As shown in Figure 9A, compound 14 partially restored sensitivity of H69AR cells against daunorubicin. As much as 5 and 10 µM of this compound reversed MDR in ABCC1-overexpressing cells by a factor of 1.8 and 2.3, respectively, although no complete sensitization could be observed. Table 4 summarizes the half-maximal growth inhibition (GI50) values of daunorubicin as well as the resistance factors of the resistant cell line, which decreased with increasing concentration (5-10 µM) of compound 14. This is illustrated in Figure 9B where the half-maximal reversal concentration (EC50) can be extracted. Compound 14 sensitizes H69AR cells halfmaximally against daunorubicin at 2.69 µM, which is in line with its inhibitory power of 5.04 µM as determined in the calcein AM assay. As other cellular effects might play a role in selected H69AR cells (enhanced GSH content, upregulation of cooperating enzymes like γ-GCS, GSS, or GST), and to directly link the MDR reversal to the overexpression of MRP1, we additionally investigated the effect of compound 14 in a transfected cell line – the MDCK II MRP1 cells. As can be seen, the thienopyrimidine derivative also sensitized this cell line half-maximally at 0.728 µM, which gave proof of a direct link between the observed MDR reversal and ABCC1 overexpression. The discrepancy between the two EC50 values of H69AR and MDCK II MRP1 cells is conspicuous, but occurs frequently when two different cell lines are compared, and has been reported for several tyrosine kinase inhibitors (e.g., apatinib vs. ABCB1,60 18 ACS Paragon Plus Environment

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doramapimod vs. ABCB1,61 nintedanib vs. ABCB1,62 trametinib vs. ABCB1,63 or afatinib vs. ABCG2).64

A 120

cell viability [%]

100 80 60 40 20 0

- -8

-7 -6 -5 daunorubicin log [M]

-4

B

resistance factor

8 6 4 2

H6 no 9A co R m pd

-6.5

-5.5

-4.5

compound 14 log [M]

H6 9

0

C 160

cell viability [%]

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


140 120 100 80 60 40 20 0

- -8

-7 -6 -5 daunorubicin log [M]


-4


D

resistance factor

8

6

4

2

-4.5

compound 14 log [M]

II

-5.5

DC K

-6.5

M

1

0 M no DCK co m II M pd R P

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Figure 9: Sensitization of ABCC1-overexpressing cancer cell lines regard to daunorubicin (10 nM – 10 µM). (A-B) selected H69AR cells, (C-D) transfected MDCK II MRP1 cells. Compound 14 was used at 0.5 µM (closed squares), 1.0 µM (closed upward triangles), 5.0 µM (closed downward triangles), and 10 µM (closed rhombs). The resulting concentration-effect curves were compared to the resistant cell lines (H69AR (A) and MDCK II MRP1 (C), respectively) without compound addition (closed circles) as well as the sensitive H69 (A) and MDCK II (C) cells, respectively (open circles). Shown is mean of at least three independent experiments with duplicate measurements ± SEM. (B) The resulting resistance factors as given in Table 4 were plotted against the used compound concentrations. The half-maximal reversal concentration (EC50) of compound 14 in H69AR cells (A) was 2.69 µM, while transfected MDCK II MRP1 cells (D) were half-maximally sensitized at 0.728 µM.


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Table 4: Summary of biological data obtained in the MDR reversal assay. Half-maximal growth inhibition (GI50) values as well as the resistance factors (rf) of the resistant cell lines (H69AR and MDCK II MRP1) incubated with daunorubicin either without or with compound 14 supplementation at concentrations between 0.5 and 10 µM are depicted. The resistance factors were calculated by taking the GI50 values of the sensitive cell lines (H69 and MDCK II, respectively) into account. The half-maximal reversal concentration (EC50) was calculated from the resistance factors and the used concentrations of compound 14. Shown is mean of at least three independent experiments with duplicate measurements ± SEM. cell line

GI50 ± SEM [µM]

EC50

daunorubicin + compound 14 at

[µM]

0.0 µM

0.5 µM

1.0 µM

5.0 µM

10 µM

0.0 µM

H69AR

2.71 ± 0.22

n.e.

n.e.

