(M1230-06-32) Identification of Small Molecule APJ Receptor Antagonists for Treatment of Cancers

Purpose: The APJ receptor, a G-protein coupled receptor (GPCR), and its ligand, apelin, are over-expressed in different cancer types^{[1, 2]}. Apelin increases the expression of several factors involved in angiogenesis, cell proliferation, migration, and metastasis, promoting tumor progression^[3-5]. High expression of apelin and APJ receptor are associated with decreased overall survival in different types of cancer^{[6, 7]}. In ovarian cancer, high APJ expression was correlated with a significantly reduced median overall survival by 14.7 months^[8]. Inhibition of the APJ receptor led to decreased tumor growth in various in vitro and in vivo models^{[9, 10]}. Given the role of apelin and APJ receptor in cancer, targeting the apelin-APJ pathway presents an attractive therapeutic strategy. Despite the discovery of a few APJ antagonists^[11] used in vitro and in vivo, each has limitations such as low solubility, stability, and potency, which hinder their ability to fully test the potential of APJ inhibition and further development for clinical evaluation. ML221, the only identified small molecule APJ inhibitor to date, showed undetectable levels in liver microsomal stability test after 5 minutes. Therefore, our objective is to identify selective, stable, and potent small molecule APJ antagonists to better characterize the effects of pharmacological inhibition of APJ in various preclinical cancer models.

Methods: We conducted a medicinal chemistry campaign to synthesize a series of small molecules structurally related to ML221, aiming to improve metabolic stability without compromising selectivity. The β-arrestin recruitment assay was developed using PRESTO-Tango^[12] platform to facilitate the rapid screening of candidate compounds against APJ receptor. In-vitro stability was tested in both plasma and liver microsomes. The cell viability of active compounds was determined using ovarian cancer cell-line (OVCAR8) with APJ expression. The in vivo pharmacokinetics of the selected compounds were evaluated in mice, and appropriate dosing regimens were identified. For hit compounds with the highest stability and activity, their in vivo antitumor efficacy was studied in comparison to ML221 using an orthotopic cell-line based xenograft mouse model. Tumor burden was measured using bioluminescence signal using the IVIS system.

Results: From the compounds tested, nine showed varying levels of activity against the APJ receptor. Compounds 7 and 9 were found to have the highest inhibitory potency and efficacy. The active compounds demonstrated greater liver microsomal stability compared to ML221, with in-vitro half-lives ranging from 5 minutes to 15 minutes. The compounds showed stability in human plasma over 72 hours. Compounds 7 and 9 exhibited the longest in-vitro half-lives. Compound 7 was more potent and more effectively inhibited OVCAR8 cell growth compared to ML221. In the in vivo ovarian cancer model, compound 7 significantly decreased tumor progression compared to the control group. The difference for the ML221 with control was not significant, presumably due to ML221’s in vivo instability. During the study, one mouse in each of the control and ML221 groups died due to ascites formation, while no mouse in the compound 7 group died.

Conclusion: We successfully identified metabolically stable small molecule APJ inhibitors with comparable activity to ML221. In the in-vivo ovarian model, compound 7 showed promising activity by decreasing cancer progression and increasing the survival of mice compared to the control and ML221 groups. The improved compound 7 will facilitate the exploitation of the therapeutic potential of pharmacological APJ inhibition in cancer.

References: 1. Berta, J., et al., Apelin expression in human non-small cell lung cancer: role in angiogenesis and prognosis. J Thorac Oncol, 2010. 5(8): p. 1120-9.
2. Picault, F.X., et al., Tumour co-expression of apelin and its receptor is the basis of an autocrine loop involved in the growth of colon adenocarcinomas. Eur J Cancer, 2014. 50(3): p. 663-74.
3. Yang, L., et al., ERK1/2 mediates lung adenocarcinoma cell proliferation and autophagy induced by apelin-13. Acta Biochim Biophys Sin (Shanghai), 2014. 46(2): p. 100-11.
4. Ran, J., et al., Apelin enhances biological functions in lung cancer A549 cells by downregulating exosomal miR-15a-5p. Carcinogenesis, 2021. 42(2): p. 243-253.
5. Podgórska, M., K. Pietraszek-Gremplewicz, and D. Nowak, Apelin Effects Migration and Invasion Abilities of Colon Cancer Cells. Cells, 2018. 7(8).
6. Hao, Y.Z., et al., APJ Is Associated with Treatment Response in Gastric Cancer Patients Receiving Concurrent Chemoradiotherapy and Endostar Therapy. Cancer Biother Radiopharm, 2017. 32(4): p. 133-138.
7. Uribesalgo, I., et al., Apelin inhibition prevents resistance and metastasis associated with anti-angiogenic therapy. EMBO Mol Med, 2019. 11(8): p. e9266.
8. Neelakantan, D., et al., Multifunctional APJ Pathway Promotes Ovarian Cancer Progression and Metastasis. Mol Cancer Res, 2019. 17(6): p. 1378-1390.
9. Harford-Wright, E., et al., Pharmacological targeting of apelin impairs glioblastoma growth. Brain, 2017. 140(11): p. 2939-2954.
10. Chen, H., et al., APLN promotes hepatocellular carcinoma through activating PI3K/Akt pathway and is a druggable target. Theranostics, 2019. 9(18): p. 5246-5260.
11. Huang, Z., et al., Targeting drugs to APJ receptor: From signaling to pathophysiological effects. J Cell Physiol, 2018. 234(1): p. 61-74.
12. Kroeze, W.K., et al., PRESTO-Tango as an open-source resource for interrogation of the druggable human GPCRome. Nat Struct Mol Biol, 2015. 22(5): p. 362-9.

Acknowledgements: The chemical structure of the APJ inhibitors designed and synthesized in this project are not disclosed for this abstract as there is a plan to apply for patency. This work was funded by the following grant (R01CA258546)

β-arrestin (PRESTO-Tango assay) Recruitment Assay

In-vitro Stability Using Human Liver Microsomes

The effect of APJ inhibition using ML221 and compound 7 in orthotopic ovarian in-vivo mice model