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  • To investigate the mechanism of

    2021-06-17

    To investigate the mechanism of action of 2,4,5-trisubstituted azoles, we profiled selected 2,4,5-trisubstituted azoles against a panel of 97 kinases to identify potential targets that could be involved in cardiogenesis. We narrowed down the identified targets to the casein kinase 1 (CK1) family due to its involvement in the Wnt/β-catenin signaling pathway, which is known to be important for cardiogenesis. Cardiac differentiation is regulated by Wnt induced mesoderm formation [39] and correlates with post mesoderm Wnt inhibition [5]. Wnt signaling is regulated by the CK1 family [40], and we were particularly interested in the CK1δ/ε isoforms because the inhibition of CK1δ/ε with 2,4,5-trisubstituted azoles such as SB203580, SB202190 [13], [41] and TAK-715 [42] has been shown to inhibit Wnt signaling. Furthermore, SB203580 induced cardiac differentiation has been shown to be most efficient when applied after mesoderm development [29], consistent with a mechanism based on Wnt inhibition. Thus, we hypothesized that CK1ε/δ inhibition by 2,4,5-trisubstituted azoles induce post mesoderm cardiac differentiation via Wnt/β-catenin signaling pathway (Fig. 4). In addition, 2,4,5-trisubstituted azoles show a biphasic biologic activity on cardiac differentiation. At low concentrations they are able to induce cardiac differentiation but restrict cardiac development at high concentration when applied before mesoderm development. The β-catenin/Wnt signaling can induce Endo-Mesoderm development [43]. However, the limited kinase profile data lacks appropriate data to support Wnt induced mesoderm differentiation. Therefore, we cannot rule out that other kinases and other enzyme Cyclic Pifithrin-α hydrobromide such as Phosphorylases and Phosphatases might influence initial mesoderm development. Further research would be necessary to uncover the biphasic behavior of certain 2,4,5-trisubstituted azoles. The full details of the Wnt/β-catenin signaling pathway have been elucidated and reviewed extensively elsewhere [24], [44]. In our experiments we were able to show that the affinities of 2,4,5-trisubstituted azoles to CK1δ/ε correlated with their post mesoderm cardiac differentiation profile. 2,4,5-trisubstituted azoles also caused de-phosphorylation of Dishevelled2, but not LRP6 within the Wnt/β-catenin signaling pathway, indicating the involvement of CK1δ/ε [45]. Several Wnt pathway-associated phosphorylated β-catenin proteins were also strongly decreased in the signaling downstream, indicating β-catenin proteolysis. Further we showed that TCF/LEF and Wnt transcription targets were strongly decreased with 2,4,5-trisubstituted azoles treatment. Although we acknowledge that the role of CK1 family proteins in Wnt/β-catenin signaling is complex [24] and not fully elucidated in differentiating stem cells, our evidence strongly suggests that 2,4,5-trisubstituted azoles may act as negative regulators of the Wnt/β-catenin signaling via CK1δ/ε inhibition in the Wnt associated membrane protein complex (Fig. 4). Cardiogenesis via CK1ε/δ inhibition was not limited to 2,4,5-trisubstituted azole alone. Structurally unrelated molecules such as CK1 VIII and DRF053 enhanced cardiogenesis. However, other tested CK1 inhibitors were either cytotoxic or less cardiogenic, highlighting the sensitivity of cardiogenesis to changes in compound structure. We speculate that the lack of cardiogenic activity for LH846 could be due to its distinct off-target profile as a result of its widely differing molecular geometry. In support of this idea, the kinase profiles of structurally unrelated p38α MAPK inhibitors SB203580, BIRB-796, and VX745 have shown that they have very different off-target profiles [21]. However the kinase profile of LH846 [46] is limited, making it difficult for us to draw a firm conclusion in this case. The CK1 inhibitor PF670462 has a similar CK1δ/ε affinity as TA-01 (Supplemental Tables S1 & S2) but showed a reduced cardiogenic potential. Although there are structural differences between PF670462 and the 2,4,5-trisubstituted azoles in this study, their kinome profiles are similar [47]. However, TAs such as SB203580, SB202190, and SB431542 are known to inhibit TGF-β receptors [48]. TA-01 and TA-02 especially inhibit TGFBR-2 (Supplemental Table S3) while PF670462 shows a very low affinity towards TGFBR-2 [49] when compared to high cardiogenic TAs (e.g. SB203580, TA-01…). Inhibition of TGFBR-2 had been reported to enhance the differentiation of uncommitted mesoderm to cardiomyocytes [50]. This could point to an additional mechanism of cardiogenesis by 2,4,5-trisubstituted azoles. Our results showed that SB431542, a commercial inhibitor of TGF-β signaling weakly induced cardiomyogenesis. However, its cardiogenic potential correlated with its CK1δ/ε IC50 values. Hence, TGF-β is less likely the driving mechanism of cardiac differentiation, but an additive effect cannot be ruled out. It is possible that cross reactions with multiple kinases such as CK1δ/ε and TGF-β might induce cardiogenesis efficiently, which would explain the structural advantage of 2,4,5-trisubstituted azoles over other forms of trisubstituted azoles. Further research would be required to understand the relationship between CK1δ/ε, Wnt and TGF-β regulated cardiogenesis.