Pharmacologic characterization of SHR8443, a novel dual inhibitor of phosphatidylinositol 3-kinase and mammalian target of rapamycin

Abstract

Dysregulation of the phosphatidylinositol 3-kinase (PI3K) pathway occurs frequently in human cancer and contributes to resistance to antitumor therapy. Inhibition of key signaling proteins in this pathway therefore represents an attractive targeting strategy for cancer therapy. Here, we show that SHR8443, an imidazo [4,5-c] quinoline derivative, inhibited mammalian target of rapamycin (mTOR) kinase and PI3K, especially PI3Kα/δ/γ isoforms with picomolar potency, by binding to the ATP subunits of the respective enzymes. Inhibition of PI3K/AKT/mTOR signaling by SHR8443 induced G₁ phase arrest, autophagy and apoptosis, and resulted in broad anti-proliferative activity against a panel of cancer cells with different genetic backgrounds. Furthermore, SHR8443 overcame resistance to RAF/MEK inhibitors and exhibited synergistic antitumor activity in combination with RAF/MEK inhibitors in vitro. Compared with the well-known PI3K/mTOR inhibitor BEZ235, SHR8443 showed broader and stronger efficacy against carcinoma xenografts, including those resistant to anti-HER2 antibody trastuzumab, in association with the inhibition of AKT and S6 phosphorylation in tumor tissues, and also caused no noticeable toxicity. Thus, our preclinical data show that SHR8443 is a dual PI3K/mTOR inhibitor with pharmaceutical properties favorable for use as an anticancer agent.

Keywords: BEZ235; PI3K; SHR8443; drug resistance; mTOR.

Figure 1

(A) Chemical structure of SHR8443. (B) The binding modes of BEZ235 and SHR8443 with PI3Kα. The protein was represented as a ribbon diagram (green); SHR8443 (yellow) and BEZ235 (magenta), as well as residues that interacted with these compounds, were shown in stick form. Hydrogen bonds were shown as dashed lines (SHR8443, yellow; BEZ235, cyan) between heavy atoms. (C) The binding mode of SHR8443 within mTOR. SHR8443 was represented by wheat-colored sticks; mTOR and PI3Kα were shown as cyan and green ribbon diagrams, respectively. The key residues of mTOR and PI3kα were shown as sticks. Hydrogen bonds were shown as dashed lines (yellow) between heavy atoms.

Figure 2. Effects of SHR8443 on PI3K/mTOR signaling in cancer cell lines with different genetic backgrounds

(A) Breast cancer cell lines (MCF7, BT-474 and MDA-MB-468) and (B) KRAS or BRAF mutated cell lines (COLO205, SW620 and A549) were treated with increasing concentrations of SHR8443 or 100 nM BEZ235 for 3 h. (C) U-87MG cells were treated with 100 nM SHR8443 for the indicated time. (D) Reversibility of PI3K/mTOR signaling. MDA-MB-468 cells were treated with 100 nM SHR8443 for 2 h, with or without further culture in drug-free medium for the indicated time after washing with PBS. Whole-cell lysates were analyzed by Western blotting using the indicated antibodies.

Figure 3. Cell profile of SHR8443 cytotoxicity

(A) Anti-proliferative activity of SHR8443 against a panel of cancer cell lines with different tissues of origin and genetic backgrounds. Tumor cell lines harboring a PIK3CA mutation (♦), HER2 amplification (●), PTEN deficiency (□), or RAS/RAF mutation (^*) were treated with different concentrations of SHR8443 for 72 h. Cell viability was determined by SRB assay. IC₅₀ values were presented as means ± SD of three independent experiments. (B) Melanoma cell lines A375, A375/TR (trametinib-resistant), and A375/DR (dabrafenib-resistant) were treated with different concentrations of SHR8443, dabrafenib, or trametinib for 72 h. Cell viability was determined by SRB assay. (C) Melanoma cell lines A375, A375/TR and A375/DR were treated with SHR8443, dabrafenib or trametinib for 2 h. Whole-cell lysates were analyzed by Western blotting using the indicated antibodies.

Figure 4. SHR8443 causes cell cycle arrest, autophagy, and apoptosis

(A) Cell-cycle phase histograms of MCF7, MDA-MB-468, COLO205 and A549 cell lines following treatment with SHR8443 or BEZ235 at the indicated concentration for 24 h. (B) MCF7, MDA-MB-468 and A549 cells were treated with SHR8443 or BEZ235 at the indicated concentrations for 72 h, and then analyzed by annexin V-FITC/PI staining and flow cytometry. (C) After treatment of cells with SHR8443 or BEZ235 for 72 h, whole-cell lysates were immunoblotted with an anti-PARP antibody. (D) A549 cells were treated with SHR8443 (left), BEZ235, or the combination of SHR8443/BEZ235 (100 nM) with E64d/pep (10 mg/mL) for 48 h. Whole-cell lysates were analyzed by immunoblotting with an anti-LC3 antibody.

Figure 5. Combined treatment of BRAF mutant A375 and COLO205 cells with SHR8443 and dabrafenib or trametinib

(A) A375 and COLO205 cells were incubated with the indicated concentrations of each compound or their combinations for 72 h. Cell viability was determined by SRB assay. CI, combination index. (B) A375 and COLO205 cells were incubated with SHR8443 (100 nM), trametinib (10 nM), dabrafenib (100 nM), or their combinations for 72 h, and analyzed by annexin V-FITC/PI staining and flow cytometry. ^*P < 0.05, versus either single agent treatment. (C) A375 and COLO205 cells were incubated with SHR8443 (100 nM), trametinib (10 nM), dabrafenib (100 nM), or their combinations for 2 h. Whole-cell lysates were analyzed by Western blotting using the indicated antibodies.

Figure 6. In vivo study of SHR8443 in mouse xenograft models

(A, B) Pharmacokinetic/pharmacodynamic studies of SHR8443. U-87MG tumor-bearing mice were orally administered a single 10-mg/kg dose of SHR8443 and sacrificed at the indicated times. The concentration of SHR8443 in blood plasma and tumor tissues was determined (A). In parallel, tumor extracts were analyzed by Western blotting (B). (C) Antitumor activity of SHR8443 against xenografts. U-87MG, PC-3, A549, BT-474 and BT-474/trastuzumab tumor-bearing mice were orally administered BEZ235 or SHR8443 daily, or intravenously injected with trastuzumab twice a week. Tumor volumes were measured twice a week. ^*P < 0.05, ^**P < 0.01 versus vehicle.