1.26 ± 0.12

0.975 ± 0.093

-

rf

8.2

-

-

3.8

3.0

1.0

H69

-

-

-

-

-

0.330 ± 0.030

0.931 ± 0.97

0.608 ± 0.063

-

MDCK II MRP1 2.94 ± 0.31 1.97 ± 0.25 1.41 ± 0.18

a

rf

7.0

4.7

3.4

2.2

1.4

1.0

MDCK II

-

-

-

-

-

0.421 ± 0.044

n.e. = no effect 21 ACS Paragon Plus Environment

2.69

0.728


140


120 100 80 60 40 20 0

25

24

23

22

21

20

19

18

17

16

15

14

13

12

11

10

-20

9

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 69

Figure 10: Depiction of screening results obtained in the calcein AM assay using ABCB1-overexpressing A2780/ADR cells. Cyclosporine A has been used as reference inhibitor, giving 100% (full inhibition at 10 µM); 0% was determined without compound. Compounds with an inhibition level (+ SEM) of at least 20% inhibition at 10 µM were investigated for their IC50 value (compounds 12-14, 18-23, 25. Shown is mean of at least three independent experiments with duplicate measurements ± SEM.

Calcein AM Assay for ABCB1 Inhibition. All compounds identified from virtual screening were additionally evaluated for their inhibitory potency against ABCB1. Here, the same assay as described for ABCC1 inhibition was employed, but using the A2780/ADR human ovarian carcinoma cells overexpressing ABCB1.22,23,24 Figure 10 displays the effect values of the corresponding compounds, while Table 5 gives the corresponding IC50 values of compounds that reached an inhibition level (+ SEM) of at least 20%. Several compounds were found to be active with scaffolds which have already been in association with ABCB1 inhibition, like pyrimidines, 65,66 (is)oxazoles and (benzo)thia(di)azoles,26,27,32,67,68 quinolines,30,69,70 pyrrolotriazines,71,72 triazoles or tetrazoles.68,73,74 Again, thienopyrimidine 14 is a unique representative regarding ABCB1 inhibition, as it was already in case of ABCC1. It inhibited ABCB1 with even 22 ACS Paragon Plus Environment

Page 23 of 69

greater potency than ABCC1 (IC50 = 1.73 ± 0.31 µM). The corresponding concentration-effect curve is shown in Figure 11 in comparison to the ABCB1 standard inhibitor cyclosporine A. The second most potent inhibitor – compound 22 – belongs to the same thienopyrimidine class (IC50 of 2.81 ± 0.31 µM). Taken together, this scaffold is a promising lead for future ABCB1 inhibitors.


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


120 100 80 60 40 20 0

-

-7

-6

-5

log [M]

Figure 11: Concentration-effect curves of compound 14 (closed squares) obtained in the calcein AM assay using A2780/ADR cells (ABCB1; IC50 = 1.73 ± 0.31 µM; Hill slope: 1.2). Cyclosporine A (closed circles; IC50 = 0.676 ± 0.020 µM; Hill slope: 2.9) was used as reference compound. Shown is mean of at least three independent experiments with duplicate measurements ± SEM.

