Summary /

The homology model of Clk4 was developed by utilizing the X-ray structure of Clk1 as the template (86% sequence identity at the catalytic domain), while the Dyrk1B homology model was built based upon the highly homologous Dyrk1A (77% sequence identity) using MOE molecular modeling software (Figure 1A) (MOE).  Several of our lead compounds were then docked into the ATP binding domains of these Clk and Dyrk1 models to achieve an optimal binding pose using FRED (OpenEye Scientific Software suite)(Figure 1B) (FRED).  The resulting docking poses were considered in the context of the experimentally determined IC50 and Kd values. In agreement with our previous docking results, the quinazoline core adopted a common pose within the ATP binding pocket forming previously validated hydrogen bonds with the hinge region (Figure 1B highlights the docking of 46 with Clk4). As previously discussed, when an alkyl group was added to the 4-position amine (either a methyl or ethyl) activity generally improved. Our model rationalizes this result due to a small hydrophobic pocket (as indicated by a white line) in which the alkyl group is oriented which would likely increase specified van der Waals interactions and lock the inhibitor in a preferred conformation (Figure 1C). Interestingly, the SAR surrounding the amine side-chain suggests that several variations are well tolerated. This model suggests that the primary role of this moiety is space-filling rather than interacting with specific protein residues via H-bonding or electrostatic interactions. A primary hope was that these models might yield insight into defining more selective inhibitors of the Clk and Dyrk1 targets. One insight surrounds Clk4, which is nearly identical to Clk1 at the catalytic domain, but shows a key divergence in residue Asp248. Models docking 63 at Clk4 and Clk1 suggest that this molecule’s hydroxyl group is within an appropriate proximity to Asp248 (~3.2 Å) to form a hydrogen bond (Figure 1D). This potential interaction would help rationalize the potency for 63 versus Clk4 (136 nM) as compared to Clk1 (1522 nM). In the Dyrk1A crystal structure and the Dyrk1B homology model, the Asp residue at the same position shifted more than 6 Å away from the hydroxyl group in analogue 63 due to a backbone conformational change. This distance is too far for a viable hydrogen bond and offers a basis for the potency decrease versus Dyrk1A and Dyrk1B (>10,000 nM and 4420 nM, respectively). This interaction also highlights a potential route to furthering the SAR of this chemotype.

Figure 1. (A) Ribbon representation of the catalytic clefts in the Clk1 crystal structure (green; PDB 1Z57), Clk4 homology model (cyan), Dyrk1a crystal structure (orange, PDB 2VX3) and Dyrk1b homology model (purple). The ligand shown is the co-crystal ligand in Dyrk1A. (B) Docking model of 46 in the Clk4 catalytic cleft. The binding pocket is depicted by molecular surface and the hydrogen bonds are labeled as yellow dotted lines. (C) The close view of the small hydrophobic pocket (as indicated by a white line) in which the methyl group is sitting in the docking model of 46 within the Clk4 catalytic cleft. (D) Docking model of 63 in the Clk4 catalytic cleft superimposed with Clk1, Dyrk1a and Dyrk1b. The hydrogen bond from the hydroxyl group to Asp248 is labeled as a yellow dotted line. Clk4 kinase domain is a homology model derived from the X-ray structure of Clk1 (PDB code: 1Z57). This figure was prepared with the program VIDA (OpenEye Scientific Software).

Summary /

To assure that these analogues still possessed the high selectivity for the Clk and Dyrk1 class of kinases we selected four representative inhibitors 45 (ML197), 46 (ML195), 54 (ML196), and compound 63 to examine in the same kinome-wide assay (possessing 442 kinases at the time of the profile). The kinome scan records activity as a percentage of kinase bound to an immobilized ligand in the presence and absence of each compound. In accordance with our previous work, activities beyond a selected threshold were submitted for K_d determination. The resulting K_d values further validated the selectivity of 45 and 54 for the Clk and Dyrk classes of kinases (Figure 3). Compound 46, although slightly less selective, is highly active against the desired targets as well as undesired kinases, Mek5 (K_d = 47 nM), a potential prostate cancer target⁴¹, and the kinase encoded by PIK3C2G (PI3K family)(K_d = 40 nM), which is involved in the pathophysiology of diabetes.⁴² The results for 63 suggested that this agent is somewhat promiscuous across several kinases and not acceptable as a probe of Clk and Dyrk1 activity (and highlights the utility of these profiles).

Figure 2. Dendrogram representation of the human kinome demonstrating kinase selectivity of reported inhibitors over a panel of 442 kinases. Activity for 45: Clk1 = 50 nM, Clk2 = 380 nM, Clk4 = 43 nM, Dyrk1A = 82 nM, PIP5K2C protein = 280 nM. Activity for 46: Clk1 = 18 nM, Clk2 = 59 nM, Clk4 = 5 nM, Dyrk1A = 13 nM, Dyrk1B = 300 nM, Dyrk2 = 480 nM, Erk8 = 430 nM, Mek5 = 47 nM, PIK3C2B protein = 340 nM, PIK3C2G protein = 40 nM, PIK3CG protein = 370 nM, PIP5K2C protein = 360 nM, Ysk4 = 190 nM. Activity for 54: Clk1 = 72 nM, Clk2 = 320 nM, Clk4 = 30 nM, Dyrk1A = 27 nM, PIK3C2B protein = 410 nM, PIK4CB protein = 430 nM, PIP5K2C protein = 310 nM. Data from DiscoveRx (http://kinomescan.com).

Summary /

The kinetic aqueous solubility and Caco-2 permeability data for ML197 (compound 45), ML195 (compound 46), and ML196 (compound 54) were obtained via a commercial provider (Analiza, Inc.) and (Cyprotex PLC). We further examined the stability of these agents in aqueous buffered environment for 48 hours (results are based upon retention of UV/Vis signal and mass assessment within a standard HPLC gradient). The outcome of these studies is shown in Table 1. ML197 and ML195 were found to possess only modest aqueous solubility while ML196 was highly soluble. The Caco-2 profile suggested that each agent was capable of passive membrane permeability and had favorable efflux ratios. Finally, each agent was highly stable in an aqueous environment for up to 48 hours.

Table 1. Aqueous solubility and stability and Caco-2 permeability data for selected agents. (a) Solubility measurements done at Analiza, Inc (http://analiza.com). (b) Caco-2 permeability was measured at Cyprotex (www.cyprotex.com/adme). (c) Aqueous stability measured over 48 h in pH 7.4 DPBS buffer.