LAUSR

Introduction: Transforming growth factor β (TGF-β) plays a key role in promoting tumor progression mainly through facilitating angiogenesis, epithelial-mesenchymal transition (EMT) and also inducing immunosuppression in tumor microenvironment, allowing cancer cells to escape immune surveillance. Activin receptor-like kinases (ALKs) consisting of seven type I receptor serine/threonine kinases are responsible for TGF-β family signal transduction by phosphorylating Smad proteins, which can activate target gene expression. Therefore, ALKs are attractive drug targets for intervention in TGF- β signaling pathways as cancer immunotherapy. Materials and methods: We have produced seven human recombinant ALK proteins (ALK1-7). The in vitro kinase assays for all ALKs have been developed using TR-FRET system. To evaluated cellular potency, TGF-β-dependent Smad3 phosphorylation assays in A549 (human lung adenocarcinoma cell line) and NMuMG (murine normal mammary gland cell line) cells were established. A TGF-β-mediated Treg differentiation assay was performed as an ex-vivo cellular assay using naive CD4+ T cell isolated from mouse spleen. In vivo anti-tumor efficacies of selected compounds, alone or in combination with anti-PD-1 antibody were assessed in a CT26.WT murine colon carcinoma syngeneic model. Results: We have implemented a high-throughput screening (HTS) against human ALK5 over our in-house compound library. Hit compound 1 was identified from the HTS, and the subsequent Hit-to-Lead study led to a novel thiazole analog 2 as the starting point of lead optimization. Our lead optimization efforts led to advanced lead compound AL1, which exhibited strong inhibitory potency for ALK5 with an IC50 value of single digit nanomolar. Unfortunately AL1 showed very low oral bioavailability in dogs, we continued the optimization study and identified compound AL2 having improved ADME property. In cellular assays, AL2 strongly suppressed the TGF-β-dependent Smad3 phosphorylation in A549 cells. In addition, treatment with AL2 decreased in the percentage of mouse CD4+/CD25+/Foxp3+ Treg cells dose-dependently in the ex vivo TGF-β-mediated Treg differentiation assay. As a monotherapy and combined with an anti-PD-1 antibody, oral administration of AL2 demonstrated robust in vivo antitumor efficacy in the CT26.WT murine colon carcinoma syngeneic model. Conclusions: On the basis of its in vitro potencies, PK profiles, and in vivo efficacies, AL2 is currently being further evaluated in preclinical studies. Citation Format: Mai Arai, Mitsuharu Hanada, Hideki Moriyama, Hiroshi Ohmoto, Kazuhito Naka, Masaaki Sawa. Discovery of a novel activin receptor-like kinases (ALKs) inhibitor targeting TGF-β signaling pathways. [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 5336.