# Identification of an N-methylpyrazole derivative as a selective inhibitor of breast cancer resistance protein (BCRP/ABCG2)

Andrezza Braiane Ferreira Siridó, Arthur Henrique Gomes de Oliveira, Julia Poletto, Thales Kronenberger, Fabiane Gomes de Moraes Rego, Geraldo Picheth, Marc Le Borgne, Ahcène Boumendjel, Fernanda Andreia Rosa, Glaucio Valdameri, et al.

► To cite this version:

Andrezza Braiane Ferreira Siridó, Arthur Henrique Gomes de Oliveira, Julia Poletto, Thales Kronenberger, Fabiane Gomes de Moraes Rego, et al. Identification of an N-methylpyrazole derivative as a selective inhibitor of breast cancer resistance protein (BCRP/ABCG2). Archives of Biochemistry and Biophysics, 2026, 779, pp.110761. 10.1016/j.abb.2026.110761. hal-05528887

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# Identification of an N-methylpyrazole derivative as a selective inhibitor of breast cancer resistance protein (BCRP/ABCG2)

Andrezza Braiane Ferreira Siridó<sup>a</sup>, Arthur Henrique Gomes de Oliveira<sup>a,b</sup>, Julia Poletto<sup>c</sup>, Thales Kronenberger<sup>b,g</sup>, Fabiane Gomes de Moraes Rego<sup>d</sup>, Geraldo Picheth<sup>d</sup>, Marc Le Borgne<sup>e</sup>, Ahcène Boumendjel<sup>f</sup>, Fernanda Andreia Rosa<sup>c</sup>, Glaucio Valdameri<sup>a,∗</sup>, Vivian Rotuno Moure<sup>a,∗∗</sup>

## ARTICLE INFO

### Keywords:

ABCG2 inhibition

Multidrug resistance

N-methylpyrazoles derivatives

ABC transporters

## ABSTRACT

Multidrug resistance (MDR) mediated by ATP-binding cassette (ABC) transporters remains a major obstacle to cancer chemotherapy, particularly at later disease stages with metastases. Among the 48 human ABC proteins, P-glycoprotein (P-gp/ABCB1), multidrug resistance-associated protein 1 (MRP1/ABCC1), and breast cancer resistance protein (BCRP/ABCG2) are the most studied ABC transporters associated with MDR. Inhibition of ABC transporters has been considered as one possible strategy to overcome MDR. In this study, twelve N-methyl-pyrazole derivatives were evaluated as inhibitors of ABCB1, ABCC1, and ABCG2. Several compounds selectively inhibited ABCG2 while showing no activity against ABCB1 or ABCC1. The most potent derivative, **11**, inhibited more than 50% of ABCG2 activity at 10 µM and displayed substrate-independent inhibition, with IC₅₀ values ranging from 1.6 to 3.8 µM depending on the fluorescent probe used. Compound **11** exhibited only mild cytotoxicity and was not transported itself by ABCG2. Mechanistic studies revealed that **11** induced conformational changes in ABCG2, as evidenced by increased binding of the 5D3 conformational antibody. Combination assays with established ABCG2 inhibitors, including chromone 4a and indeno-[1,2-b]indole 5e, showed neither synergistic nor antagonistic effects. Induced-fit docking simulations supported the experimental data, indicating that **11** binds within the central transmembrane cavity of ABCG2, engaging key residues such as Phe439 and stabilizing inward-facing conformations. Finally, co-treatment with **11** restored sensitivity of ABCG2-overexpressing cells to the anticancer drug SN38, effectively reversing the MDR phenotype. Collectively, these results identify N-methylpyrazole derivatives as promising selective inhibitors of ABCG2.

## 1. Introduction

The pharmacological treatment of cancer is commonly based on the use of chemical agents. However, the use of anticancer drugs in oncological patients, particularly those with metastases, often results in low therapeutic efficacy due to multidrug resistance (MDR) [1,2]. Several

mechanisms of MDR are observed in oncological patients, including the overexpression of ABC transporters [3]. The MDR mediated by ABC transporters is not specific for a single type of cancer, but it is ubiquitously observed [4]. This resistance mechanism can be classified as intrinsic or acquired, and it is triggered by the exposure of cancer cells to anticancer drugs [2].

<footnote><sup>a</sup> Graduate Program in Pharmaceutical Sciences, Laboratory of Cancer Drug Resistance, Federal University of Parana, Curitiba, 80210-170, PR, Brazil</footnote>

<footnote><sup>b</sup> Interfaculty Institute of Microbiology and Infection Medicine (IMIT), DZIF Tübingen Partner Site. University Hospital Tübingen, Tübingen, Germany</footnote>

<footnote><sup>c</sup> Laboratory for Synthesis of Heterocycles (SINTHET), Chemistry Department, State University of Maringá- UEM, Maringá, Paraná 87020-900, Brazil</footnote>

<footnote><sup>d</sup> Graduate Program in Pharmaceutical Sciences, Federal University of Parana, Curitiba, 80210-170, PR, Brazil</footnote>

<footnote><sup>e</sup> Gastroenterology and Technologies for Health Team, Centre de Recherche en Cancérologie de Lyon, Centre Léon Bérard, CNRS 5286, INSERM 1052, Université Claude Bernard Lyon 1, Univ Lyon, 69373, Lyon, France</footnote>

<footnote><sup>f</sup> Université Grenoble Alpes, INSERM, LRB, 38000, Grenoble, France</footnote>

