Megumi Murakami’s research while affiliated with Northern Inyo Hospital and other places

Human P‐glycoprotein (P‐gp) utilizes energy from ATP hydrolysis for the efflux of chemically dissimilar amphipathic small molecules and plays an important role in the development of resistance to chemotherapeutic agents in most cancers. Efforts to overcome drug resistance have focused on inhibiting P‐gp‐mediated drug efflux. Understanding the features distinguishing P‐gp inhibitors from substrates is critical. Cryo‐electron microscopy has revealed distinct binding patterns, emphasizing the role of the L‐site or access tunnel in inhibition. We substituted 5–9 residues of the L‐site with alanine to investigate whether the binding of a second inhibitor molecule to the L‐site is required for inhibiting drug efflux. We reveal, for the first time, that mutations in the L‐site affect the drug efflux activity of P‐gp, despite their distance from the substrate‐binding pocket (SBP). Surprisingly, after the mutations were introduced, inhibitors such as tariquidar and zosuquidar still inhibited drug efflux by mutant P‐gps. Communication between the transmembrane helices (TMHs) and nucleotide‐binding domains (NBDs) was evaluated using the ATPase assay, revealing distinct modulation patterns by inhibitors for the mutants, with zosuquidar exhibiting substrate‐like stimulation of ATPase. Furthermore, L‐site mutations abolished ATP‐dependent thermal stabilization. In silico molecular docking studies corroborated the altered inhibitor binding due to mutations in the L‐site residues, shedding light on their critical role in substrate transport and inhibitor interactions with P‐gp. These findings suggest that inhibitors bind either to the SBP alone, and/or to alternate site(s) when the L‐site is disabled by mutagenesis.

MTX-211 is a first-in-class dual inhibitor of epidermal growth factor receptor (EGFR) and phosphoinositide-3 kinase (PI3K) signaling pathways with a compelling pharmaceutical profile and could enhance the effectiveness of mitogen-activated protein kinase kinase (MEK) inhibitor therapy in colorectal tumors with KRAS mutations. However, the specific mechanisms contributing to the acquired resistance to MTX-211 in human cancers remain elusive. Here, we discovered that the overexpression of the ATP-binding cassette (ABC) drug transporter ABCG2, a prevalent mechanism associated with multidrug resistance (MDR), could diminish the effectiveness of MTX-211 in human cancer cells. We showed that the drug efflux activity of ABCG2 substantially decreased the intracellular accumulation of MTX-211 in cancer cells. As a result, the cytotoxicity and effectiveness of MTX-211 in suppressing the activation of the EGFR and PI3K pathways were significantly attenuated in cancer cells overexpressing ABCG2. Moreover, the enhancement of the MTX-211-stimulated ATPase activity of ABCG2 and the computational molecular docking analysis illustrating the binding of MTX-211 to the substrate-binding sites of ABCG2 offered a further indication for the interaction between MTX-211 and ABCG2. In summary, our findings indicate that MTX-211 acts as a substrate for ABCG2, underscoring the involvement of ABCG2 in the emergence of resistance to MTX-211. This finding carries clinical implications and merits further exploration.

Backgrounds: Reserpine is an agent that is used for the treatment of hypertension. ABCG2 also known as breast cancer resistance protein (BCRP) is a member of the ABC transporters associated with multidrug resistance (MDR). Our high-throughput screening assay has revealed that reserpine analog inhibits transport function of ABCG2. Aim: To investigate the effect of reserpine analog on ABCG2 function. Results: Flow cytometry assay showed that reserpine analog blocked the efflux of Pheophorbide a (PhA: fluorescent substrate of ABCG2) from ABCG2-expressing HCT-116/BCRP colon cancer cells in a concentration-dependent manner. Furthermore, cell proliferation assay (MTS assay) revealed that 5 and 10 μM reserpine analog sensitized HCT-116/BCRP cells to SN38 with IC50 value 0.61 + 0.12 μM and 0.22 + 0.036 μM, respectively. Finally, reserpine analog did not stimulate ABCG2-mediated ATP hydrolysis, but rather inhibited it (concentration required for 50% inhibition = 2.7 μM), suggesting that reserpine analog might prevent conformational changes required for ATP hydrolysis. Conclusion: Reserpine analog could be developed as a potent modulator to overcome ABCG2-mediated MDR in cancer cells. Citation Format: Shinobu Ohnuma, Shoji Kokubo, Norihiko Sugisawa, Megumi Murakami, Yuuri Hatsuzawa, Minoru Kobayashi, Taiki Kajiwara, Hideyuki Suzuki, Hideaki Karasawa, Kazuhiro Watanabe, Takayuki Doi, Suresh V. Ambudkar, Takashi Kamei, Michiaki Unno. Reserpine analog modulates transport function of ABCG2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4674.

The high expression of the ATP-binding cassette (ABC) drug transporter ABCG2 in cancer cells contributes to the emergence of multidrug resistance (MDR) in individuals afflicted with either solid tumors or blood cancers. MDR poses a major impediment in the realm of clinical cancer chemotherapy. Recently, substantial endeavors have been dedicated to identifying bioactive compounds isolated from nature capable of counteracting ABCG2-mediated MDR in cancer cells. Imperatorin, a natural coumarin derivative renowned for its diverse pharmacological properties, has not previously been explored for its impact on cancer drug resistance. This study investigates the chemosensitizing potential of imperatorin in ABCG2-overexpressing cancer cells. Experimental results reveal that at sub-toxic concentrations, imperatorin significantly antagonizes the activity of ABCG2 and reverses ABCG2-mediated MDR in a concentration-dependent manner. Furthermore, biochemical data and in silico analysis of imperatorin docking to the inward-open conformation of human ABCG2 indicate that imperatorin directly interacts with multiple residues situated within the transmembrane substrate-binding pocket of ABCG2. Taken together, these results furnish substantiation that imperatorin holds promise for further evaluation as a potent inhibitor of ABCG2, warranting exploration in combination drug therapy to enhance the effectiveness of therapeutic agents for patients afflicted with tumors that exhibit high levels of ABCG2.

ATP-binding cassette transporters, including ABCB1 (P-glycoprotein) and ABCG2 (BCRP/MXR/ABCP), are pivotal in multidrug resistance (MDR) development in cancer patients undergoing conventional chemotherapy. The absence of approved therapeutic agents for multidrug-resistant cancers presents a significant challenge in effectively treating cancer. Researchers propose repurposing existing drugs to sensitize multidrug-resistant cancer cells, which overexpress ABCB1 or ABCG2, to conventional anticancer drugs. The goal of this study is to assess whether furmonertinib, a third-generation epidermal growth factor receptor tyrosine kinase inhibitor overcomes drug resistance mediated by ABCB1 and ABCG2 transporters. Furmonertinib stands out due to its ability to inhibit drug transport without affecting protein expression. The discovery of this characteristic was validated through ATPase assays, which revealed interactions between furmonertinib and ABCB1/ABCG2. Additionally, in silico docking of furmonertinib offered insights into potential interaction sites within the drug-binding pockets of ABCB1 and ABCG2, providing a better understanding of the underlying mechanisms responsible for the reversal of MDR by this repurposed drug. Given the encouraging results, we propose that furmonertinib should be explored as a potential candidate for combination therapy in patients with tumors that have high levels of ABCB1 and/or ABCG2. This combination therapy holds the potential to enhance the effectiveness of conventional anticancer drugs and presents a promising strategy for overcoming MDR in cancer treatment.