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Antimitotics that target tubulin are among the most useful chemotherapeutic drugs, but their clinical activity is often limited by the development of multidrug resistance. We recently discovered the novel small-molecule DJ101 as a potent and metabolically stable tubulin inhibitor that can circumvent the drug efflux pumps responsible for multidrug resistance of existing tubulin inhibitors. In this study, we determined the mechanism of action of this drug. The basis for its activity was illuminated by solving the crystal structure of DJ101 in complex with tubulin at a resolution of 2.8Å. Investigations of the potency of DJ101 in a panel of human metastatic melanoma cell lines harboring major clinically relevant mutations defined IC50 values of 7–10 nmol/L. In cells, DJ101 disrupted microtubule networks, suppressed anchorage-dependent melanoma colony formation, and impaired cancer cell migration. In melanoma-bearing mice, DJ101 administration inhibited tumor growth and reduced lung metastasis in the absence of observable toxicity. DJ101 also completely inhibited tumor growth in a paclitaxel-resistant xenograft mouse model of human prostate cancer (PC-3/TxR), where paclitaxel was minimally effective. Our findings offer preclinical proof of concept for the continued development of DJ101 as a next-generation tubulin inhibitor for cancer therapy. Significance: These findings offer preclinical proof of concept for the continued development of DJ101 as a next-generation antitubulin drug for cancer therapy. Cancer Res; 78(1); 265–77. ©2017 AACR.

Background: The androgen receptor (AR) is the most highly expressed steroid receptor in breast cancer with 75-95% of estrogen receptor (ER)-positive and 40-70% of ER-negative breast cancers expressing AR. Prior studies have shown that women with metastatic breast cancer who have been previously treated with tamoxifen and progress have responded to non-tissue-selective androgens like fluoxymesterone, medroxyprogesterone and danazol, with overall response rates ranging from 20 to 60%. Although these non-tissue-selective androgens have been used to treat breast cancer, the unwanted virilizing side effects, including facial and body hair, enlargement of voice box, acne, and edema, have limited their widespread clinical use. Nonsteroidal, tissue- selective androgen receptor modulators (SARMs) like enobosarm and GTx-027 may provide a novel targeted approach to exploit the therapeutic benefits of androgen therapy in breast cancer without the unwanted virilizing side effects or concerns related to their conversion by aromatase to estrogens. Methods: MDA-MB-231 triple-negative and MCF-7 triple-positive breast cancer cells stably expressing AR (MDA-MB-231-AR and MCF-7-AR, respectively) were used to evaluate the in vitro and in vivo anti-proliferative and gene expression effects of bicalutamide (an AR antagonist), dihydrotestosterone (DHT), enobosarm, and GTx-027. Phase II clinical studies examined the pharmacologic effects (lean body mass and muscle strength), tissue selectivity (skin, endometrium, and hair growth) and safety of enobosarm, a first-in-class SARM, in postmenopausal women. Results: DHT and SARMs, but not bicalutamide, inhibited the proliferation of MDA-MB-231-AR and MCF-7-AR cells. DHT and the SARMs functioned as AR agonists in transactivation assays in MDA-MB-231 cells, indicating that AR agonists, but not antagonists, elicit anti-proliferative effects in breast cancer. MDA-MB-231-AR and MCF-7-AR cells were implanted subcutaneously in nude mice and were treated orally with vehicle or 30 mg/kg/day GTx-027. GTx-027 reduced the tumor growth significantly with greater than 75% tumor growth inhibition observed in MDA-MB-231-AR tumors and greater than 50% tumor growth inhibition observed in MCF-7-AR tumors, compared to vehicle-treated tumors. GTx-027 also inhibited tumor weights by greater than 50% concurring with the tumor volume observation. GTx-027 inhibited the intratumoral expression of genes and pathways that promote breast cancer development through its actions on the AR. Interestingly, GTx-027 and other AR agonists elicited opposite effects on various AR target genes in ER-positive versus ER-negative breast cancer cells, indicating a potential cross talk between AR and ER signaling pathways on the promoter of these genes. Phase II clinical studies showed that enobosarm was generally well tolerated in postmenopausal women. Statistically significant improvements in lean body mass and physical function were observed after 12 weeks of treatment with 3 mg enobosarm, with no significant changes in sebaceous gland volume (biopsy), hair growth, endometrial stripe thickness (transvaginal ultrasound) or bleeding (medroxyprogesterone challenge). Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-09-21.

Despite the success of medical strategies to reduce androgen levels in the treatment of prostate cancer, this disease invariably relapses to a castrate-resistant state that is generally fatal. Although it had been thought that androgen-insensitive cancers no longer relied on the androgen receptor (AR) for growth and survival, it is now clear that this is not the case. Because relapses are known to occur by many mechanisms that keep the AR functionally active, strategies to block AR accumulation in the nucleus may be therapeutically useful. Here, we report the discovery of a selective nuclear androgen receptor exporter (SNARE) that functions to exclude AR from the nucleus. SNARE-1 binds wild-type and mutant ARs and efficiently inhibits their transactivation activity and ability to induce PSA gene expression. SNARE-1 inhibits the androgen-sensitive growth of LNCaP cells and tumor xenografts. Quantitative subcellular localization studies suggest that SNARE-1 inhibits nuclear translocation of AR, but also facilitates export of nuclear AR that has been translocated by an agonist. Mechanistic studies indicate that SNARE-1 rapidly phosphorylates p38 mitogen-activated protein kinase (MAPK) and Ser650 of the AR. Additionally, SNARE-1 was found to promote ubiquitination of AR in LNCaP cells. Lastly, SNARE-1 functions as a tissue-selective AR inhibitor, as it fails to phosphorylate p38 MAPK in U2OS bone cells that are stably transfected with AR. In summary, SNARE-1 inhibits AR function by a mechanism that is distinct from clinically available antiandrogens, such that it might inform novel methods to block AR function in androgen-independent prostate cancer. Cancer Res; 70(2); 842–51