<footnote><sup>g</sup> School of Pharmacy, Faculty of Health Sciences, University of Eastern Finland, P.O. Box 1627, FI-70211 Kuopio, Finland</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>** Corresponding author.
E-mail addresses: gvaldameri@ufpr.br (G. Valdameri), vivian.moure@ufpr.br (V.R. Moure).</footnote>
The human genome encodes 48 ABC proteins, most of which are transporters that span the entire cell membrane [5,6]. The most important ABC transporters that are responsible for causing the MDR phenotype are P-glycoprotein (P-gp/ABCB1), multidrug resistance-associated protein 1 (MRP1/ABCC1), and breast cancer resistance protein (BCRP/ABCG2) [7]. P-glycoprotein, which is also known as MDR1, is encoded by ABCB1 gene and was the first ABC transporter discovered [8]. The second ABC transporter was the MRP1, encoded by the ABCC1 gene [9]. BCRP/ABCG2 was the third ATP-binding cassette transporter identified in the context of cancer-associated MDR [10–12], and it was simultaneously described by three different groups. It was named as BCRP for identification in a resistant breast cancer cell line [10], MXR for its ability to confer resistance to mitoxantrone [11], and ABCP for its high expression in the placenta [12]. Nowadays, this protein, encoded by the ABCG2 gene, is commonly referred to as the ABCG2 transporter [13].

For instance, MRP7/ABCC10 has been linked to resistance to taxanes and other anticancer agents, particularly in certain solid tumors [14]. In addition, members of the ABCA subfamily (e.g., ABCA3 and, in some contexts, ABCA2) are associated to MDR through intra-cellular/lysosomal drug sequestration rather than classical efflux at the plasma membrane [15].

Inhibition of ABC transporters has been considered as one possible strategy to overcome MDR [1]. Although inhibition of ABCB1 (specific inhibitors) and ABCB1/ABCG2 (dual inhibitors) has been explored in clinical trials, none of these inhibitors has progressed to clinical approval, highlighting the complexity of translating transporter inhibition into effective therapeutic interventions [16]. The first ABCG2 inhibitor identified was the fungal toxin fumitremorgin C (FTC) [17], but it exhibited neurotoxic effects. The FTC analogue, Ko143, was later recognized as a potent ABCG2 inhibitor, being considered as a reference inhibitor for in vitro studies [18]; However, its clinical use has been limited by rapid metabolic hydrolysis to inactive metabolites [19], as well as by its absence of selectivity toward ABCG2, since Ko143 has been reported to inhibit other ABC transporters [20]. Several classes of other compounds appeared as promising specific ABCG2 inhibitors, including heterocyclic compounds, such as indeno [1,2-b]indoles [21] and chromones [22], as well as chalcones, flavonoids, stilbenes, and porphyrins [13,23,24].

The pyrazole scaffold is widely used in medicinal chemistry, being a fundamental framework in the chemical structure of various protein kinase inhibitors (PKI) [3], such as erdafitinib, a fibroblast growth factor receptor (FGFR) inhibitor [25]. Erdafitinib was also shown to inhibit ABCB1 activity, reversing the MDR phenotype without affecting ABCG2 [26]. Increasing evidence indicates that pyrazole derivatives may act as promising modulators of ABC transporter-mediated MDR. Based on computational-guided studies, pyrazole derivatives named 29 and its variants inhibited ABCB1 [27], while derivatives 22 and 24 reduced both ABCB1 and ABCG2 activity by approximately 50% at 10 µM [28]. Moreover, pyrazolo-pyrimidine derivatives have been reported to inhibit ABCC1 [29] and ABCG2 [30]. In this work, twelve previously synthesized 3,4- and 4,5-disubstituted N-methylpyrazoles [31] were evaluated against ABCB1, ABCC1, and ABCG2. These compounds were specific toward ABCG2, and the mechanism of inhibition for the best derivative was investigated using different cell-based approaches.

## 2. Material and methods

### 2.1. Chemicals

The pre-synthesized N-methylpyrazole derivatives **1a–1** were obtained through a collaboration with the SINTHET research group (Laboratory for Synthesis of Heterocycles), Department of Chemistry, State University of Maringá (UEM), Maringá, Paraná, Brazil. These compounds had been previously prepared by the SINTHET group using established methodologies [31] and were supplied for use in the present

study. All reagents not available commercially were prepared and rigorously characterized within the SINTHET laboratory to guarantee reproducibility and high purity standards.

Structural assignments for derivatives **1a–1** were confirmed by ¹H and ¹³C NMR spectroscopy. The spectra were consistent with the proposed structures and showed no detectable impurities. High-resolution mass spectrometry (HRMS) analyses were performed, with ESI(+)-MS and tandem ESI(+)-MS/MS data acquired on a hybrid high-resolution, high-accuracy microTOF (Q-TOF) mass spectrometer (Bruker®). Routine purity assessment was carried out by thin-layer chromatography (TLC) on silica gel (Merck, Germany), using compound-specific mobile phases; under these conditions, each compound afforded a single spot, indicating high purity. In addition, melting points were measured in triplicate using a Melting Point M – 560 apparatus (BUCHI Labortechnik AG); the observed melting point values were consistent and aligned with those expected for pure substances, further supporting both purity and structural integrity.

