(T1530-05-29) Discovery of a Deazaflavin Analog as Potent and Selective Inhibitor of Multidrug Resistance-Associated Protein 1 (MRP1)

Purpose: Overexpression of ATP binding cassette (ABC) efflux transporters, such as multidrug resistance-associated protein 1 (MRP1), is a main mediator of multidrug resistance (MDR) in cancers and therefore, their selective inhibition is an attracting approach to enhance the efficacy of chemotherapeutics. Previously, we have identified some deazaflavin analogs that can potentiate the action of topoisomerase II (TOP2) poisons in cancer cells with a significant increase in drug accumulation ^{1, 2}. Hence, we hypothesize that the deazaflavin analogs might be competitive substrates or modulators of drug efflux toward TOP2 poison ETP and/or other anticancer drugs. To test our hypothesis, a highly potent analog 11a¹ was selected and screened against the major ABC efflux transporters in vesicular transport assays and ATPase activity assays. The analog was also evaluated in the multi-drug resistant small lung cancer H69AR cells and the sensitive parental NCI-H69 cells for its cancer sensitizing effect toward ETP and doxorubicin (DOX).

Methods: The inhibitory effect of 11a on transporter activity was determined in the “inside-out” membrane vesicles expressing individual ABC transporters, including P-gp, BCRP, and MRP1-3, by measuring the uptake of corresponding substrates at different conditions. ATPase activity assays using a colorimetric method were also performed to validate the results from vesicular transport assays and to further elucidate the mechanism of action of 11a. The efficacy of 11a in potentiating the cytotoxic effect of ETP and DOX was evaluated in the multi-drug resistant H69AR cells and the parental NCI-H69 cells using Cell Counting Kit-8. The intracellular levels of ETP and DOX in the presence or absence of 11a were also determined in these two cell lines using LC/MS/MS.

Results: Results from the vesicular transport assays revealed that 11a had a greater inhibitory effect on MRP1 than other transporters. While 11a displayed an IC₅₀ of 0.5 μM on inhibiting the transport activity of MRP1, its IC₅₀ values on inhibiting other transporters (P-gp, BCRP, MRP2 and MRP3) were all shown to be greater than 50 μM, suggesting an excellent MRP1 selectivity index of > 100. 11a also exhibited a much lower IC₅₀ than that of the positive control MK-571 against MRP1 (IC₅₀s: 0.5 μM vs 8.7 μM). However, unlike MK-571, the inhibitory activity of 11a is essentially dependent on the presence of glutathione (GSH), an abundant endogenous molecule and weak substrate known to facilitate MRP1 transport of some substrates. Interestingly, further mechanistic exploration showed that 11a did not increase the baseline ATPase activity of MRP1 with or without the presence of GSH, suggesting that it may not be a substrate of MRP1. Nevertheless, MRP1 inhibition by 11a is validated in the ATPase activity assays using different substrates including N-ethylmaleimide-glutathione conjugate (NEM-GSH), GSH, and glutathione disulfide (GSSG). Moreover, 11a greatly potentiated the cytotoxic effect of ETP and DOX in the MRP-1 overexpressing H69AR cells by lowering their CC₅₀s from 161.7 μM to 2.9 μM and 8.7 μM to 0.2 μM, respectively, while 11a itself did not show much cytotoxicity at the selected concentration (cell viability > 90% at 10 μM). In contrast, 11a only moderately potentiated the action of ETP in the parental NCI-H69 cells by reducing their CC50s from 4.5 μM to 1.7 μM and 0.3 μM to 0.1 μM, respectively. Importantly, intracellular levels of ETP and DOX were shown to be much lower in the multi-drug resistant H69AR cells than in NCI-H69 cells, and 11a was able to boost their concentrations in H69AR cells by 30-fold and 2.2-fold, respectively. In NCI-H69 cells, a lesser extent of drug accumulation stimulated by 11a was observed with ETP ( < 8-fold) but not with DOX. Collectively, these results strongly suggest that MRP1 inhibition by 11a is an important contributing factor to its potent cancer sensitizing effect toward ETP and DOX.

Conclusion: In this study, we discovered that the deazaflavin analog 11a is a potent and selective inhibitor of MRP1, and its inhibitory activity is GSH dependent. In addition, we demonstrated that 11a is highly effective in sensitizing MRP1-overexpressing H69AR small lung cancer cells toward ETP and DOX with increased drug accumulation. These findings suggest that the deazaflavin analog may represent a new and exciting chemotype for selectively modulating MDR related MRP1 activity and support further investigation of the analog as a long-sought chemo-sensitizing agent for therapeutics against cancers with high MRP1 expression.

References: 1. Kankanala, J.; Ribeiro, C. J. A.; Kiselev, E.; Ravji, A.; Williams, J.; Xie, J.; Aihara, H.; Pommier, Y.; Wang, Z., Novel Deazaflavin Analogues Potently Inhibited Tyrosyl DNA Phosphodiesterase 2 (TDP2) and Strongly Sensitized Cancer Cells toward Treatment with Topoisomerase II (TOP2) Poison Etoposide. J Med Chem 2019, 62 (9), 4669-4682.
2. Kiselev, E.; Ravji, A.; Kankanala, J.; Xie, J.; Wang, Z.; Pommier, Y., Novel deazaflavin tyrosyl-DNA phosphodiesterase 2 (TDP2) inhibitors. DNA Repair (Amst) 2020, 85, 102747.

Acknowledgements: We thank the Center for Drug Design in College of Pharmacy at University of Minnesota for supporting this research. The authors declare no conflicts of interest.

Figure 1. Inhibitory effect of 11a on ABC efflux transporters in vesicular transport assays. Data are shown as mean ± SD. (A) Inhibition of vesicular transport activity by 11a in membrane vesicles expressing different ABC efflux transporters. Values are expressed as % of inhibition relative to uptake activity in the presence of ATP without inhibitor (n ≥ 3); (B) Inhibition of E217βG uptake by 11a in MRP1-expressing vesicles in the presence or absence of GSH. One-way ANOVA: **p < 0.01, ***p < 0.001, n = 3; (C) Inhibitory potencies of 11a and positive control MK-571 on E217βG uptake by MRP1-expressing vesicles (n = 2); (D) Inhibitory potencies of 11a and positive control MK-571 on E217βG uptake by MRP2-expressing vesicles (n = 2).

Figure 2. Effect of 11a on vesicular MRP1 ATPase activity. Data represent vanadate-sensitive ATPase activity in MRP1 expressing membrane vesicles and are shown as mean ± SD (n = 2). (A) ATPase stimulation by 11a in the presence or absence of GSH; (B) Inhibition of NEM-GSH stimulated ATPase activity by 11a in the presence or absence of GSH; (C) Inhibition of GSH stimulated ATPase activity by 11a; (C) Inhibition of GSSG stimulated ATPase activity by 11a. One-way ANOVA: *p < 0.05, **p < 0.01.

Figure 3. Effect of 11a on chemosensitivity of small lung cancer cells. Top: Intracellular drug uptake and representative dose-response curve of H69AR cells (A) and NCI-H69 cells (B) treated with ETP alone or in combination with 10 µM 11a. Bottom: Intracellular drug uptake and representative dose-response curve of H69AR cells (C) and NCI-H69 cells (D) treated with DOX alone or in combination with 10 µM 11a. One-way ANOVA: **p < 0.01, ***p < 0.001. Data are expressed as mean ± SD (n ≥ 3).