To understand the kinase selectivity profile

To understand the kinase selectivity profile of this series, GKT137831 , as a representative analog from this series, was profiled against 216 purified protein kinases representing the tyrosine and serine/threonine kinase families using an in-house Caliper EZ Reader mobility shift assay. Assays were conducted with the concentration of ATP at for each individual kinase with compound at a concentration of 1.0μM. Among 216 kinase screened, compound showed ∼100% inhibition of ACK1 and >80% inhibition of 12 other kinases. Of the 12 off-target kinases, none were considered major cell growth drivers such as receptor tyrosine kinases or cell-cycle regulating kinases. The selectivity was further confirmed by KINOM profiling against an even larger panel of 451 kinases, where compound showed a selectivity score of (10)=0.08, which represents a relatively high degree of selectivity. Compound possesses favorable drug-like properties as indicated by its physicochemical and ADMET properties, as summarized in . A convergent synthesis of compound is illustrated in . Phenol was converted to di-phenyl ether though a Cu(II) mediated Chan–Lam coupling, the subsequent palladium catalyzed borylation of afforded the desired boronate . The key imidazo[1,5-]pyrazine intermediate was treated with methyl Grignard reagent MeMgCl followed by an ammonolysis to give compound , which then underwent a Suzuki coupling with boronate to provide compound . In summary, a series of imidazo[1,5-]pyrazine derived ACK1 inhibitors was identified through a combination of structure-based drug design and empirical medicinal chemistry efforts. Efforts originated from virtual screening hit , where SAR exploration and DMPK optimizations eventually led to compound , a potent and selective ACK1 inhibitor with favorable in vitro physicochemical and ADMET properties. Furthermore, compound demonstrated good in vivo PK properties, which makes it a valuable compound to probe the in vivo biology of ACK1. Acknowledgments
Acknowledgments
Results and discussion Ack proteins are a new family of intracellular tyrosine kinases which, in addition to their catalytic properties, bind to the GTPase Cdc42 through a specific domain of 42 amino acids called Cbd (Cdc42 binding domain) (Mott et al., 1999; Cerione, 2004). These molecules also have two other protein–protein interaction sequences: an SH3 domain and a proline-rich region. The Ack family members that have been described to date are: human Ack1 (Manser et al., 1993), bovine Ack2 (Yang and Cerione, 1997), and mouse Tnk2 (Strausberg, 2002) in mammals; Drosophila Ack (Sem et al., 2002; Worby et al., 2002) and Caenorhabditis elegans Ark (Hopper et al., 2000) in invertebrates. The sequence differences between these members correspond mainly to the proline-rich region located at the C-terminal tail of the molecule, being about 200 amino acids shorter for bovine Ack2 than for Ack1 and Tnk2. Ack molecules are highly expressed in the brain (Manser et al., 1993; Yang and Cerione, 1997), although it is also present in other tissues. In a previous work, we studied the expression of the mouse homologue of Ack1 in different tissues and at the subcellular level (Ureña et al., 2005), and showed its presence both in pre-synaptic vesicles and dendritic spines. Here, we describe the expression pattern of mouse Ack1 in CNS throughout development. Mouse Ack1 mRNA was detected in early stages of development at embryonic day 10 (E10, Table 1) and embryonic day12 (E12, Table 1, Figs. 1H and 2E) and its expression continued and increased in all areas of CNS during embryonic and postnatal stages. Ack1 mRNA showed preferential expression in certain areas such as hippocampus, neocortex, cerebellum, and brainstem (Table 1). Overall, the maximum transcription levels were located in some regions of the telencephalon (Fig. 1A–C) and the signal was particularly high at postnatal stages. In the forebrain, Ack1 was already observed at E12, E14, and E16 (Figs. 1A, D, H, I, J, M, N).