### 2.2. Cell lines

The human HEK293 cell line was stably transfected to overexpress ABCG2 (HEK293-ABCG2) as previously described [32]. The murine NIH3T3 cell line was stably transfected to overexpress ABCB1 (NIH3T3-ABCB1) as previously described [33]. The hamster BHK21 cell line was stably transfected to overexpress ABCC1 (BHK21-ABCC1) as previously described [34]. Those cells were kindly provided by Dr. Attilio Di Pietro (IBCP, Lyon, France). The human non-small lung cancer H460 cells [35] and mitoxantrone-induced H460 cells overexpressing ABCG2 (H460MX20) [36] were provided by Dr. Robert W. Robey and Dr. Michael Gottesman (NCI/NIH, Bethesda, MD, USA). All cell lines were maintained in Dulbecco's Modified Eagle's Medium supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, and maintained at 37 °C and 5% CO₂ under controlled humidity. The stably transfected cell lines were additionally treated with 0.75 mg mL⁻¹ of G418 (HEK293-ABCG2), 60 ng mL⁻¹ of colchicine (NIH3T3-ABCB1), or 0.1 mg mL⁻¹ methotrexate (BHK21-ABCC1).

### 2.3. Inhibition assays

We carried out the transport inhibition using HEK293-ABCG2, NIH3T3-ABCB1 and BHK21-ABCC1 cell lines. Confluent cells were detached by trypsinization and seeded at 1.5 × 10⁵ cells/well onto 24-well plates. The plates were then incubated for 48 h at 37 °C under 5% CO₂ atmosphere for cellular adhesion. After incubation, the medium was removed, and the cells were treated with N-methylpyrazole derivatives at 1 and 10 µM. Hoechst 33342 (3 µM), rhodamine 123 (5 µM), and calcein-AM (0.15 µM) were used as substrates of ABCG2, ABCB1, and ABCC1, respectively. Cells exposed only to the substrates were used as the negative control (baseline transport activity). The reference inhibitors Ko143 (1 µM), GF120918 (1 µM), and MK571 (50 µM) were used as positive controls for inhibition, respectively, and were defined as 100% transport inhibition. The cells were treated with compounds, substrates, and inhibitors for 30 min at 37 °C and 5% CO₂, to allow the drug efflux mediated by ABC transporters [13]. After incubation, the culture medium was removed, the cells were washed with PBS, collected by trypsinization, and resuspended in ice-cold PBS. Intracellular fluorescence was assessed using a FACS Celesta flow cytometer (BD Biosciences), and at least 10,000 events were acquired for each condition. The fluorescent emission of each substrate was detected as follows: ultraviolet laser and 450-50 nm filter for Hoechst 33342, blue laser and 530-30 nm filter for both rhodamine 123 and calcein-AM. Transport inhibition was calculated based on intracellular fluorescence values, expressed as median fluorescence intensity (MFI). Specifically, 100% inhibition corresponds to the difference between the MFI obtained in the presence of the reference inhibitor and the MFI measured in substrate-only conditions. The inhibitory effect of test compounds was
expressed as a percentage relative to this maximal inhibition.

## 2.4. IC₅₀ value determination and bimodulation assay

HEK293-ABCG2 cells were used for both assays, which were conducted according to the inhibition assay protocol previously described [21], but with different treatment conditions. For the IC₅₀ assay, cells were treated with increasing concentrations of **11** (0.2 - 50 µM) and Hoechst 33342 (3 µM), mitoxantrone (10 µM), and pheophorbide *a* (10 µM) as fluorescent ABCG2 substrates. For bimodulation assays, cells were simultaneously treated with one or two combined inhibitors as follows: full ABCG2 inhibitor chromone **C4a** (0.1 and 10 µM) [37] and partial ABCG2 inhibitor indeno[1,2-b]indole **5e** (10 µM) [21] alone or in combination with **11** (10 µM). For both assays, the obtained results were expressed as a percentage of ABCG2 transport inhibition related to the positive control (reference inhibitor Ko143 at 1 µM).

## 2.5. Cell viability assays

Cell viability assays were conducted according to 3-(4,5-dimethylthiazol-2-yl)-2,5-phenyltetrazolium bromide (MTT) colorimetric protocol [38]. Parental HEK293 and HEK293-ABCG2 cells were used for both cytotoxicity and reversion (sensitization) assays. The second assay also used parental H460 and H460MX20 cells. Confluent cells were seeded onto 96-well plates at 1.5 × 10⁴ cells/well density and kept overnight at 37 °C under 5% CO₂ for cellular adhesion. After incubation, and for cytotoxicity assessment, the cells were treated with increasing concentrations of **11** (0.19 – 100 µM) for 72 h under the same incubation conditions. For reversion assay, HEK293-ABCG2 cells were treated with 0.1 µM of SN-38 alone or in combination with either Ko143 (1 µM) – used as a positive control of ABCG2 transport activity inhibition – or **11** (10 µM). In addition, H460 and H460MX20 cells were treated with increasing concentrations of SN-38 (0.005 – 10 µM) in the presence or absence of **11** at 10 µM. After treatment, the culture medium was removed and 100 µL of a 0.5 mg mL⁻¹ of MTT solution was added. After incubating for 4 h at 37 °C under 5% CO₂, the formazan crystals were dissolved with a solution of DMSO/EtOH (1:1) and the absorbance was measured at 595 nm using the microplate reader iMark (Bio-Rad USA). The results were expressed as a percentage of viable cells related to control cells (0.1% DMSO v/v), assumed as 100%.