Table 5: Inhibitory potency (IC50) and scaffold class of compounds investigated in calcein AM assay using ABCB1-overexpressing A2780/ADR cells. The concentrationeffect curves had to be constrained to the maximum effect value of cyclosporine at 10 µM (100%). Shown is mean of at least three independent experiments with duplicate measurements ± standard SEM.



compound

scaffold class

IC50 ± SEM [µM]

12

quinoline

27.8 ± 0.6

13

oxadiazole

31.0 ± 2.0

14

thienopyrimidine

1.73 ± 0.31

18

pyrrolotriazine

17.7 ± 1.7

19

triazole

15.6 ± 1.3

20

tetrazole

26.4 ± 2.3

21

thiazole

15.4 ± 1.9

22

(pyrido)thienopyrimidine

2.81 ± 0.17

23

(pyrido)pyrazolopyrimidine

14.5 ± 0.6

25


4.83 ± 0.66

A 120 100 80 60 40 20 0

B

H N

O

O

N

O NH

O O

Ko143 (compound 27)


25

24

23

22

21

20

19

18

17

16

15

14

13

12

11

10

-20

9


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 69

Page 25 of 69 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 12: Depiction of screening results against ABCG2 determined in the pheophorbide A assay using ABCG2-overexpressing MDCK II BCRP cells. Ko143 [(3S,6S,12aS)-1,2,3,4,6,7,12,12a-Octahydro-9-methoxy-6-(2-methylpropyl)-1,4dioxopyrazino[1′,2′:1,6]pyrido[3,4-b]indole-3-propanoic acid 1,1-dimethylethyl ester compound 27; (B)] was used as reference inhibitor, giving 100% (full inhibition at 10 µM); 0% was determined without compound. Compounds with an inhibition level (+ SEM) of at least over 20% at 10 µM were investigated for their IC50 value (compounds 12-14, 16-18, 21-23, 25). Shown is mean of at least three independent experiments with duplicate measurements ± SEM.

Pheophorbide A Assay for ABCG2 Inhibition. As ABCG2 is also a major key player in ABC transporter-related MDR, we routinely screened the compounds against this transporter using ABCG2-transfected MDCK II BCRP cells and the pheophorbide A accumulation assay as described earlier.22,23,24 Figure 12 highlights the effect values of the 17 virtual screening-derived compounds, while Table 6 gives the corresponding IC50 values of the representatives that reached an inhibition level (+ SEM) of at least 20% compared to the maximal inhibition as caused by the ABCG2 standard inhibitor 36 at 10 µM. Here, several compound classes were found that were already connected to ABCG2 inhibition, such as pyrimidines,65,66 quinolines,30,69 (benzo)-(is)oxa(di)azoles and (benzo)thiazoles67,68 pyrrolotriazines,71,72 as well as pyrazoles75,76 and triazoles.68 Thienopyrimidine 14 possessed an inhibitory power even higher than against ABCC1 (IC50 = 2.38 ± 0.47 µM). Figure 13 displays the corresponding concentration-effect curve of compound 14 in comparison to the ABCG2 standard inhibitor compound 27. Within the 17 evaluated compounds, 14 is the second most active ABCG2 inhibitor, being slightly less active than the pyrimidine and benzothiazole derivative 25 (IC50 = 25 ACS Paragon Plus Environment


1.39 ± 0.21 µM). Furthermore, it is a new scaffold that was not reported before regarding ABCG2 inhibition.77

120


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 69

100 80 60 40 20 0

-

-7

-6

-5

log [M]

Figure 13: Concentration-effect curves of compound 14 (closed squares) obtained in the calcein AM assay using MDCK II BCRP cells (ABCG2; IC50 = 2.38 ± 0.47 µM; Hill slope: 1.1). Compound 27 (closed circles; IC50 = 0.202 ± 0.001 µM; Hill slope: 1.4) was used as reference inhibitor. Shown is mean of at least three independent experiments with duplicate measurements ± SEM.

Table 6: Inhibitory potency (IC50) and scaffold class of compounds that reached an inhibition level (+ SEM) of at least 20% in the screening using ABCG2-overexpressing MDCK II BCRP cells in the pheophorbide A assay. The concentration-effect curves had to be constrained to the maximum effect value of compound 27 at 10 µM (100%). Shown is mean ± SEM.


