Biochemical, Cellular Target Engagement, and Functional Assay Data for ALK2 Inhibitors

Diffuse Intrinsic Pontine Glioma (DIPG) is a rare and aggressive pediatric cancer located in the pons region of the brainstem, classified as a broader class of H3 K27M mutant Diffuse Midline Gliomas (DMG). Traditional drug development models struggle to address rare diseases like DIPG due to small patient populations and high risks. M4K Pharma focuses on developing an ALK2 enzyme inhibitor, the first therapeutic designed specifically for DIPG. We are leveraging highly potent, selective, and drug-like molecules targeting this protein to create a promising therapeutic option for children affected by this devastating disease. The following datasets evaluate the potency, selectivity, and cellular target engagement of more than 500 ALK2 inhibitor compounds using a combination of biochemical kinase assays and NanoBRET-based cellular target engagement assays. Biochemical radiometric kinase assays were used to measure ATP-dependent substrate phosphorylation and generate dose-response curves and IC₅₀ values, enabling quantitative comparisons of inhibitor activity across multiple ALK2 variants and related ALK receptor kinases. Compounds were assessed against wild-type (WT) ALK2, clinically relevant ALK2 (ACVR1) mutations (G328V, R206H, and R258G) and for selectivity relative to related TGF-β family receptors, including ALK1, ALK3, ALK4, ALK5 and ALK6. Cellular activity target engagement of ALK2 in HEK-293 cells was determined using a NanoBRET assay, providing insight into intracellular potency. In addition, a dual luciferase reporter (DLA) assay was used to evaluate functional activity of compounds against wild type ALK2- and ALK5- mediated signaling in cells. Late-stage development candidates M4K2281 and M4K2308 were also evaluated using NanoBRET assays in HEK-293 cells expressing clinically relevant ALK2 (ACVR1) mutations (G328V, R206H, and R356D), as well as the constitutively active Q207D mutant. Both compounds demonstrated low single-digit nanomolar potency across all ACVR1 mutant cell lines tested. Procedures and protocols for the biochemical ALK kinase, NanoBRET, and dual-luciferase reporter (DLA) assays have been described previously in J. Med. Chem. 2020, 63, 10061–10085 (https://dx.doi.org/10.1021/acs.jmedchem.0c01199). Together, these datasets provide an integrated assessment of biochemical potency, mutant target coverage, kinase selectivity, and cellular target engagement, supporting the lead optimization and development of selective ALK2-targeted therapeutics for diffuse intrinsic pontine glioma (DIPG) and other ALK2-driven diseases. Table of key terms and definitions of the project. Keyword Definition ALK2 / ACVR1 A bone morphogenetic protein (BMP) type I receptor kinase that regulates cellular growth and differentiation. Activating mutations in ALK2 are implicated in diseases such as Fibrodysplasia Ossificans Progressiva (FOP) and Diffuse Intrinsic Pontine Glioma (DIPG). NanoBRET Assay A live-cell bioluminescence resonance energy transfer assay used to quantify intracellular target engagement of small-molecule inhibitors with ALK2. DLA Assay A biochemical assay used to evaluate compound activity against ALK2 and related kinase targets, providing measurements of inhibitor potency and selectivity. Radiometric Kinase Assay An in vitro assay that measures ATP-dependent substrate phosphorylation to determine kinase activity and inhibitor potency. IC₅₀ The concentration of a compound required to inhibit 50% of kinase activity or target engagement, commonly used to compare inhibitor potency. R206H / G328V / G356D Disease-associated ALK2 mutations evaluated to compare inhibitor potency and target engagement across clinically relevant receptor variants.