Abstract
Clks have been shown by recent studies to be promising targets for cancer therapy, as they are considered key regulators in the process of pre-mRNA splicing, which in turn affects every aspect of tumor biology. In particular, Clk1 and -4 are overexpressed in several human tumors. Most of the potent Clk1 inhibitors reported in the literature are non-selective, mainly showing off-target activity towards Clk2, Dyrk1A and Dyrk1B. Herein, we present new 5-methoxybenzothiophene-2-carboxamide derivatives with unprecedented selectivity. In particular, the introduction of a 3,5-difluoro benzyl extension to the methylated amide led to the discovery of compound 10b (cell-free IC50 = 12.7 nM), which was four times more selective for Clk1 over Clk2 than the previously published flagship compound 1b. Moreover, 10b showed an improved growth inhibitory activity with T24 cells (GI50 = 0.43 µM). Furthermore, a new binding model in the ATP pocket of Clk1 was developed based on the structure-activity relationships derived from new rigidified analogues.
Highlights
Pre-mRNA splicing represents a critically important step for various processes such as development and differentiation
The bladder carcinoma cell line T24 was identified as a sensitive system for testing the cellular activity of Clk1/4 inhibitors, where 1b showed a GI50 value of 0.63 μM [2]. This was in accordance with the Oncomine data from several studies (Oncomine.org) which had revealed Clk1 mRNA overexpression in bladder cancer
Among the tested non-Clk kinases, only Dyrk1A and Dyrk1B were appreciably inhibited; the IC50 determination for Dyrk1A revealed that 10b was 28 times more potent for Clk1; this selectivity factor was higher than that reported for many highly potent Clk1 inhibitors in the literature: e.g., the quinazoline derivatives 34 and 35 described in [25], with selectivity factors of 2.8 and 2.7 respectively; the aminopyrimidine derivatives 15 and 17 reported in Ref. [26], with selectivity factors of 3.25 and 2, respectively; the azaindole derivative 10c reported in Ref. [27], with a selectivity factor of 3.2; TG003 reported in [28], which showed higher potency against Dyrk1A than against Clk1 (IC50s of 12 and 20 nM, respectively); and KH-CB19 reported in [29], with a selectivity factor of 2.8
Summary
Pre-mRNA splicing represents a critically important step for various processes such as development and differentiation. 1b demonstrated great ability in inhibiting the growth of various types of tumor cells through selective depletion of cancer-relevant proteins, an effect that is mediated through the modulation of pre-mRNA splicing, which in turn leads to the nonsense-mediated decay of the mRNAs of genes that are essential for cell growth and survival [2]. These aimed at the stabilization of the biologically active.
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