The family of transforming growth factor-β (TGF-β) significantly impacts the regulation of many physiological processes. Mammalian cells expressed three isoforms of TGF-β isoforms (i.e., TGF-β1, TGF-β2 and TGF-β3) encoded by unique genes [1]. TGF-β1 is the most significant prototypic member of this cytokine's family, transducing signals via the type I and type II TGF-β receptors, two significantly conserved single transmembrane serine/threonine kinases. The type I receptor, i.e., activin receptor-like kinase 5 (ALK5), is phosphorylated and binds to Smad2/Smad3 proteins, which are more specifically phosphorylated. Phosphorylated Smad2/Smad3 proteins form a heteromeric complex with Smad4 [2]. Such a complex is translocated into the nucleus, assembles with particular co-modulators and DNA-binding cofactors, and then binds to TGF-β target genes' promoters in extracellular matrix (ECM) production, cell migration, anti-apoptosis, proliferation, differentiation, as well as immune suppression [[3], [4], [5]]. Development of inhibitors targeting the TGF-β/ALK5/SMAD pathway provides a promising clinical therapeutic strategy for diverse diseases, including myelofibrosis, intestinal fibrosis, cardiac fibrosis [6,7], progressive renal diseases [8] and various cancer types [[9], [10], [11], [12], [13], [14], [15], [16], [17], [18]].

Accordingly, the development of potent and selective inhibitors targeting ALK5 would provide potential treatment reagents for various diseases. Increasing pharmaceutical companies have focused on developing efficient and selective ALK5 inhibitors. Recently, four compounds were currently at different clinical study stages (Fig. 1), which were LY2157299 (phase III, myelodysplastic syndromes) [19], EW-7197 (phase II, solid tumor) [20], LY3200882 (phase I, Metastatic Colorectal Carcinoma) [21] and GFH018 (phase I, Non-small cell lung cancers) [22]. Besides, considerable medicinal research groups developed numbers of ALK5 inhibitors with different structures for various adaptational diseases [[23], [24], [25], [26], [27], [28], [29]]. Moreover, some co-crystal structures of ALK5 pockets and ligands were reported [25,[30], [31], [32], [33], [34], [35], [36], [37]], which would make design new ALK5 inhibitors more feasible.

In this study, we developed an alternative conformation-similarity based virtual screening (CSVS) combined with fragment-based drug designing (FBDD) to efficiently identify ALK5 inhibitors with novel chemical structures. And after SAR study and structural optimization, a potential drug candidate compound was discovered as a promising ALK5 inhibitor both in vitro and in vivo.