Cancer remains one of the leading causes of mortality worldwide. While current therapies are effective in treating early stage cancers, their efficacy against advanced cancers, especially multidrug-resistant cancers, is limited. Our lab recently reported a set of 2-aryl-4-benzoyl-imidazoles (ABI) derivatives with potent anti-proliferative activity against melanoma and prostate cancer cell lines and xenografts. Further optimization has led to the synthesis of a new series of 4-aryl-2-benzoyl-imidazoles with the aryl group and the benzoyl group reversed as compared to the previous ABI structure. The reverse ABI (RABI) compounds were evaluated for biological activity against 8 cancer cell lines including some which were multidrug-resistant. The in vitro results showed that several RABI compounds had excellent antiproliferative properties with IC50 values in the low namomolar range, comparable to existing tubulin-targeting agents such as paclitaxel, colchicine, or vinblastine, but with the ability to effectively circumvent Pgp-mediated drug resistance. The IC50 of the most active compound 12a was 9 nM. The mechanism of action (MOA) of the RABI compounds was investigated using cell cycle analysis, tubulin polymerization assays and molecular docking studies. These studies showed that their antitumor activity was achieved through inhibition of tubulin polymerization at the colchicine binding site. Our results suggest that the novel RABI compounds may be developed as a promising antitumor agent for advanced cancers. Citation Format: Min Xiao, Sunjoo Ahn, Jianjun Chen, Jin Wang, Duane D. Miller, James T. Dalton, Wei Li. Target tubulin polymerization with novel reverse ABI analogs interacting with the colchicine binding site. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5684. doi:10.1158/1538-7445.AM2013-5684

A novel indole antimitotic compound, GTx-230, was identified in cell-based screening assays that binds to the colchicine site in tubulin and destabilizes microtubules. In this study, the pharmacokinetics, antitumor efficacy, neurotoxicity, and vascular disrupting ability of GTx-230 were evaluated. GTx-230 inhibited the in vitro growth of a number of human cancer cell lines including DU-145, LNCaP, PC-3, and PPC-1 prostate cancer cells with low nanomolar IC50 values. Following a 10 mg/kg intravenous (i.v.) dose to mice, the plasma clearance (CL), volume of distribution at steady state (Vdss), and terminal half-life (T1/2) of GTx-230 were 19 ml/min/kg, 2.9 l/kg, and 1.7 h, respectively. Oral bioavailability of GTx-230 was 36% in mice. In vivo, GTx-230 (6.7 mg/kg, qd, p.o.) was effective with near 100% tumor growth inhibition in xenograft models using PC-3/TXR cells over-expressing P-glycoprotein. In contrast, docetaxel, a known antimitotic agent, was not effective in the PC-3/TXR xenograft model. In vivo studies of mouse performance on an accelerating rotarod and hotplate showed that GTx-230 did not induce any symptoms of peripheral neuropathy. In addition, we evaluated the effect of GTx-230 on in vitro vascular permeability using a transwell system with confluent HUVEC monolayers. GTx-230 resulted in increased permeability after 1 h incubation and showed potency similar to CA4. In summary, the potent in vitro and in vivo antitumor activities of GTx-230 suggest that it may represent a new antimitotic agent for management of solid malignancies, particularly for patients with drug resistant cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2788. doi:1538-7445.AM2012-2788

With rapidly rising incidences of metastatic melanoma and its high resistance to current therapeutic agents, developing more effective drugs is urgently needed. We recently discovered a novel class of 2-aryl-4-benzoyl-imidazoles (ABI) analogs. These compounds showed great antiproliferative activity on melanoma cells and the IC50 is as low as 8 nM. ABIs showed equal potency against multidrug resistant melanoma cells and the sensitive parent cells, suggesting that they can effectively overcome P-gp meditated multidrug resistance. ABIs inhibited capillary network formation which indicates high potential for anti-angiogenesis. Cell cycle analysis showed ABIs arrested cells in the G2/M phase and inhibited tubulin polymerization. ABIs bind to the colchicine binding site in the α/β-tubulin heterodimer as indicated by a number of evidences including competitive binding and molecular modeling studies. Immunofluorescence experiments revealed that microtubule modality was dramatically changed with ABIs treatment. In vivo studies using both B16-F1/C57BL model and A375/SHO model showed significant inhibition of melanoma tumor growth by ABIs. These novel analogs hold great promise as new selective agents for the improved treatment of malignant melanoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1350. doi:10.1158/1538-7445.AM2011-1350

Inhibition of tubulin dynamics has been a fruitful drug target for cancer treatment. Out the three ligand binding sites in tubulin, drugs targeting taxane binding site (e.g. taxol and epithilones) and vinca alkaloids binding site (e.g. vinblastine) are clinically very successful drugs. However, no FDA-approved cancer drugs targeting the colchicine binding site in tubulin currently exist. In our ongoing efforts to search for effective drugs for the treatment of advanced melanoma that are highly resistant to all existing cancer treatment, we have discovered a new class of compounds that are not substrates of P-glycoprotein (Pgp) and are highly efficacious against human melanoma tumors in A375 xenograft models. At relatively low doses (5∼15 mg/kg), these compounds inhibit melanoma tumor growth more effectively than DTIC (the gold standard drug for advanced melanoma) at 60 mg/kg. Extensive biological and molecular modeling studies suggest that these compounds work by competitively binding to the colchicine site in tubulin. Further optimization of these compounds hold great promise in providing an effective agent for the treatment of advanced melanoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 730.