Page 27 of 69 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


compound

scaffold class

IC50 ± SEM [µM]

12

quinoline

16.0 ± 0.6

13

oxadiazole

14.4 ± 0.6

14

thienopyrimidine

2.38 ± 0.47

16

pyrazole

15.4 ± 2.4

17

pyrazole

10.4 ± 0.85

18

pyrrolotriazine

10.2 ± 0.4

21

thiazole

11.2 ± 0.4

22

(pyrido)thienopyrimidine

8.68 ± 0.52

23

(pyrido)pyrazolopyrimidine

11.8 ± 0.2

25


1.39 ± 0.21

Reversal of ABCB1- and ABCG2-mediated MDR. Since compound 14 had not only moderately good inhibitory power against ABCC1, but also against two other MDR related ABC transporters, namely ABCB1 and ABCG2, we additionally evaluated this compound for its ability to reverse ABCB1- as well as ABCG2-mediated MDR using either selected A2780/ADR and MCF-7/MX cells or transfected MDCK II MDR1 (ABCB1) and MDCK II BCRP (ABCG2) cells, respectively. The results are summarized in Table 7, and illustrated in Figures 14A and C as well as 15A and C, respectively. In addition, the decrease of the resistance factors of A2780/ADR and MCF-7/MX as well as MDCK II MDR1 and MDCK II BCRP cells are visualized in Figures 14B and D as well as 15B and D, respectively. At this point it must be stated that the data related to the MCF-7/MX cell line must be taken with care, as this cell line was highly resistant toward SN-38, and the EC50 calculated is only an approximation to the real sensitizing concentration value. Nevertheless, taking all data into account, compound 14 is a new 27 ACS Paragon Plus Environment


triple inhibitor of ABCC1, ABCB1, and ABCG2, half-maximally sensitizing selected H69AR, A2780/ADR, and MCF-7/MX as well as transfected MDCK II MRP1, MDCK II MDR1, and MDCK II BCRP cells at 2.69 µM, 0.748 µM, and 2.73 µM, respectively, and 0.728 µM, 0.361 µM, and 1.31 µM, respectively.

A 120

cell viability [%]

100 80 60 40 20 0

-

-8

-7

-6

-5

daunorubicin log [M]

B

resistance factor

50 40 30 20 10

-6.5

-5.5

-4.5

compound 14 log [M]


A2 78 0

0 A2 no 780 co /AD m pd R

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 69

Page 29 of 69

C 160

cell viability [%]

140 120 100 80 60 40 20 0

-

-8

-7

-6

-5

-4

daunorubicin log [M]

D

resistance factor

20

15

10

5

0 -4.5

compound 14 log [M]

II

-5.5

DC K

-6.5

M

M no DCK co I m I M pd D R1

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 14: Sensitization of ABCB1-overexpressing selected A2780/ADR (A) as well as transfected MDCK II MDR1 (C) cells toward daunorubicin (10 nM – 10 µM). Compound 14 was used at 0.5 µM (closed squares), 1 µM (closed upward triangles), 5 µM (closed downward triangles), and 10 µM (closed rhombs). The resulting concentration-effect curves were compared to the resistant cell lines (A2780/ADR (A) and MDCK II MDR1 (C), respectively) without compound (closed circles) as well as the sensitive A2780 (A) and MDCK II (C) cells, respectively (open circles). Shown is mean of at least three independent experiments with duplicate measurements ± SEM. The resulting resistance factors as given in Table 7 were plotted against the compound 29 ACS Paragon Plus Environment


concentrations. The half-maximal reversal concentration (EC50) for compound 14 in selected A2780/ADR cells (B) was 0.748 µM (B), while transfected MDCK II MDR1 cells (D) were half-maximally sensitized at 0.361 µM.

