Discovery of a benzimidazole-based dual FLT3/TrKA inhibitor targeting acute myeloid leukemia

Abstract

FMS-like tyrosine kinase 3 (FLT3) enzyme overexpression and mutations are the most common molecular abnormalities associated with acute myeloid leukemia (AML). In addition, recent studies investigated the role of tropomyosin receptor kinase A (TrKA) enzyme fusions in promoting AML growth and survival. Based on these premises, targeting both kinases using dual inhibitors would constitute a promising therapeutic approach to target resistant AML. Guided by ligand-based design and structure simplification of the FLT3 inhibitor, quizartinib, we developed a benzimidazole-based small molecule, 4ACP, that exhibited nanomolar activity against wild-type FLT3, FLT3-Internal tandem duplications (FLT3-ITD), and FLT3-D835Y (FLT3-TKD) mutation (IC₅₀ = 43.8, 97.2, and 92.5 nM respectively). Additionally, 4ACP demonstrated potent activity against colon cancer KM12 cell line (IC₅₀ = 358 nM) and subsequent mechanistic deconvolution identified TrKA enzyme as a second plausible target (IC₅₀ = 23.6 nM) for our compound. 4ACP manifested preferential antiproliferative activity against FLT3-ITD positive AML cell lines (MV4-11 IC₅₀ = 38.8 ± 10.7 nM and MOLM-13 IC₅₀ = 54.9 ± 4.1 nM), while lacking activity against FLT3-ITD negative AML cell lines. Western blot analysis confirmed 4ACP ability to downregulate ERK1/2 and mTOR signaling downstream of FLT3-ITD in AML cells. Furthermore, 4ACP prompted apoptotic and necrotic cell death and G0/G1 cell cycle arrest as indicated by cell cycle analysis. 4ACP did not show cytotoxic effects on normal BNL and H9c2 cells and demonstrated decreased activity against c-Kit enzyme, hence, indicating lower probability of synthetic lethal toxicity and a relatively safer profile. In light of these data, 4ACP represents a novel FLT3/TrKA dual kinase inhibitor for targeted therapy of AML.

Keywords: Acute myeloid leukemia; Benzimidazole; Dual kinase inhibitor; FLT3; Scaffold hopping; TrKA.