## 2.6. Conformational antibody binding (5D3)

Confluent HEK293-ABCG2 cells were detached by trypsinization, collected with PBS, and divided into aliquots of 300 µL at a cellular density of 3 × 10⁵ cells per tube. Cells were then centrifuged (1000×g for 3 min), and the pellets were suspended in 100 µL of PBS containing 4 µL of a BSA solution (1 mg mL⁻¹). Cells were treated either with Ko143 (2 µM) – used as positive control – or **11** (10 µM). Negative control was established with untreated cells. After treatment, the 5D3 primary antibody (Purified mouse anti-human CD338, BD Pharmigen – 1:100) was added and cells were then incubated at 37 °C for 30 min. After incubation, cells were submitted to the same centrifugation conditions, and the pellets were suspended with 100 µL of PBS containing the secondary antibody conjugated with phycoerythrin (PE Goat anti-mouse IgG, Abcam – 1:200), followed by incubation at 37 °C for 30 min. After incubation, centrifugation was performed under the same conditions, and the pellets were suspended in 300 µL of ice-cold PBS. The flow cytometric analysis was performed as previously described, with a FACS Celesta flow cytometer (BD Biosciences) using a blue laser and 575/25 nm filter. The raw data analysis and histogram overlays were created using FlowJo v.7.6.2 software.

## 2.7. Molecular docking

The cryo-EM structure of human ABCG2 in the inward-facing

conformation (PDB ID: 6VXJ) was used as the receptor model for all docking calculations. The compounds were sketched in Maestro and subsequently processed with LigPrep [39] to obtain three-dimensional structures, assign ionization states consistent with physiological pH (7.4), and compute partial atomic charges using the OPLS4 [40] force field. Molecular docking was carried out in Glide (v7.7) [41,42] under the induced-fit docking protocol in extra-precision (XP) mode. In this procedure, receptor side chains within the binding pocket were relaxed by a local minimization step performed with Prime [43] to accommodate ligand flexibility. Docking grids were generated by centering on the centroid of the co-crystallized ligand. For each molecule, five poses were produced, and the resulting conformations were evaluated by a combination of scaffold similarity criteria and visual inspection.

# 3. Results and discussion

## 3.1. Screening of N-methylpyrazole derivatives as inhibitors of ABC transporters

We evaluated a series of twelve N-methylpyrazole derivatives [31] as inhibitors of the three ABC transporters: ABCB1, ABCC1, and ABCG2. Stably transfected cells overexpressing each ABC transporter were used, and the inhibition capacity was evaluated by flow cytometry. For the screening, we tested two concentrations of each N-methylpyrazole **1a-1** (1 and 10 µM). As shown in Table 1 and Fig. S1, none of these 12 compounds was able to inhibit either ABCB1 or ABCC1, whereas six of these compounds caused an ABCG2 inhibition higher than 20% at 10 µM. Interestingly, among these six derivatives, only one of them, the compound **1f**, are to the series of the 4,5-disubstituted derivatives (**1a-f**), while the other five compounds **1h-1** are 3,4-disubstituted derivatives, including the N-methylpyrazole derivative **11** (Fig. 1A) that emerged as the most promising, showing an inhibition higher than 50%.

ABCG2 inhibitors are generally classified as selective toward a single transporter or dual-/pan-inhibitors [13]. Dual inhibitors are commonly observed to inhibit ABCB1 and ABCG2 (e.g., GF120918) [44] and rarely for ABCC1 and ABCG2 (e.g., some indeno[1,2-b]indoles) [23,45,46]. Recent reports describe that some small molecules behave as pan-ABC transporter inhibitors, showing activity across multiple ABC transporters [46,47]. Lopinavir [48] and curcumin [49] are well-studied pan-inhibitors, since these drugs can inhibit ABCB1, ABCC1 and ABCG2. In our study, the N-methylpyrazole derivatives were selective toward ABCG2 (Table 1 and Fig. 1B–D), which is an advantage, because a single target is a desirable feature for the development of new drugs. This selective ABCG2 inhibition was also described for other compound classes, such as porphyrins [50] and chromones [51,52].

## 3.2. IC₅₀ values of ABCG2 inhibition caused by **1l**

To compare the potency of inhibition among different ABC transporter inhibitors, the IC₅₀ values (compound concentrations giving a half-maximal inhibition) obtained by cell-based assays were considered. In addition, specifically for ABCG2, the curve of inhibition allows the identification of *I*<sub>MAX</sub> values, which correspond to the maximal inhibition effect [13]. Using Hoechst 33342, that was the same substrate used during the screening, **1l** showed a IC₅₀ value of 3.80 µM (Fig. 2A). To evaluate if the inhibition caused by **1l** is substrate-specific or not, two additional ABCG2 substrates were studied: mitoxantrone, a chemo-therapeutic that is also transported by ABCB1 and ABCC1, and pheophorbide *a*, a porphyrin derivative that is transported only by ABCG2. Compound **1l** also inhibited the mitoxantrone and pheophorbide *a* efflux (Fig. 2B and C), showing IC₅₀ values of 1.60 and 2.42 µM, respectively. These results highlight that **1l** can inhibit the ABCG2 activity independently of the substrate.

