Fig 1: SiRNA reduction of Clk 1, 2 and 4 levels by specific siRNA reveals that Clk1 inhibition regulates levels of UCP1 and PGC1a. (A) siRNA to Clk1 reduced Clk1 levels by 50% while UCP1 mRNA levels increased fourfold, and PGC1a increased 3.5-fold. (B) siRNA to Clk2 reduced Clk2 levels by 40%, but only increased UCP1 mRNA by 20% while PGC1a was only increased 50%. (C) siRNA to Clk4 reduced Clk4 by 70%; it reduced UCP1 mRNA by 70%, and PGC1a was increased only 15%. A–C. TG003 (50 nM) treatment consistently increased UCP1 by 3–4 fold, PGC1a by twofold, and had minimal effects on lowering Clk levels. (**p < 0.01 for siClk1,2 and 4 or TG003 vs. scrambled siRNA which was equivalent to control in all cases as evaluated using ANOVA). (D) Oxygen Consumption Rates (OCR) for untreated and TG003-treated 3T3-L1 adipocytes. Basal OCR increased 25 pmol/min in TG003-treated cells vs. control. Proton leak was increased 2.3-fold with TG003 treatment. Spare receptor capacity increased 50 pmol/min while ATP production was reduced approximately 45% in TG003-treated cells. Data shown are mean ± SEM (**p < 0.01 for 5 separate experiments performed in duplicate and analysed by Friedman's paired t-test)
Fig 2: Binding of TG003 and PGC1a to Clk 1. (A) Steady state SPR of ~1800 RU Clk1 affixed to an Ni-NTA chip and titrated with TG003 in HBS-N with 1% DMSO plotted in GraphPad Prism using an one-site specific binding model with a KD of 25.24 with a 95% confidence interval of 12.93 to 48.53 nM. (B) Steady state SPR of ~400 RU Clk1, ~1000 RU of Clk4 and ~1000 RU Clk2 attached to a CM5 chip via NHS/EDC chemistry indicating binding of Clk1 and Clk4 to TG003 greater than binding of Clk2 to TG003. (C) Steady state SPR of Clk1, Clk2 and Clk4 as in 7B, indicating affinity of Clk1 to PGC1a of 240 nM +/- 28 nM, Clk2 to PGC1a of 980 nM +/- 89 nM, and Clk4 to PGC1a of 509 nM +/- 73 nM. (D) Ligand interaction diagram of TG003 5 bound to Clk1 (PDB ID: 1Z57) generating grid from ligand in crystal structure. (E) 100 ns simulation of Clk1 in the absence of ligand prepared using Schrodinger Protein Preparation Wizard on PDB ID: 1Z57 and NAMD run using CHARMM-GUI Solution Builder with RMSD of aligned backbone taken over the 1000 frames of the trajectory with each frame equal to 100 ps. (F) ClusPro predicted binding of PGC1a fragment from (PDB ID: 5UNJ) minimized with Protein Preparation Wizard and bound to receptor Clk1 from 7D aligned and one CLK deleted indicating proximal binding to nearby region of Clk1. (G) Heatmap to visualize inhibition of kinases by TG003
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