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  • SPR was used to measure biomolecular kinetic interactions


    SPR was used to measure biomolecular kinetic interactions between TERRA G-quadruplex and ligand in the present study. SPR is a widely used method, but cannot tell which specific type of interaction happened. Compounds with similar structures probably have similar mechanism of interaction, while compounds with different structures might have different mechanism of interaction. Therefore, in the present study, we used several other well established methods including FRET and MST to confirm biomolecular interactions. In the present study, we found that CK1-14, a Quindoline derivative had strong interaction with TERRA G-quadruplex, and could effectively induce acute cell growth arrest in U2OS cancer cells. Besides, TERRA G-quadruplex stabilized by CK1-14 could bind to TRF2 more tightly, resulting in disruption of the interaction between TRF2 and telomeric DNA in a dose-dependent manner both in vitro and in cells, with a graphical figure as shown in Fig. 8. The delocalization of TRF2 from telomere induced DNA damage response at telomere region. In another words, TERRA G-quadruplex bound with ligand CK1-14 is a novel allosteric modulator of TRF2, which could elicit a potent and rapid telomeric t-loop uncapping effect, and cause DNA damage response. CK1-14 could effectively inhibit the proliferation of osteosarcoma cancer cells, cause the cell G2/M phase arrest, and induce the cell apoptosis. Our present mechanistic studies indicated that CK1-14 induced DNA-damage response in U2OS Copanlisib through stabilization of TERRA G-quadruplex resulting in TRF2 delocalization from telomeric DNA. These findings illustrated that the binding of TRF2 with telomeric DNA could be controlled by a TERRA G-quadruplex binding ligand for regulating telomere protection in ALT cell line. CK1-14 had a selectivity index of 7.5 in USO2 versus mesangial primary cells, which could be further optimized through chemical structural modifications. Various side chains could be introduced into the pharmacophore skeleton through combinatorial chemical synthesis. CK1-14 could become a lead compound for further development with a new target for cancer treatment.
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    The authors have noted that there was a mix-up in the picture for CK1δwt in Fig. 2. The original picture is now presented in the corrected figure. Furthermore, to clearly indicate which CK1δ mutant has been used CK1δmt is now named CK1δmt(Rev).
    Introduction Casein kinase 1 (CK1) is a widely expressed Ser/Thr kinase in eukaryotic organisms [[1], [2], [3]], and seven mammalian CK1 isoforms (α, β, γ1–3, δ and ε) have been cloned and characterized to date [[4], [5], [6], [7]]. CK1 isoforms (CK1s) play important roles in various cellular processes (e.g., centrosome-associated processes, DNA damage-related signal transduction, circadian rhythm and apoptosis) [[8], [9], [10]]. Therefore, the physiological roles of CK1s and their enzymatic properties have recently received increased attention by researchers in various fields. The regulatory mechanism of CK1s is of great interest: the kinase activity is inactivated by autophosphorylation, which occurs especially in the C-terminal domain [5,[11], [12], [13], [14]]. In particular, the downregulation mechanism of CK1δ and ε has been well studied, and the autophosphorylated C-terminal domain is considered to act as a pseudo-substrate resulting in inhibition of the kinase. Recombinant CK1s prepared by the Escherichia coli protein expression system are highly autophosphorylated, and thus inactivated [11,13,14]. These preparations are unfavorable for enzymatic characterization of bona fide kinase activity because CK1s are actively maintained in the dephosphorylated/highly active form by cellular protein phosphatases (PPase) in vivo [15]. Therefore, to obtain unphosphorylated and highly active CK1s, bacterially expressed CK1s are needed to be treated with PPase in vitro. Conventionally, recombinant CK1s have been dephosphorylated by PPase such as λ protein phosphatase (λPPase) or the catalytic subunit of protein phosphatase 2A (PP2Ac), and then used for in vitro kinase assays after overriding the effects of PPase by adding a PPase inhibitor [11,14,16]. C-terminal deletion mutants [CK1s(ΔC)] that lack autophosphorylation and the inhibitory region are commercially available and used for various studies on CK1s in vitro and in vivo [[17], [18], [19]]. Particularly in recombinant CK1ε(ΔC) prepared from E. coli, however, it has been reported that in vitro phosphatase treatment led to further activation of the kinase, suggesting that CK1s(ΔC) have additional inhibitory autophosphorylation sites in the catalytic domain [14,16]. Thus, CK1s(ΔC) may not be appropriate for evaluation of the full activity of CK1s, especially in in vitro studies.