Preclinical studies of the triazolo[1,5- a]pyrimidine derivative WS-716 as a highly potent, specific and orally active P-glycoprotein (P-gp) inhibitor

Abstract

Multidrug resistance (MDR) is the main cause of clinical treatment failure and poor prognosis in cancer. Targeting P-glycoprotein (P-gp) has been regarded as an effective strategy to overcome MDR. In this work, we reported our preclinical studies of the triazolo[1,5-a]pyrimidine-based compound WS-716 as a highly potent, specific, and orally active P-gp inhibitor. Through direct binding to P-gp, WS-716 inhibited efflux function of P-gp and specifically reversed P-gp-mediated MDR to paclitaxel (PTX) in multiple resistant cell lines, without changing its expression or subcellular localization. WS-716 and PTX synergistically inhibited formation of colony and 3D spheroid, induced apoptosis and cell cycle arrest at G2/M phase in resistant SW620/Ad300 cells. In addition, WS-716 displayed minimal effect on the drug-metabolizing enzyme cytochrome P4503A4 (CYP3A4). Importantly, WS-716 increased sensitivity of both pre-clinically and clinically derived MDR tumors to PTX in vivo with the T/C value of 29.7% in patient-derived xenograft (PDX) models. Relative to PTX treatment alone, combination of WS-716 and PTX caused no obvious adverse reactions. Taken together, our preclinical studies revealed therapeutic promise of WS-716 against MDR cancer, the promising data warrant its further development for cancer therapy.

Keywords: ATP-Binding cassette; Cancer therapy; Drug combination; Multidrug resistance (MDR); P-gp inhibitors; Preclinical studies; Triazolo[1,5-a]pyrimidine.

Graphical abstract

Figure 1

WS-716 reversed multidrug resistance mediated by P-glycoprotein (P-gp). (A) Chemical structure of WS-716. (B) P-gp was up-regulated in SW620/Ad300 cells. (C) WS-716 and verapamil (VPM) below 30 μmol/L had little inhibitory effect on the viability of SW620/Ad300 cells. (D) WS-716 significantly increased the sensitivity of SW620/Ad300 cells to paclitaxel (PTX) at 20 μmol/L. (E) P-gp was up-regulated in KB-C2 cells. (F) P-gp was over-expressed in HEK293/ABCB1 cells. β-Actin was used as the loading control. Data are presented as the mean ± SD, n = 3.

Figure 2

WS-716 increased the intracellular accumulation of paclitaxel (PTX) by intercepting its efflux. (A) WS-716 increased intracellular PTX in SW620/Ad300 cells. (B) WS-716 increased the accumulation of [³H]-PTX. (C) WS-716 slightly reduced the efflux of PTX in SW620 cells. (D) WS-716 significantly reduced the efflux of PTX in SW620/Ad300 cells. Data are presented as the mean ± SD, n = 3. ^#P < 0.05, ^##P < 0.01.

Figure 3

WS-716 stimulated ATP hydrolysis without changing the expression or subcellular localization of P-glycoprotein (P-gp). (A) WS-716 did not change the protein level of P-gp in SW620/Ad300 cells. (B) Effect of WS-716 on the subcellular localization of P-gp (green) after treatment for 76 h. The cell nuclei (blue) were stained by Hoechst 33258. The scale bar is 10 μm. (C) WS-716 stimulated ATP hydrolysis and reached the maximal value at 20 μmol/L. Data are presented as the mean ± SD, n = 3.

Figure 4

WS-716 binds directly to P-glycoprotein (P-gp). (A) The interaction of WS-716 and P-gp was assessed through cellular thermal shift assay. (B) The ribbon diagram of P-gp with the binding location of WS-716 at the internal cavity. (C) The 2D bonding interaction of WS-716 within P-gp (PDB code: 6FN1). (D) The 3D ligand–receptor interaction diagram of WS-716 and P-gp.

Figure 5

WS-716 had little effect on the activity of cytochrome P4503A4 (CYP3A4). (A) The effect of WS-716, VPM and ketoconazole on the reaction kinetics of fluorogenic substrate metabolism in human liver microsomes containing CYP3A4 during 40 min at 37 °C. (B) Dose–response curve of WS-716, VPM, and ketoconazole on CYP3A4 at 37 °C. Data are presented as the mean ± SD, n = 3.