A 140

cell viability [%]

120 100 80 60 40 20 0

-

-8

-7

-6

-5

-4

-3

-2

SN-38 log [M]

B

resistance factor

800

600

400

200

0 -4.5

compound 14 log [M]

CF -7

-5.5

M

no CFco 7/M m X pd

-6.5

M

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 69


Page 31 of 69

C

cell viability [%]

140 120 100 80 60 40 20 0

-

-8

-7

-6

-5

SN-38 log [M]

D

resistance factor

30

20

10

0 -4.5

compound 14 log [M]

II

-5.5

DC K

-6.5

M

M no DCK co m II B pd CR P

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 15: Sensitization of ABCG2-overexpressing selected MCF-7/MX (A) as well as transfected MDCK II BCRP (C) cells toward SN-38 (10 nM – 10 µM). Compound 14 was used at 0.5 µM (closed squares), 1 µM (closed upward triangles), and 5 µM (closed downward triangles). The resulting concentration-effect curves were compared to the resistant cell lines (MCF-7/MX (A) and MDCK II BCRP (C), respectively) without compound (closed circles) as well as the sensitive MCF-7 (A) and MDCK II (C) cells, respectively (open circles). Shown is mean of at least three independent experiments with duplicate measurements ± SEM. The resulting resistant factors as given in Table 7 are plotted against the used compound concentrations (B and D). The half-maximal 31 ACS Paragon Plus Environment


reversal concentration (EC50) of compound 14 in selected MCF-7/MX cells (B) was 2.73 µM while transfected MDCK II BCRP cells (D) were half-maximally sensitized at 1.32 µM.


Page 32 of 69

Page 33 of 69 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46


Table 7: Summary of biological data obtained in the MDR reversal assay using selected ABCB1-overexpressing A2780/ADR and transfected MDCK II MDR1 cells as well as ABCG2-overexpressing selected MCF-7/MX and transfected MDCK II BCRP cells, respectively. Half-maximal growth inhibition (GI50) values as well as the resistance factors (rf) of the resistant cell lines either without or with compound 14 at concentrations between 0.5 and 10 µM are depicted. The resistance factors were calculated by taking the GI50 values of the sensitive cell lines (A2780, MDCK II, and MCF-7, respectively) into account. The half-maximal reversal concentration (EC50) was calculated from the resistance factors and the used concentrations of compound 14. Shown is mean of at least three independent experiments with duplicate measurements ± SEM. cell line

A2780/ADR

ABC

GI50 ± SEM [µM]

transporter

daunorubicin (ABCB1) or SN-38 (ABCG2) + compound 14 at

ABCB1

rf A2780

-

EC50

0.0 µM

0.5 µM

1 µM

5 µM

10 µM

0.0 µM

0.710 ±

0.466 ±

0.292 ±

0.0903 ±

0.0589 ±

-

0.034

0.023

0.015

0.0042

0.0025

49

32

20

6.3

4.1

1.0

-

-

-

-

-

0.0144 ± 0.0004


0.748

Journal of Medicinal Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46

MDCK II

ABCB1

Page 34 of 69

7.01 ± 0.71

2.88 ± 0.33

1.74 ± 0.20

0.376 ± 0.034

0.189 ± 0.017

-

17

7.1

4.3

0.9

0.5

1.0

0.361

MDR1 rf MDCK II MCF-7/MX

0.402 ± 0.037 ABCB1

rf

333 ± 750

n.e.a

n.e.a

789

75.4 ± 32.8

22.4 ± 4.3

-

179

53

1.0

MCF-7

MDCK II

0.422 ± 0.051

ABCG2

3.98 ± 0.42

3.08 ± 0.32

2.44 ± 0.25

0.662 ± 0.101

27

21

17

4.5

n.t.b

-

BCRP rf

n.e. = no effect

b

n.t. = not tested

1.0 0.146 ± 0.022

MDCK II a

2.73


1.31

Page 35 of 69

Determination of Mode of Inhibition. For further compound characterization, thienopyrimidine 14 was analyzed regarding its mode of inhibition. Investigated were the transport of calcein AM by ABCC1 and ABCB1, as well as the ABCG2-mediated transport of pheophorbide A. In case of ABCC1- and ABCB1-mediated transport of calcein AM, Lineweaver-Burk analysis resulted in a competitive mode of action (Figure 16A and B). While the ABCG2-mediated transport of pheophorbide A, was found to be non-competitive (Figure 16C). Competitive inhibitors of either ABCC1- or ABCB1mediated calcein AM transport are rare observations in the literature.