The IC₅₀ values obtained for **1l** are in the same order of magnitude for the three substrates used, and the values are similar to those reported for other ABCG2 inhibitors, such as N-desmethyl enzalutamide (2.3 µM)
<table><caption>**Table 1**

Screening of the 4,5-disubstituted N-methylpyrazoles 1a-f and 3,4-disubstituted N-methylpyrazoles 1g-l as inhibitors of ABC transporters.</caption><thead><tr><th rowspan="2">N-methylpyrazole derivatives</th><th rowspan="2"></th><th rowspan="2">Substituents</th><th colspan="2">ABCB1 (% ± SD)</th><th colspan="2">ABCC1 (% ± SD)</th><th colspan="2">ABCG2 (% ± SD)</th></tr><tr><th>1 µM</th><th>10 µM</th><th>1 µM</th><th>10 µM</th><th>1 µM</th><th>10 µM</th></tr></thead><tbody><tr><td></td><td></td><td>R<sub>1</sub>/R<sub>2</sub></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td rowspan="5">1a<br>1b<br>1c<br>1d<br>1e<br>1f</td><td rowspan="5">4,5-disubstituted<br><img src="chemical_structure_1a.png" alt="Chemical structure of 1a"></td><td>Cl</td><td>Me</td><td>1.5 ± 3.0</td><td>4.4 ± 4.7</td><td>-8.3 ± 13.1</td><td>-8.5 ± 9.6</td><td>5.4 ± 11.4</td><td>-1.5 ± 0.9</td></tr><tr><td>Cl</td><td>Ph</td><td>3.8 ± 4.3</td><td>1.3 ± 1.0</td><td>-7.4 ± 8.4</td><td>10.8 ± 14.2</td><td>0.8 ± 4.0</td><td>5.3 ± 3.9</td></tr><tr><td>Cl</td><td>4-MeO-C<sub>6</sub>H<sub>5</sub></td><td>5.6 ± 3.9</td><td>4.7 ± 4.7</td><td>-8.7 ± 8.7</td><td>-5.7 ± 11.5</td><td>2.8 ± 0.7</td><td>15.8 ± 10.1</td></tr><tr><td>Br</td><td>Me</td><td>-0.2 ± 1.2</td><td>-1.5 ± 1.5</td><td>6.7 ± 5.2</td><td>9.4 ± 8.5</td><td>-1.4 ± 4.0</td><td>-4.0 ± 3.5</td></tr><tr><td>Br</td><td>Ph</td><td>3.6 ± 3.0</td><td>1.2 ± 1.1</td><td>8.6 ± 14.6</td><td>4.5 ± 8.0</td><td>-2.7 ± 1.6</td><td>-2.3 ± 3.9</td></tr><tr><td></td><td></td><td>Br</td><td>4-MeO-C<sub>6</sub>H<sub>5</sub></td><td>2.7 ± 2.4</td><td>1.8 ± 5.8</td><td>-13.0 ± 13.1</td><td>1.2 ± 1.0</td><td>6.5 ± 5.6</td><td><strong>22.1 ± 11.0</strong></td></tr><tr><td rowspan="5">1g<br>1h<br>1i<br>1j<br>1k<br>1l</td><td rowspan="5">3,4-disubstituted<br><img src="chemical_structure_1g.png" alt="Chemical structure of 1g"></td><td>R<sub>3</sub>/R<sub>4</sub></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Cl</td><td>H</td><td>2.7 ± 2.4</td><td>2.4 ± 3.1</td><td>7.0 ± 8.1</td><td>18.3 ± 10.9</td><td>1.2 ± 1.6</td><td>17.9 ± 4.0</td></tr><tr><td>Cl</td><td>OMe</td><td>1.2 ± 4.9</td><td>2.1 ± 2.4</td><td>-5.7 ± 6.7</td><td>3.3 ± 1.0</td><td>3.8 ± 1.6</td><td><strong>26.7 ± 11.5</strong></td></tr><tr><td>Cl</td><td>NO<sub>2</sub></td><td>2.5 ± 3.0</td><td>4.8 ± 8.3</td><td>-3.0 ± 4.1</td><td>-9.2 ± 10.8</td><td><strong>21.8 ± 13.2</strong></td><td><strong>34.7 ± 11.9</strong></td></tr><tr><td>Br</td><td>H</td><td>0.9 ± 3.3</td><td>1.4 ± 7.7</td><td>15.3 ± 7.6</td><td>10.7 ± 4.8</td><td>3.5 ± 2.7</td><td><strong>26.2 ± 6.7</strong></td></tr><tr><td></td><td></td><td>Br</td><td>OMe</td><td>3.1 ± 3.6</td><td>2.5 ± 8.6</td><td>-12.4 ± 8.5</td><td>-4.2 ± 4.6</td><td>6.7 ± 2.5</td><td><strong>35.5 ± 8.2</strong></td></tr><tr><td></td><td></td><td>Br</td><td>NO<sub>2</sub></td><td>3.7 ± 2.0</td><td>4.2 ± 6.2</td><td>-3.3 ± 6.6</td><td>4.9 ± 2.3</td><td><strong>22.7 ± 14.7</strong></td><td><strong>55.4 ± 14.5</strong></td></tr></tbody></table>

Note: the inhibition effect was evaluated in ABCB1-, ABCC1- and ABCG2-transfected cells. Cells were exposed to N-methylpyrazole derivatives (1 and 10 µM) and substrates Rhodamine 123 (ABCB1), Calcein-AM (ABCC1) and Hoechst 33342 (ABCG2) for 30 min. The intracellular fluorescence was recorded by flow cytometry. The percentage inhibition was determined using a reference inhibitor for each ABC transporter: GF120918 for ABCB1, MK571 for ABCC1 and Ko143 for ABCG2. The data represents the mean ± SD of at least three independent experiments.