Figure 6

WS-716 enhanced the inhibitory effect of paclitaxel (PTX) on the formation of colonies and 3D spheroids. (A) The colony-formation of SW620/Ad300 cells after treatment with WS-716 (20 μmol/L), PTX (1 μmol/L), or their combination on Day 7. (B) The 3D spheroid-formation of SW620/Ad300 cells after treatment of WS-716 (20 μmol/L), PTX (1 μmol/L), or their combination on Day 5 and Day 10. The scale bar is 200 μm. Data are presented as the mean ± SD, n = 3. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 vs. NC, ^#P < 0.05.

Figure 7

WS-716 enhanced the apoptosis induced by paclitaxel (PTX) through the mitochondrial pathway in SW620/Ad300 cells. SW620/Ad300 cells were treated with WS-716 (20 μmol/L, 4 h), PTX (1 μmol/L, 2 h）or combination of WS-716 and PTX. (A) Damaged cell nucleus of SW620/Ad300 was detected by Hoechst 33258 staining. The scale bar is 100 μm. (B) Apoptosis was measured by FCM. (C) Expression of cleaved caspase-9, -3, -7, PARP, NLRP3, cleaved caspase-1, Gasdermin D, and LC3B was measured by Western blot. (D) The mitochondrial membrane potential was evaluated by FCM. (E) Bax, Bcl-2, Bcl-xL, and cytochrome c in cytoplasm expression were analyzed by Western blot at 24 h. β-Actin was used as a loading control. Data are represented as mean ± SD, n = 3. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 vs. NC, ^#P < 0.05.

Figure 8

WS-716 potentiates the ability of paclitaxel (PTX) to induce cycle arrest at G2/M phase. SW620/Ad300 cells were treated with WS-716 (20 μmol/L, 4 h)，PTX (1 μmol/L, 2 h) or combination of WS-716 and PTX. (A, B) The distribution of cell cycle was examined by FCM. (C) Expression of p-CDK2, total CDK2, p-CDK1, total CDK1, p21, p27, cyclin H, and CDK7 were analyzed by Western blot at 24 h. β-Actin was used as a loading control. Data are represented as mean ± SD, n = 3. ∗P < 0.05, ∗∗∗P < 0.001 vs. NC, ^#P < 0.05.

Figure 9

WS-716 significantly improves the inhibitory efficacy of paclitaxel (PTX) in vivo. Mice bearing SW620/Ad300 xenograft were treated with vehicle (10 mL/kg/day), WS-716 (100 mg/kg/day), PTX (5 mg/kg/3 days), PTX plus VPM (4 mg/kg/day) or PTX plus WS-716 for 15 days. (A) Tumor volume during the therapy. (B) Tumor weight on Day 15. (C) Photographs of tumors on Day 15. (D) Body weight of mice during the therapy. (E) HE and TUNEL analysis of tumor and liver tissues. Data are presented as the mean ± SD (n = 5). ∗P < 0.05 vs. vehicle. Scale bar is 100 μm.

Figure 10

WS-716 enhances the antitumor activity of paclitaxel (PTX) in the multidrug resistance (MDR) patient-derived xenograft (PDX) models. Mice bearing PDX were treated with vehicle (10 mL/kg/day), PTX (5 mg/kg/3 days), WS-716 (100 mg/kg/day) or PTX plus WS-716. Tumor volume and animal body weight were measured periodically. (A) Workflow of establishment of MDR PDX models. (B) HE staining was used for pathological validation and IHC analysis was used for detection of P-gp expression in tumor tissues from the patient (primary tumor), F0 (F0 tumor), and F2 xenografts (F2 tumor). (C) Expression of P-gp in F0 and F2 tumor was detected using Western blot. (D) Tumor volume during treatment. (E) Tumor weight on Day 13. (F) The relative tumor proliferation rate (T/C) of treatment groups (The red dotted line: 40%). (G) Photographs of tumors on Day 13. (H) Body weight during treatment. Data are presented as the mean ± SD, n = 5. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 vs. vehicle, ^#P < 0.05, ^#P < 0.01. Scale bar is 100 μm.

Figure 11

Hepatic and renal functions were evaluated at the end of treatment. (A) Concentrations of total protein, albumin, direct bilirubin, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase in serum of the mice were used to evaluate hepatic function of mice. (B) The concentrations of creatinine and urea were used to evaluate renal function of mice. Data are presented as mean ± SD, n = 3.