A

1/v (ABCC1)

6 4 2

10 -2

20

30

1/c (calcein AM)

B 10 8

1/v (ABCB1)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


6 4 2

-20

20 -2

40

60

1/c (calcein AM)


80


C 1/v (ABCG2)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 36 of 69

4

2

-8

-6

-4

-2

2

4

6

8

-2 1/c (pheophorbide A)

Figure 16: Depiction of mode of inhibition by compound 14. The transport of calcein AM either by ABCC1 using H69AR cells or by ABCB1 using A2780/ADR cells is depicted in graphics (A) and (B), respectively. The transport of pheophorbide A by ABCG2 using MDCK II BCRP cells is shown in graphic (C). (A) Concentrations of compound 14 were 0.316 µM (closed circles), 0.562 µM (closed squares), 1.00 µM (closed upward triangles), 1.78 µM (closed downward triangles), and 0.0 µM (control, open circles). (B) Concentrations of compound 14 were 0.056 µM (closed circles), 0.100 µM (closed squares), 0.178 µM (closed upward triangles), 0.316 µM (closed downward triangles), and 0.0 µM (control, open circles). (C) Compound 14 concentrations were 0.178 µM (closed circles), 0.316 µM (closed squares), 0.562 µM (closed upward triangles), 1.00 µM (closed downward triangles), and 0.0 µM (control, open circles). (A) Calcein AM was used at concentrations of 0.25 µM, 0.3 µM, 0.45 µM, 0.6 µM, and 0.8 µM. (B) Calcein AM was used at concentrations of 0.2 µM, 0.3 µM, 0.45 µM, 0.6 µM, and 0.8 µM (C) Pheophorbide A was used at concentrations of 0.5 µM, 0.6 µM, 0.8 µM, 1.0 µM, and 1.5 µM. Shown are three representative experiments out of three different experiments.


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Cytotoxicity Assay. Finally, a MTT-based cytotoxicity assay was applied as already reported before to determine the intrinsic cytotoxicity of compound 14. 22,23,24 As shown in Table 8, compound 14 can be considered as rather nontoxic regarding the used cell lines, having therapeutic ratios between ~7-15.

Table 8: Summary of half-maximal growth inhibition (GI50) values of compound 14 obtained in the cytotoxicity assay using either H69AR, MDCK II MRP1, MDCK II, A2780/ADR, A2780, MDCK II MDR1, MCF-7/MX, MCF-7, and MDCK II BCRP cells. In addition, the therapeutic ratios have been calculated for those cell lines that were used in the inhibition assays (H69AR, A2780/ADR, MDCK II BCRP; see Tables 3, 5, and 6). Except for the transfected MDCK II BCRP cell line, the obtained GI50 values were at least 25 µM or higher, which made compound 14 beside the stated exception a rather nontoxic broad-spectrum ABCC1, ABCB1, and ABCG2 inhibitor.

an.c.

cell line

transporter

GI50 ± SEM [µM]

therapeutic ratio

H69AR

ABCC1

56.9 ± 0.2

11.3

MDCK II MRP1

ABCC1

42.4 ± 0.9

n.c.a

MDCK II

-

27.0 ± 1.0

n.c.a

A2780/ADR

ABCB1

25.8 ± 0.3

14.9

A2780

-

24.6 ± 0.6

n.c.a

MDCK II MDR1

ABCB1

25.0 ± 0.2

n.c.a

MCF-7/MX

ABCG2

47.0 ± 0.2

n.c.a

MCF-7

-

57.8 ± 3.1

n.c.a

MDCK II BCRP

ABCG2

16.4 ± 0.2

6.9

= not calculated.



