br Materials and methods br Results
Materials and methods
Discussion We initially found that ISL, a natural product with a linear scaffold (2-propenone), is able to inhibit CK2 (IC50 = 17.3 μM). Then we have investigated the inhibitory potential of a novel series of compounds with linear scaffolds by combining fragment-hybrid computational design with in vitro assays. Compound 8b, containing an amino-substituted 2-amino-1H-imidazole and a m-benzoic INCB3344 on the 2-propenone scaffold, exhibited improved inhibitory activity against CK2 (IC50 = 0.6 μM) and HepG2 cell proliferation (IC50 = 17.4 μM). Molecular docking results elucidated the binding modes of novel compounds with CK2, illustrating the importance of hydrogen bonds with the hinge region for binding and recognition. These results provide new insights for further optimization of CK2 inhibitors with linear scaffolds.
Conflicts of interest
Acknowledgments This study was supported by the Beijing Natural Science Foundation (No.7192015), the Great Wall scholars program of Beijing Municipal Education Commission (No.CIT&TCD20180308) and Beijing Municipal Commission of Education Research Projects (Grant No. KM201610005031).
Introduction Heat shock proteins (HSPs) represent a group of molecular chaperones involved in protein homeostasis maintenance and cell survival, whose expression is induced in response to a range of different stress stimuli. The primary role of this class of proteins is to counteract or inhibit denaturation or unfolding of cellular proteins. Furthermore, HSPs have the ability to antagonise death pathways including caspase-dependent apoptosis, and promoting survival under stressful conditions . Thus, while in normal cells the increased expression of HSPs in response to stress has a cytoprotective function, their dysregulated accumulation can contribute to the development of several pathologies including cancer . Accordingly, high levels of different HSPs have been detected in many types of cancer  where an higher demand of chaperone machinery arises during malignant transformation and tumour progression. For this reason, HSPs may represent therapeutic targets for cancer treatment, inspiring the development of novel chemotherapeutic agents . However, to date none of the HSP inhibitors has been approved for the treatment of patients with cancer. HSPs are categorized according to their molecular weight. Among them, the small-heat shock proteins family, characterized by a low molecular mass (~15–30 kDa), includes ten member in human. Small HSPs display a highly conserved α-crystallin domain with a non-conserved N- and C-terminus sequence. Unlike other HSPs, these proteins are ATP-independent chaperones (reviewed in [5,6]). One of the best-studied member of this family is HSP27 (also named HspB1 or Hsp28). Its ability to interact with multiple molecules is tightly regulated by phosphorylation that allows the supra-structural reorganization of HSP27 in small oligomers . HSP27 plays a role in various diseases, including neurodegenerative diseases, ischemia, and atherosclerosis. It is particularly important in the regulation of the development, progression and metastasis of cancer as well as cell apoptosis and drug resistance [7,8]. Noteworthy, HSP27 is overexpressed in a variety of cancers and it has been suggested as a biomarker in tumour diagnosis and prognosis [7,8]. Many different studies have showed that the lowering of HSP27 levels enhances anti-tumour drug's efficacy and reduces cancer cell resistance to the combination of surgery and chemotherapy, suggesting HSP27 as a potential target in cancer treatment [7,8]. However, the absence of an ATP binding domain that is instead present in other HSPs such as HSP90 and HSP70, hampers the development of small molecules as inhibitors of HSP27. Our results, obtained using CRISPR/Cas9 gene editing, siRNA transfections as well as specific kinase inhibitors, show that HSP27 protein expression is regulated by CK2, a ubiquitous serine/threonine protein kinase involved in many signalling pathways including transcription, proteins stability, differentiation, cell cycle and survival [, , ]. CK2 exists as a stable hetero-tetrameric complex composed of two catalytic (CK2α and CK2α′), and two regulatory subunits (CK2β) forming the complexes α2β2, α′2β2, or αα'β2. The two catalytic subunits are linked through the β subunits that are not essential for the activity but affect kinase substrate targeting . CK2 expression and activity are increased in many tumour cell lines and its overexpression is often associated with worse prognosis [, , ]. Accordingly, CK2 inhibition, alone or in combination with other targets, may represent an important tool to treat human cancer [16,17]. The recent availability of a mammalian cell line knocked out for both the catalytic or the regulatory CK2 subunits, namely α/α′ and β, discloses new perspectives to explain its real contribution to different signal transduction pathways [18,19]. Here we show that the availability of catalytically active CK2 holoenzyme is a necessary pre-requisite for the expression of a normal level of HSP27. Thus, CK2 can represent a valuable pharmacological target when a reduction in HSP27 level is desirable.