Structural Basis of Activin Receptor-Like Kinase 2 (R206H) Inhibition by Bis-heteroaryl Pyrazole-Based Inhibitors for the Treatment of Fibrodysplasia Ossificans Progressiva Identified by the Integration of Ligand-Based and Structure-Based Drug Design Approaches

[Image: see text] Fibrodysplasia ossificans progressiva (FOP) is a rare but severe genetic disorder in which acute inflammation elicits progressive heterotopic ossification in the muscles, tendons, and ligaments. Classic FOP is caused by the R206H mutation in ALK2/ACVR1. While several activin receptor-like kinase 2 (ALK2) inhibitors were found to be efficacious in animal models of FOP, most of the ALK2 (R206H) inhibitors lacked sufficient oral bioavailability for efficacy. Previously, the synthesis of a series of novel bis-heteroaryl pyrazole-based ALK2 (R206H) inhibitors that achieved both substantial potency and an improved ADMET profile was reported. In the present study, the detailed procedure of the in silico approach employed to identify the initial bis-heteroaryl pyrazole-based ALK2 (R206H) inhibitor RK-59638 and the analysis of the ALK2 (R206H) RK-59638 complex structure to guide the synthetic optimization of the chemical series, obtaining RK-71807 showing improved potency and metabolic stability, were described. According to the initial in silico screening, the screening efficiencies and chemical diversity of the hit compounds of both ligand-based and structure-based methods were evaluated. Then, X-ray structures of ALK2 (R206H) and the inhibitors were analyzed to assess the structure–activity relationships of the synthesized compounds. The 3D-RISM analysis indicated the existence of the additional hydrogen bond via water molecules restricting the attachment point in the pyrazole scaffold. The quantum mechanics calculation of the newly determined ALK2 (R206H) RK-71807 complex structure using a fragment molecular orbital method and pair interaction energy decomposition analysis was employed to evaluate the interaction energies between the inhibitor and each of the amino acid residues and decompose them to electrostatic, exchange-repulsion, and charge transfer energies. The pattern of decomposed interaction energies was then compared to that formed by RK-59638 and LDN-193189 to investigate the structural basis of ALK2 (R206H) inhibition.