Page 38 of 69

Conclusion The aim of our work was the classification of known inhibitors of the ABC transporter ABCC1 to develop a computational approach that can predict putative inhibitors out of a database of chemical structures. Through virtual screening using similarity search and pharmacophore modeling, we evaluated 17 compounds for ABCC1 inhibition. As much as four of these compounds gave promising results, leading to inhibitors with moderate to good inhibition values in the low micromolar concentration range. Three new compound classes – thienopyrimidines, pyrrolotriazines, and benzothiazoles – were identified, which have not been reported in literature as inhibitors of ABCC1 before.19 The most promising candidate 14 (IC50

(ABCC1)

= 5.04 µM) belongs to the

structure class of thienopyrimidines. Certain representatives of this compound class were shown to activate ABCB1-mediated efflux, such as QB13 (2-(phenethylthio)5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-amine;

28)78

and

thienopyrimidine 9 (29).79 As far as we know, there has only been one report of thienopyrimidinones as inhibitors of membrane bound transporters until now (MCT1; compound 1; 30; Figure 17).80 Certain thienopyrimidines were reported as kinase inhibitors,81,82 a compound class that is known to reverse MDR in ABC transporteroverexpressing cell lines.83,71,72 However, no report stated thienopyrimidines as inhibitors of ABC transport proteins in general or ABCC1 in particular, nor have any kinase inhibitors with this scaffold been associated with ABC transporters. This makes the thienopyrimidine structure a new scaffold regarding ABCC1 inhibition.


Page 39 of 69 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


S

S

N

S

S

N

N

N

NH2

NH2

28 (QB13; Kondratov et al., 2001)

29 (thienopyrimidine 9; Häcker et al., 2009)

S

N

O N

HO

S

O

30 (thienopyrimidinone 1; Murray et al., 2005)

Figure 17: Thienopyrimidines associated with ABCB1 transport activation (left and middle), as reported by Kondratov et al. in 2001 (QB13; 2-(phenethylthio)-5,6,7,8tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-amine; compound 28)78 and Häcker et al. in 2009 (thienopyrimidine 9; compound 29).79 Right: the only known thienopyridinone that interferes with transporters in an inhibitory way (compound 1 (compound 30) against MCT1 as reported by Murray et al. in 2005).

Compound 14 partially restored sensitivity of ABCC1-overexpressing H69AR cells against daunorubicin with a half-maximal sensitization concentration (EC50) of 2.69 µM. Furthermore, full sensitization against daunorubicin could be observed using transfected MDCK II MRP1 cells with an EC50 of 0.728 µM. Such discrepancy between cell lines has commonly been observed in the literature,60,61,62,63,64 and can be attributed to different expression levels of the transporter, varying GSH content, differing equipment with MDR-associated enzymes of the cells (e.g., γ-GCS, GSS, GST), or distinct morphology of the cells.84,85,86,87,88



Page 40 of 69

Further screening revealed that compound 14 was not specific as it had even greater inhibitory power against two other MDR related ABC transporters, ABCB1 (IC50 = 1.73 µM), and ABCG2 (IC50 = 2.38 µM). Exploration of its capability to reverse MDR in ABCB1- and ABCG2-overexpressing cells showed that its half-maximal reversal concentration was 0.748 µM and 0.361 µM using ABCB1-overexpressing selected A2780/ADR and transfected MDCK II MDR1 cells, respectively, as well as 2.73 µM and 1.31 µM using ABCG2-overexpressing selected MCF-7/MX and transfected MDCK II BCRP cells, respectively. This highlights compound 14 as an exceptional triple ABCC1/ABCB1/ABCG2 inhibitor that proved to sensitize ABCC1-, ABCB1-, and ABCG2-overexpressing cells in the low and sub-micromolar concentration range. As far as we know, only three compounds have been found to reverse MDR caused by all three stated transporters – the tyrosine kinase inhibitors pelitinib and sunitinib (31 and 32; Figure 18)89,90 as well as the 9-deazapurine derivative 4 (Figure 2). Considering this, thienopyrimidine derivative 14 is a very rare and important finding.

