Polymerization of tubulin dimers to form microtubules is one of the key events in cell proliferation. The inhibition of this event has long been recognized as a potential treatment option for various types of cancer. Compound 1e was previously developed by our team as a potent inhibitor of tubulin polymerization that binds to the colchicine site. To further improve the potency and therapeutic properties of compound 1e, we hypothesized based on the X-ray crystal structure that modification of the pyrimidine dihydroquinoxalinone scaffold with additional hetero-atom (N, O, and S) substituents could allow the resulting new compounds to bind more tightly to the colchicine site and display greater efficacy in cancer therapy. We therefore synthesized a series of new pyrimidine dihydroquinoxalinone derivatives, compounds 10, 12b−c, 12e, 12h, and 12j−l, and evaluated their cytotoxicity and relative ability to inhibit proliferation, resulting in the discovery of new tubulin-polymerization inhibitors. Among these, the most potent new inhibitor was compound 12k, which exhibited high cytotoxic activity in vitro, a longer half-life than the parental compound in liver microsomes (IC 50 = 0.2 nM, t 1/2 = >300 min), and significant potency against a wide range of cancer cell lines including those from melanoma and breast, pancreatic, and prostate cancers. High-resolution X-ray crystal structures of the best compounds in this scaffold series, 12e, 12j, and 12k, confirmed their direct binding to the colchicine site of tubulin and revealed their detailed molecular interactions. Further evaluation of 12k in vivo using a highly taxane-resistant prostate cancer xenograft model, PC-3/TxR, demonstrated the strong tumor growth inhibition at the low dose of 2.5 mg/kg (i.v., twice per week). Collectively, these results strongly support further preclinical evaluations of 12k as a potential candidate for development.
Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy with high mortality. It is the 4th leading cause of cancer-related death in the U.S. and has the lowest 5-year survival rate among all cancer types. One of the main hurdles for the treatment is limited effective chemotherapy and rapidly acquired resistance against it. Advanced PDAC research and various treatment options only showed minimal enhancement to the overall survival outcomes. Hence more effective drugs and therapies are in need for improving overall therapeutic outcomes for PDAC patients. Recently, we reported the discovery of a new generation of tubulin inhibitors (SB-216) that targets the colchicine binding site and serves as a potent anti-cancer agent for various cancer types. Here, we evaluated the in vitro effects of SB-216 on the PDAC cell lines in comparison with one of the first-line treatment paclitaxel. We also established luciferase labeled Mia PaCa-2 cell line, Mia PaCa-2-Luc, for live monitoring tumor progression and evaluating the anti-tumor effect in subcutaneous mouse models. The results showed that SB-216 effectively inhibited cell proliferation, colony formation, and cell migration dose-dependently at low nanomolar concentrations. Immunoblotting also confirmed SB-216 induced cell apoptosis in a dose-dependent manner. Cell cycle arrest assay confirmed that SB-216 can arrest cells in the G2/M phase. In vivo study suggested that SB-216 inhibited tumor growth significantly in Mia PaCa-2-Luc subcutaneous xenograft model with low toxicity. Our preclinical data demonstrated the potential of SB-216 to inhibit proliferation and induce apoptosis in PDAC cells, indicating a promising chemotherapy agent in treating PDAC. However, further evaluations are warranted in orthotopic and PDX tumor models for clinical significance. Citation Format: Rui Wang, Satyanarayana Pochampally, David Hamilton, Duane D. Miller, Wei Li. Evaluation of anti-tumor activity of SB-216, a novel potent tubulin inhibitor, in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4052.
Colchicine binding site inhibitors have proven to have potent anti-cancer efficacy in many cancer types. Inhibition of tubulin polymerization, a popular mechanism of action for anti-cancer activities, will be activated once the inhibitor interacts with the colchicine binding site. We have previously developed and reported a potent tubulin inhibitor (SB-216). One of its derivatives, the compound SP-1-39, a pyrimidine dihydroquinoxalinone derivative, has been recently designed to further improve the potency and therapeutic properties. Here, we examined the impact of SP-1-39 on preclinical prostate cancer models in vitro and in vivo. EBI competition assay confirmed that SP-1-39 binds to colchicine-binding site and inhibits the formation of EBI:β-tubulin adduct. The anti-proliferation abilities of SP-1-39 was tested against melanoma, breast cancer, pancreatic cancer, prostate cancer, and paclitaxel-resistant prostate cancer cell lines by MTS assay, and defined IC50 values of 0.2-4 nmol/L. Other in vitro results showed SP-1-39 disrupted microtubule network, induced cell cycle arrest at G2/M phase, and caused apoptosis in paclitaxel-resistant human prostate cancer cells (PC-3/TxR). For in vivo study, 2.5mg/kg SP-1-39 significantly attenuated tumor growth in PC-3/TxR xenograft mouse model, inhibited angiogenesis, and overcame taxane resistance. Our findings demonstrate the preclinical therapeutic efficacy and safety of SP-1-39, and provide support for further exploration of novel tubulin inhibitors as anti-cancer agent for cancer therapy. Citation Format: Rui Wang, Kelli L. Hartman, Satyanarayana Pochampally, Duane D. Miller, Wei Li. SP-1-39 as a novel tubulin inhibitor overcomes taxane resistance in castration-resistant prostate cancer model. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4943.
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