(W1230-03-21) Biological Evaluation of zq-1-68, a Novel Dual-Targeting Tubulin/HDAC Inhibitor in Metastatic Castration-Resistant Prostate Cancer

Purpose: Prostate cancer is the 2^nd most prevalent cancer in men in the United States, with over 300,000 new cases projected to be diagnosed in 2025. Although most patients respond well to androgen deprivation therapy, 10-20% of patients are either nonresponsive or develop resistance to androgen deprivation therapy and advance to castration-resistant prostate cancer. Metastatic castration-resistant prostate cancer (mCRPC) remains incurable with a five-year survival rate of < 40%. Treatment options for mCRPC include more advanced hormone therapies such as enzalutamide or abiaterone, often in combination with chemotherapy. Microtubule-targeting agents (MTAs) such as taxanes are common but are susceptible to acquired drug resistance. There is a clinical need for effective, well-tolerated prostate cancer drugs that can bypass taxane resistance and androgen independence. Colchicine binding site inhibitors (CBSIs) can bypass many mechanisms of acquired drug resistance and are therefore emerging as potential alternatives to taxanes. As epigenetic dysregulation is an emerging hallmark of cancer, there is also rising interest in the development of epigenetic-targeting drugs for cancer treatment. In particular, histone deacetylase (HDAC) enzymes are emerging as a promising drug target. In this study, we developed a small molecule dual tubulin/HDAC inhibitor, zq-1-68, which has potent antiproliferative activity in a variety of cancer models. Zq-1-68 has on-target anti-HDAC6/8 activity, high affinity for the colchicine binding site of tubulin, and strong anticancer activity in vitro. In vivo, zq-1-68 shows profound inhibition of tumor growth in prostate cancer models.

Methods: We designed and synthesized a series of small molecule dual tubulin/HDAC inhibitors and screened their antiproliferative activity in ovarian cancer (OVCAR3 and SKOV3), prostate cancer (22Rv1 and PC3), and melanoma (A375) with the MTS assay. We used tubulin polymerization assays and immunocytochemistry experiments to demonstrate that zq-1-68 inhibits tubulin polymerization and disrupts proper microtubule assembly. The EBI (N,N'-ethylene-bis(iodoacetamide)) assay was used to confirm that zq-1-68 binds tubulin at the colchicine binding site. We performed cell-based and cell-free luminescence-based assays to confirm zq-1-68’s anti-HDAC activity. We further characterized zq-1-68’s anticancer and anti-HDAC activity with western blot, wound scratch, and Transwell assays. For our in vivo studies, male NOD scid gamma (NSG) mice were subcutaneously inoculated with taxane-resistant PC3 prostate cancer cells. Mice were treated with either vehicle, zq-1-68 (20 mg/kg), its tubulin inhibitor component CH-II-77 (20 mg/kg), or HDAC6 inhibitor Tubastatin A (20 mg/kg) 3 times per week via intraperitoneal injection for 14 days. Tumor growth was measured every 2 days. At the study endpoint, mice were euthanized and their tumors and organs were harvested, fixed with 10% neutral buffered formalin, and embedded in paraffin for histological and immunohistochemical analysis. Pharmacokinetic data was obtained by administering 20 mg/kg of zq-1-68 into mice via intraperitoneal injection and determining the plasma concentration.

Results: Of the compounds tested, zq-1-68 demonstrated the strongest on-target activity in vitro and was well-tolerated in vivo. In in vitro prostate cancer models, zq-1-68 had an antiproliferative IC₅₀ of 6.2-8.4 nM. EBI assay confirmed that zq-1-68 has a strong affinity for the colchicine binding site. The HDAC-Glo assay indicated that it selectively inhibits the HDAC6 and HDAC8 isoforms. Western blot experiments showed that zq-1-68 induces apoptosis and has potent anti-HDAC6 activity. We also found that zq-1-68 significantly inhibits tumor cell invasion and migration at concentrations as low as 2 nM. In vivo, zq-1-68 is metabolically stable with a half-life of 5.69 hours. It significantly inhibited primary tumor growth in a taxane-resistant PC3 prostate cancer models. Interestingly, zq-1-68’s dual activity appeared to have a synergistic effect on primary prostate tumor growth compared to single-acting CH-II-77 and Tubastatin A, with average tumor volumes of 264 mm³, 604 mm³, and 518 mm³, respectively. Immunohistochemistry analysis showed that tumors in the zq-1-68 treatment group had significantly less expression of proliferation marker Ki67 and 7-fold higher induction of apoptosis relative to the tumors in the vehicle control group.

Conclusion: We identify zq-1-68 as a potent anticancer agent that inhibits tumor growth and metastasis, induces apoptosis, and has on-target anti-HDAC activity in prostate cancer both in vitro and in vivo. These preclinical findings support zq-1-68’s potential as a therapeutic candidate for metastatic castration-resistant prostate cancer.

References: 1. McManus HD, Dorff T, Morgans AK, Sartor O, Shore N, Armstrong AJ. Navigating therapeutic sequencing in the metastatic castration-resistant prostate cancer patient journey. Prostate Cancer Prostatic Dis. Published online October 17, 2024. doi:10.1038/s41391-024-00906-z
2. McLoughlin EC, O’Boyle NM. Colchicine-Binding Site Inhibitors from Chemistry to Clinic: A Review. Pharmaceuticals (Basel). 2020;13(1):8. doi:10.3390/ph13010008
3. Liang T, Wang F, Elhassan RM, et al. Targeting histone deacetylases for cancer therapy: Trends and challenges. Acta Pharm Sin B. 2023;13(6):2425-2463. doi:10.1016/j.apsb.2023.02.007

Acknowledgements: This work is supported by the National Institutes of Health (NIH) National Cancer Institute (NCI) grant R01CA148706 and the University of Tennessee Health Science Center College of Pharmacy Drug Discovery Center. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of NIH. All animal experiments were performed in accordance with the protocol approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Tennessee Health Science Center (protocol 23-0454, Li).

Western blot of the EBI competitive binding assay shows that zq-1-68 blocks formation of the ß-tubulin:EBI adduct, indicating that zq-1-68 binds tubulin at the colchicine binding site.

Zq-1-68 selectively inhibits HDAC6 and HDAC8, with minimal inhibition of the other isoforms

Zq-1-68 effectively inhibits growth of primary prostate cancer tumors in vivo. Tumor growth in the zq-1-68 treatment group was significantly inhibited compared to the CH-II-77 (tubulin inhibitor) and Tubastatin A (HDAC6 inhibitor) treatment groups