H N

F H N

N O

O

HN

N

O H N

Cl

F

H N

N

N

31 (pelitinib)

O

32 (sunitinib)

Figure 18: structural formulas of the tyrosine kinase inhibitors pelitinib (31) and sunitinib (32).


Page 41 of 69 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Further screening of the compounds selected through virtual screening showed that several of them inhibited ABCB1- and ABCG2-mediated transport, although the majority had only moderate activity and most of the compound classes had already been discovered earlier in literature to affect ABC transport proteins. Strikingly, except for thienopyrimidine 14, additional three of these compounds were also triple inhibitors of the ABC transport proteins ABCC1, ABCB1, and ABCG2: the the quinoline 12, the pyrrolotriazine 18, and the pyrimidine/benzothiazole 25 (Figure 19). In literature, only ~80 compounds are known as triple ABCC1, ABCB1, and ABCG2 inhibitors with determined

half-maximal

inhibition

concentrations

(IC50).22,23,24,28,29,30,32,33,38,67,83,91,92,93,94 Regarding potency, only 10 have a comparable high inhibitory power against the three stated transport proteins as compound 1424,29,33,38 – among these are compounds 1-4 (Figure 2). As already stated above – thienopyrimidine 14 is one of only three compounds that have been reported to overcome MDR mediated by these three transporters.

N NH HN N

N N

O

N N

S

N

S

O

N

N

N

O N H

N N

N N

N

NH

N F

compound 12

compound 18

compound 25

Figure 19: Depiction of three new compounds discovered as triple ABCC1, ABCB1, and ABCG2 inhibitors.



Although this outcome was not desired, as selective inhibitors are mostly wanted to treat the specific target-related disease(s), the discovery of several new triple ABCC1/ABCB1/ABCG2 inhibitors, having the capability to reverse ABCC1-, ABCB1-, and ABCG2-mediated MDR, might be of great use. It is known that the inhibition of a single transporter can be compensated by the overexpression of another,17,18 which is because the MDR related ABC transporters ABCC1, ABCB1, and ABCG2 have a partially overlapping substrate range (share partially the same antineoplastic substrates). In addition, besides their overlapping substrate range, these transporters have also different preferences regarding certain antineoplastic agents, which may lead to their simultaneous overexpression to act in concert to cover together an even broader substrate range.12,13,14,15,16 A triple ABCC1/ABCB1/ABCG2 inhibitor could counteract these upcoming resistance mechanisms. Furthermore, the discovery that compound 14 binds to the very same binding site as calcein AM – in case of ABCC1 as well as ABCB1 – opens new opportunities to elucidate functional aspects related to these two transporters, representing the starting point for future docking experiments. Our virtual screening approach applied in this work was of great use to extract novel scaffolds as inhibitors of ABCC1 and other MDR related ABC transporters, as the field of multi-target inhibition of ABC transporters is rather unexplored. This opens new opportunities to gain more potent, specific as well as broad-spectrum inhibitors of ABC transport proteins.


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Experimental Section. Dataset: For our analysis a data set of molecules that inhibit the human ABCC1 (MRP1) was assembled from ChEMBL.95 The data set consisted of molecules assessed by different assays and cell type with 288 molecules. Only molecules with explicit half-maximal inhibitory concentrations (IC50 values) for ABCC1 at the confidence level (confidence score 8) were considered. The molecules with approximate potency annotations (i.e., “>”, “

Identification of Thienopyrimidine Scaffold as Inhibitor of the ABC

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