Background MCL-1 is an important anti-death BCL-2 family protein and plays a key role in blocking apoptosis in cancer cells. MCL-1 gene is located in one of the most frequently amplified locus in various hematologic malignancies and solid tumors, including prostate, lung, pancreatic, breast, ovarian and cervical cancers, as well as melanoma, B-cell chronic lymphocytic leukemia (B-CLL), acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL)); moreover, MCL-1 overexpression is implicated as a resistance factor for multiple therapies including widely prescribed microtubule-targeted agents for breast cancers. Therefore, MCL-1 is an attractive therapeutic target for the treatment of cancers. In this study, we discovered the lead preclinical compound APG-3526, from its chemical synthesis optimization to potent antiproliferative and antitumor activity using multiple in vitro and in vivo xenograft models, respectively.

Methods and Experiments APG-3526 was chemically synthesized by using one convergent multistep synthesis in gram scale. The binding affinity of APG-3526 was determined by a FP assay using a fluorescein labelled peptide (FAM-Bid) which binds to the MCL-1 protein leading to an increased anisotropy measured in milli-polarization (mP) values. The effect of APG-3526 on cell viability was determined using CCK-8 assay and its antitumor activities were tested in NCI-H929 and OPM-2 multiple myeloma (MM) xenograft SCID mice model (dosed i.v. at 50 mg/kg and p.o. at 25 mg/kg).

Results Our biochemical study has demonstrated that APG-3526 binds to MCL-1 with a nanomolar affinity (IC50 = 7 nM). It is highly potent in inhibition of cell growth in MCL-1-dependent multiple myeloma cell lines (NCI-H929 cells: IC50 = 14 nM; OPM-2 cells: IC50 = 62 nM). Administration of APG-3526 (p.o. at 25 mg/kg, QDx14D, or i.v. at 50 mg/kg, once) achieved complete tumor regression in both NCI-929 and OPM-2 multiple myeloma xenograft models. The pharmacodynamics (PD) study using tumor samples further revealed caspase 3 activation and PARP cleavage triggered by APG-3526. MCL-1 complex MSD assay confirmed that APG-3526 disrupted MCL-1:BIM binding thus freeing BIM to initiate the apoptotic cascade. In conclusion, we have developed a novel and highly potent MCL-1 inhibitor, APG-3526, which displays clinically relevant pharmacokinetic properties and elicits potent antiproliferative and antitumor activities via disrupting MCL-1 complex and triggering caspase activation, especially in MCL-1 driven MM models. These results support APG-3526 as a promising MCL-1inhibitor for further clinical development.

Citation Format: Jianyong Chen, Chengzhe Wu, Lingling Jiao, Leilei Zhao, Yunlong Zhou, Dongbo Li, Guozhi Tang, Shoulai Gu, Jing Deng, Guangfeng Wang, Douglas D. Fang, Shaomeng Wang, Dajun Yang, Yifan Zhai. Development of APG-3526 as a novel and highly efficacious MCL-1 inhibitor [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 73.