<figure><img src="image_3.png" /><figcaption>**Fig. 1.** 1l and the representative histograms of ABCG2 selective inhibition. (A) Chemical structure of 1l. The representative histograms are overlays of control conditions, which correspond to substrate alone and the substrate plus the reference inhibitor. The condition that corresponds to substrate plus 1l at 10 µM is also represented. Each overlay box corresponds to a different ABC transporter: (B) ABCB1. The substrate was rhodamine 123 (Rho123), and the reference inhibitor was GF120918. (C) ABCC1. Calcein-AM (Calcein) was used as substrate and MK571 as reference inhibitor. (D) ABCG2. Hoechst 33342 (Hoechst) was used as substrate and Ko143 as reference inhibitor.</figcaption></figure>

[53], nicardipine (4.8 µM) [54], flavone (2.8 µM) [55], tacrolimus (3.3 µM) [56], and porphyrin 4B (1.6 µM) [50]. In addition, the curves of
<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /></figure>

(caption on next column)

**Fig. 2.** Determination of the IC₅₀ values. The inhibition effect was evaluated in ABCG2-transfected cells. Cells were exposed to **11** (0.2 - 50 µM) and substrates (A) Hoechst 33342, (B) mitoxantrone and (C) pheophorbide *a* for 30 min. The intracellular fluorescence was recorded by flow cytometry. The percentage inhibition was determined using Ko143 as the reference inhibitor (100% of inhibition). The data represents the mean ± SD of at least three independent experiments.

inhibition allow us to obtain the I<sub>MAX</sub> values, which ranged from ~60% for Hoechst 33342 to ~80% for mitoxantrone and pheophorbide *a* (Table 2). Some ABCG2 inhibitors (e.g., Ko143) show a full inhibition [18], which means that the I<sub>MAX</sub> value reaches 100% of inhibition. In contrast, some ABCG2 inhibitors show a saturating effect between 50 and 70% (e.g., 6-prenylchrysin [55] and stilbenes [57]). Interestingly, even for the same class of compounds, it was observed that some derivatives can cause a full inhibition and others a partial effect (e.g., chromones [58], chalcones [59], and indeno[1,2-b]indoles [21]). The low solubility of the inhibitors can play some role, partially explaining this phenomenon [60], but further studies are necessary. However, the partial effect of ABCG2 inhibition is not an impediment to follow in pre-clinical studies since it was demonstrated that even partial inhibitors showing a saturating effect at 50% can fully chemosensitize cancer cells overexpressing ABCG2 [21].

### 3.3. Cytotoxicity, absence of transport, and effect on 5D3 binding caused by 1l

The absence of cytotoxic effects is a desirable feature of new inhibitors of ABC transporters [13]. However, the cytotoxicity of inhibitors can be used to investigate the transport of these compounds mediated by ABC transporters. In this study, HEK293 and HEK293-ABCG2 cells were treated with increasing concentrations of **1l** for 72 h. Compound **1l** was cytotoxic for these cells (Fig. 3A). In addition, the decrease in cell viability was similar for both cell lines, showing an GI₅₀ value (concentration that decreases 50% of cell viability) of approximately 50 µM (Fig. 3A and Table 2). The same pattern observed for these cells suggests that **1l** is not transported by ABCG2, as described for other ABCG inhibitors, such as stilbenes [57], chromones [22], and porphyrins [50].

Additionally, the GI₅₀ value, together with the IC₅₀ value, can provide valuable information about new inhibitors, the therapeutic ratio (TR). As three different substrates were tested, an IC₅₀ value for each substrate was obtained (Table 2). For all substrates, the IC₅₀ value is in the same order of magnitude, varying from 1.60 to 3.80 µM, for mitoxantrone and Hoechst 33342, respectively. The IC₅₀ value for pheophorbide *a* was 2.42 µM (Table 2). Thus, the TR for **1l** also ranged from 13 to 31. The TR of ABCG2 inhibitors can vary from the very low values of 0.26 and 0.64 for ivermectin and lopinavir, respectively [48], to very high values, such as 2000, as demonstrated for chromone **6g** [51]. Among the same class of compounds, the TR can show an enormous variation, as observed for some chalcone derivatives, which ranged from 3 to 185 for the chalcone derivatives **39** and **38**, respectively [59]. A great variation was also observed for indeno[1,2-b]indole derivatives, ranging from 5 (derivative **5n**) to 468 (derivative **6a**) [21]. The TR observed for **1l** was higher than previously described for porphyrin **4B** [50] and the magnolol derivative **11** [61]. Although TR values in this range can be considered suboptimal for direct therapeutic applications, they remain acceptable for identifying new classes of ABCG2 inhibitors.

