Enabling structure-based drug design of Tyk2 through co-crystallization with a stabilizing aminoindazole inhibitor

A comprehensive construct design effort included N- and C-terminal variations, kinase-inactive mutations, and multiple species orthologs. High-throughput cloning and expression methods were coupled with an abbreviated purification protocol to optimize protein solubility and stability. In total, 50 Tyk2 constructs were generated. Many displayed poor expression, inadequate solubility, or incomplete affinity tag processing. One kinase-inactive murine Tyk2 construct, complexed with an ATP-competitive 3-aminoindazole inhibitor, provided crystals that diffracted to 2.5–2.6 ？ resolution. This structure revealed initial “hot-spot” regions for SBDD, and provided a robust platform for ligand soaking experiments. Compared to previously reported human Tyk2 inhibitor crystal structures (Chrencik et al. (2010) J Mol Biol 400:413), our structures revealed a key difference in the glycine-rich loop conformation that is induced by the inhibitor. Ligand binding also conferred resistance to proteolytic degradation by thermolysin. As crystals could not be obtained with the unliganded enzyme, this enhanced stability is likely important for successful crystallization and inhibitor soaking methods.Practical criteria for construct performance and prioritization, the optimization of purification protocols to enhance protein yields and stability, and use of high-throughput construct exploration enable structure determination methods early in the drug discovery process. Additionally, specific ligands stabilize Tyk2 protein and may thereby enable crystallization.Janus kinases (Jaks) have broad roles in immune regulation via their action in cytokine signalling [1-3]. These non-receptor tyrosine kinases phosphorylate receptor chains, which in turn recruit and phosphorylate members of the Signal Transducer and Activator of Transcription (STAT) family [2,4]. The Jak family comprises Jak1, Jak2, Jak3 and Tyk2. These enzymes have very similar domain structures, containing a FERM domain, an SH2 domain