Fig. 1: Development and characterisation of the GWL kinase inhibitor C-604.

a Chemical structure of the lead exemplar compound 21 and the general synthetic route towards the preparation of pyrrolopyrazine GWL inhibitors. (a) for 4a, (i) NaH, SEM-Cl, DMF, 0 °C, 4 h; (ii) Arnold’s reagent, CHCl₃, 60 °C, 20 h; (iii) NaClO₂, KH₂PO₄, NH₂SO₃H, 1,4-dioxane/water, 0 °C to rt, 2 h, 35% (three steps); for 4b, (i) hexamine, TFA, MW, 80 °C, 20 min; (ii) NaH, SEM-Cl, DMF, 0 °C to rt, 16 h; (iii) NaClO₂, KH₂PO₄, NH₂SO₃H, 1,4-dioxane/water, 0 °C to rt, 1.5 h, 14% (three steps); (b) 2 M methylamine in THF, T3P, pyridine, EtOAc, 60 °C, 2-4 h, 89-97%; (c) 2-(pyrrolidin-1-yl)ethanol, CuI, L-proline, K₂CO₃, DMSO, 100 °C, 16 h, 48%; (d) TFA, DCM, rt, 16 h then ethylenediamine, DCM, rt, 3-6 h, 29-92%; (e) 1-methylpiperazine, CuI, L-proline, K₂CO₃, DMSO, 100 °C, 16 h, 77%; (f) boronate ester, Pd(dppf)Cl₂ · DCM, 1,4-dioxane/water, MW, 120 °C, 30 min, 43-81%; (g) boronate ester, Pd(dppf)Cl₂ · DCM, 1,4-dioxane/water, MW, 120 °C, 30 min, 56%; (h) amine, K₂CO₃, DMF, 95 °C, 1 h, 32-67%. b Chemical structure of the lead compound 21 (C-604). c Dose-dependent effect of compound 21 (C-604) on the in vitro GWL activity measured by an HTRF assay utilising purified full-length GWL (K72M) and ENSA. Per cent (%) inhibition was determined from fluorescence intensity values normalised to the signal measured in the control enzyme-free reaction. Red lines and shaded regions represent means and standard deviations of independently fitted four-parameter sigmoidal models. Dots represent individual measurements. Mean IC₅₀ values ± standard deviations are indicated. Numbers of independent experiments (n) are indicated. d Model engagement of compound 21 (C-604) with the GWL active site. Left, secondary structure molecular cartoon. Amino acid residues predicted to interact with compound 21 are shown in stick representation, with carbon atoms coloured magenta or green if they make hydrogen bonds or hydrophobic contact with the compound, respectively. Right, 2D interaction map. Refer to the associated key for further details. e Representative differential interference contrast (DIC) images of DMSO-treated interphase cells and STLC-treated prometaphase-arrested cells. The scale bar represents 50 µm. f A schematic outline of the experimental procedure to establish cellular EC₅₀ values of the C-604 inhibitor. g Representative Western blots showing the effect of C-604 on ENSA(S67) and ARPP19(S62) phosphorylation. ENSA and ENSA-P refer to total and phosphorylated ENSA/ARPP19 levels, respectively. Phosphorylation of PP1 at T320 (PP1-P) represents a mitotic marker. The experiment was repeated n = 3 times. h Normalised ratios of ENSA-P and ENSA levels in prometaphase-arrested cells treated with C-604. Data were normalised to the maximum value in each given set. Dots, triangles and squares indicate the results of n = 3 independent experiments. Red lines and shaded regions represent means and standard deviations of n = 3 independently fitted four-parameter sigmoidal models. Mean C-604 EC₅₀ values ± standard deviations are shown for each tested cell line. i Colony formation capacity of RPE-1 and HeLa cells transfected with siRNAs targeting GWL or B55α. Cells were treated with C-604 for 72 h and subsequently grown in a drug-free medium for 7-10 days. Representative images from one of n = 3 experiments are shown. j Quantification of data presented in (e). In each experiment, cell-line-specific colony counts were normalised to the highest recorded value. The results of n = 3 independent experiments are shown as individual rows in the heatmap.