Conformational protein changes induced by **1l** binding were investigated using the 5D3 antibody, which recognizes an extracellular loop of the protein [62]. ABCG2 inhibitors [22,63] produce a 5D3 shift, which means that the binding of these inhibitors to transmembrane domains causes conformation changes, increasing the binding of the 5D3 antibody. In sharp contrast, ABCG2 substrates do affect the 5D3 binding [13]. In accordance with previous data, compound **1l** caused an
<table><caption>**Table 2**
IC₅₀, I<sub>MAX</sub>, GI₅₀, and therapeutic ratio (TR) values.</caption><thead><tr><th>Substrate</th><th>IC<sub>50</sub> (µM ± SD)</th><th>I<sub>MAX</sub> (% ± SD)</th><th>GI<sub>50</sub> (µM)</th><th>TR</th></tr></thead><tbody><tr><td>Hoechst 33342</td><td>3.80 ± 1.00</td><td>66.34 ± 4.94</td><td>~50</td><td>13</td></tr><tr><td>Mitoxantrone</td><td>1.60 ± 0.35</td><td>80.43 ± 3.11</td><td></td><td>31</td></tr><tr><td>Pheophorbide a</td><td>2.42 ± 0.45</td><td>83.81 ± 3.72</td><td></td><td>21</td></tr></tbody></table>

Note: the IC₅₀ and I<sub>MAX</sub> values of ABCG2 inhibition were calculated for three different substrates. The therapeutic ratio (TR) was calculated using these three IC₅₀ values and the GI₅₀ value (cytotoxicity).

increase of 5D3 binding (Fig. 3D), similar to the reference inhibitor Ko143 (Fig. 3C), as well as chromone 4a [22].

### 3.4. Absence of synergistic or antagonistic effects

The observation of partial inhibition opens possibilities for investigation of synergistic or antagonistic effects. Such effects can be evaluated by combining a partial inhibitor with a full inhibitor (using the IC₅₀ value) or by testing pairs of partial inhibitors [13]. For the substrate Hoechst 33342, **11** exhibited a partial inhibition (see IC₅₀ curve – Fig. 3A). Thus, using this substrate, the effect of **11** in association with the indeno[1,2-b]indole derivative **5e** (partial inhibitor) [21] and the chromone **4a** (full inhibitor) [22,37] was investigated. As shown in Figs. 4 and 11 did not cause either a synergic or an antagonist effect under the tested conditions. This absence of effect was already reported for the association of stilbenes derivatives with 6-prenylchrysin [57] or the indeno[1,2-b]inidole derivatives **5e** and **6a** [21].

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /></figure>

**Fig. 3.** Cell viability and 5D3 assay. (A) The cytotoxic effect of **11** was evaluated in HEK293 and ABCG2-transfected cells using the MTT assay. Cells were exposed to **11** (0.2 – 100 µM) for 72 h. The data are the mean ± SD of at least three independent experiments. Representative histograms of the conformational antibody 5D3 assay, including the control condition of HEK293-ABCG2 cells in the presence of the antibody (B), the effect of Ko143 (C), and **11** (D) on 5D3 binding.

### 3.5. Chemosensitization of cells to SN38 using **11** as ABCG2 inhibitor

Before advancing to *in vivo* studies, it is strongly recommended to evaluate the effects of potential inhibitors using chemosensitization assays, in which cells are co-treated with the inhibitor and an anticancer agent for 72 h [13]. To investigate the potential of **11**, HEK293 and transfected cells overexpressing ABCG2 (HEK293-ABCG2) were treated with the active metabolite (SN38) of the chemotherapeutic irinotecan. HEK293-ABCG2 cells were less sensitive to SN38 treatment when compared to parental HEK293 cells (Fig. 5A), confirming it as an ABCG2 substrate. In addition, the co-treatment of a reference ABCG2 inhibitor (Ko143) with SN38 chemosensitized the cells, reversing the MDR phenotype. **11** also caused a chemosensitization of HEK293-ABCG2 cells to SN38 (Fig. 5A).

Finally, to confirm this effect, a resistant cancer cell line model, generated by pressure to overexpress ABCG2, was also tested [32]. H460/MX20, a human non-small cell lung cancer cell line obtained after pressure with mitoxantrone, and the parental cell line (H460) were treated with increasing concentrations of SN38. H460/MX20 were resistant to the treatment with SN38 (Fig. 5B); however, the co-treatment of **11** with SN38 fully chemosensitized the cancer cells. Together, these data confirm that **11** is an ABCG2 inhibitor able to reverse the MDR phenotype.

### 3.6. Overlapping binding sites on ABCG2

Docking places **11** in the central transmembrane cavity of ABCG2 (Fig. 6A and B), the canonical binding site of small-molecule inhibitors such as Ko143, as revealed by cryo-EM [64]. Occupation of this pocket
<figure><img src="image_11.png" /><figcaption>**Fig. 4.** Biomodulation assay using **11**. The inhibition effect was evaluated in ABCG2-transfected cells. Cells were exposed to **11** (10 µM – saturating concentration), indeno[1,2-b]indole **5e** (10 µM – saturating concentration), chromone **4a** (0.1 µM – IC₅₀ value and 10 µM – saturating concentration), and combinations of **11** (10 µM) with either **5e** or **C4a** (chemical structures on the left side). The substrate used was Hoechst 33342. The intracellular fluorescence was recorded by flow cytometry. The percentage inhibition was determined using Ko143 as the reference inhibitor (100% of inhibition). The data represents the mean ± SD of at least three independent experiments.</figcaption></figure>

