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Effects of in silico Identified Inhibitors of ABC Transporters on Drug Accumulation in Multidrug Resistant Cancer Cell Culture Models

Overexpression of ATP‐binding cassette (ABC) transporters is associated with multidrug resistance (MDR) in cancers. P‐glycoprotein (P‐gp) and breast cancer resistance protein (BCRP) are two of the most studied drug transporters related to MDR. No clinically approved drugs are currently available that inhibit these proteins, rendering chemotherapy treatment of many MDR cancers ineffective. Using computational methods, we have identified new compounds that inhibit P‐gp by affecting the nucleotide binding domains of the protein (Brewer et al., Mol. Pharmacol. 2014). Several of these compounds show successful MDR reversal in the drug resistant DU145TXR prostate cancer cell line (Follit et al., Pharmacol. Res. Perspect. 2015). Here, we show that co‐administration of identified P‐gp inhibitors, SMU‐29, 34 and 45, led to increased accumulation of the chemotherapeutic agent, daunorubicin, in the P‐gp over‐expressing ovarian cancer cell line, A2780ADR. Treatment with the in silico identified inhibitors led to ~3 fold increased accumulation of daunorubicin. Efflux studies indicated that the presence of inhibitors reduced the release of daunorubicin by nearly 50%. These experiments indicated that A2780ADR cells accumulated the chemotherapeutic as a result of inhibition of P‐gp by the novel inhibitors 29, 34 and 45. We hypothesized that the increased retention and accumulation of chemotherapeutics in the cancer cells should lead to higher efficacy of chemotherapy treatment. We tested this hypothesis using P‐gp overexpressing DU145TXR prostate tumor spheroids. Treatment of spheroids with daunorubicin in the presence of SMU‐29 increased the number of dead cells in DU145TXR spheroids compared to treatment with chemotherapeutic agent alone, resulting in significant reduction in spheroid cross sectional area. The inhibition of P‐gp by SMU‐29 was also confirmed through increased accumulation and penetration of the P‐gp substrate calcein in prostate tumor spheroids. A prolonged incubation of these spheroids with non‐toxic P‐gp inhibitor SMU‐29, after an initial 3 hour co‐administration of paclitaxel with SMU‐29, led to an increased number of dead cells highlighting the influence of increased drug accumulation and retention for therapy success.