Osteoporosis, delineated as a disorder characterized by heightened fracture susceptibility, manifests through diminished bone mass and microarchitectural degradation of both trabecular and cortical skeletal components[1], [2], [3], [4]. Sclerostin is a secreted glycoprotein that expresses predominantly in osteocytes and contains three distinct domains: loop1, loop2, and loop3[5], [6], [7], The identification of sclerostin as a pivotal inhibitor of bone formation was made by groups evaluating patients with 2 rare autosomal recessive syndromes associated with high bone mass[8], [9], and sclerostin was found inhibiting bone formation by antagonizing the Wnt/β-catenin signaling pathway[10], [11], [12], [13], [14], [15]. Consequently, sclerostin is a novel therapeutic target for bone anabolic treatment[16], [17], [18], [19].

The first sclerostin inhibitor approved by FDA is the monoclonal antibody Romosozumab[20], [21], which predominantly targeting loop2 region of sclerostin[5] and exhibiting substantial efficacy in treating osteoporosis particularly for postmenopausal women[22], [23], [24], [25]. Nonetheless, the therapeutic application of Romosozumab was found causing an escalated cardiovascular risk as evidenced in the active-controlled fracture study in postmenopausal women with osteoporosis at high risk[26], [27], [28], [29]. When compared with the treatment with alendronate, Romosozumab administration correlates with about 30% elevation in cardiovascular disease risk[18], [30], [31], [32], [33]. Thus, it is desirable to develop the new generation of sclerostin inhibitors without increasing cardiovascular risks.

Recently, Yu et al. in our group found sclerostin loop3 contributes to the inhibition effect of sclerostin on bone formation, and the protective effect of sclerostin on cardiovascular system is conversely independent of loop3 domain[34]. Thus, the development of inhibitors selectively targeting sclerostin loop3 could promote bone formation without increasing cardiovascular risk[35]. Subsequently, we developed the sclerostin loop3-selective aptamer Apc001[36], [37], [38], [39] that promotes osteogenic potential and bone formation without increasing cardiovascular risk[40]. Nonetheless, no small molecular inhibitors selectively targeting sclerostin loop3 have been reported.

In this work, a structural based virtual screening was conducted to identify potential small molecular inhibitors selectively targeting sclerostin loop3. About 20 compounds were screened out from the ZINC[41], [42] and Enamine[43] databases for further in-vitro experimental validation. Finally, 8 compounds were found binding to sclerostin, and the herbal ingredient ZINC4228235 (Tetrahydrofolic acid, THFA) exhibited a selective binding (Kd = 42.43 nM) to loop3 region of sclerostin. However, compound THFA is a natural product with poor drug-like property and synthesizability. Consequently, we designed a new compound (4-(3-acetamidoprop-1-yn-1-yl)benzoyl)glycine (AACA) with simplified structure and high synthetic feasibility by employing molecular modeling-guided modification based on the structural framework of THFA. The in-vitro experimental results demonstrated that compound AACA was a novel small molecular inhibitor targeting sclerostin loop3 and exhibited improved binding affinity (Kd = 15.4 nM) compared with THFA. In addition, compound AACA could attenuate the suppressive effect of transfected sclerostin on Wnt signaling and bone formation marker expression levels in-vitro, suggesting the potential of AACA as a candidate for development of inhibitors selectively targeting sclerostin loop3 to promote bone formation without increasing cardiovascular risk in osteoporosis imperfecta treatment.