<figure><img src="image_12.png" /><figcaption>**Fig. 5.** Reversal of resistance in the presence of **11**. (A) HEK293 and ABCG2-transfected cells (HEK293-ABCG2). (B) H460 and H460/MX20 cells (selected to overexpress ABCG2 by pressure with mitoxantrone). The cytotoxic effect of SN38 was evaluated in the absence and presence of **11** at 10 µM after 72 h of exposure. The data are the mean ± SD of at least three independent experiments.</figcaption></figure>

stabilizes the protein in the inward-facing state and prevents the conformational changes required for transport, mechanistically explaining the observed functional inhibition [65]. Aromatic residues lining the pocket, most notably Phe439, provide π-stacking and hydrophobic complementarity to planar scaffolds. This was observed in the binding pose of **11** (Fig. 6B). The ABCG2 binding pocket is separated from the extracellular vestibule by the “leucine plug” valve [66], reinforcing that central-cavity occupancy can sterically limit the inward-outward transition [67]. In addition, **11** enhanced 5D3 binding (Fig. 3D), indicating stabilization of an inward-facing conformation (Fig. 6B), an effect well described for ABCG2 inhibitors [67]. Docking analysis also showed a binding site overlap among the ABCG2 inhibitors: chromone **4a**, indeno [1,2-b]indole **5e**, and *N*-methylpyrazole **11** (Fig. 6E). This overlapped binding mode likely accounts, at least in part, for the absence of synergistic effects observed in the bimodulation assay (Fig. 4).

Selectivity among ABC transporters is driven mainly by pocket architecture and conformational dynamics, not by molecular size of the inhibitors alone. ABCG2, as a homodimer, presents a largely symmetric central cavity at the dimer interface, where ligands can be stabilized by aromatic “sandwiching/clamp” interactions, notably involving Phe439, and this binding mode is coupled to the inward-facing closed open switching described for ABCG2 [68]. In contrast, ABCB1 has a large, highly plastic polyspecific cavity with multiple partially overlapping subsites, enabling accommodation of diverse amphipathic ligands [69, 70], while ABCC1 shows a chemically biased recognition consistent with

a bipartite site that accommodates hydrophobic and polar/charged elements typical of organic anion-like substrates [71,72]. Thus, even if *N*-methylpyrazole derivatives are small, their shape/electronic pattern can match ABCG2's symmetric aromatic interface better than the broader polyspecific requirements of ABCB1 or the bipartite/anionic-leaning preferences of ABCC1.

## 4. Conclusions

This study identified a novel class of ABCG2 inhibitors, the *N*-methylpyrazoles. These compounds were selective toward ABCG2, and the best inhibitor **11** was highly potent, showing IC₅₀ values of approximately 2 µM. In addition, the TR was 10-fold higher than the IC₅₀ value, and this compound chemosensitized cells overexpressing ABCG2. **11** also inhibited ABCG2 in a substrate-independent manner, induced conformational changes in the protein, and reversed the multidrug resistance (MDR) phenotype in combination with the anticancer agent SN38. Complementary *in silico* analyses further revealed that **11** occupies the central transmembrane cavity of ABCG2, engaging key residues such as Phe439 and stabilizing inward-facing conformation, consistent with the observed 5D3 shift and functional inhibition.

### CRediT authorship contribution statement

Andrezza Braiane Ferreira Siridó: Methodology, Investigation.
Arthur Henrique Gomes de Oliveira: Methodology, Investigation.
<figure><img src="image_13.png" /><figcaption>**Fig. 6.** Proposed binding mode of N-methylpyrazole derivative **11** with the ABCG2 transporter generated by molecular docking. (A) Overall topology of ABCG2 embedded in a lipid bilayer, highlighting the transmembrane region (yellow) as well as the extracellular and intracellular compartments. (B–D) Binding poses of inhibitors within the transmembrane domain of ABCG2, showing hydrogen-bonding interactions (yellow dashed lines) and predicted binding affinities (ΔG, kcal/mol). (B) Compound **11** (salmon) interacts near Phe439 with a predicted affinity of –15.71 kcal/mol. (C) Compound **5e** (magenta) forms stabilizing interactions with Phe439, displaying the most favorable binding energy (ΔG: 18.85 kcal/mol). (D) Compound **C4a** (green) interacts within the central cavity, with a predicted affinity of –15.92 kcal/mol. (E) Superposition of docking poses, showing the overlapping binding modes of compounds **11**, **5e**, and **C4a** in the transmembrane pocket. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)</figcaption></figure>

**Julia Poletto:** Methodology, Investigation. **Thales Kronenberger:** Writing – original draft, Supervision, Conceptualization. **Fabiane Gomes de Moraes Rego:** Resources. **Geraldo Picheth:** Resources. **Marc Le Borgne:** Resources, Methodology. **Ahcène Boumendjel:** Resources, Methodology. **Fernanda Andreia Rosa:** Writing – original draft, Supervision, Methodology. **Glaucio Valdameri:** Writing – review & editing, Supervision, Project administration, Conceptualization. **Vivian Rotuno Moure:** Writing – review & editing, Supervision, Project administration, Formal analysis, Data curation, Conceptualization.

## Funding

This study was financed by Coordenação de Aperfeiçoamento de

Pessoal de Nível Superior-Brasil (CAPES) - Finance Code 001. AHGO was also supported by CAPES through a PDSE fellowship. This research was also funded by the Brazilian Research Council (CNPq 404286/2021-6) and Fundação Araucária-PPSUS 2020/2021 (SUS2020131000003). GV acknowledges the fellowship of research productivity (PQ) granted by CNPq (304699/2025-0). TK is funded by the German Federal and State Governments and the German Center for Infection Research (DZIF, TTU06.716 and TTU 08.716).

## Conflict of interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence
the work reported in this paper.

## Acknowledgments

We thank Dr Attilio Di Pietro from IBCP (France), Dr Robert W. Robey, and Dr Michael Gottesman from NCI/NIH (USA) for having provided the cell lines.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.abb.2026.110761.

## Data availability

Additional data are available from the corresponding author on reasonable request.

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