A member of the Janus kinase (JAK) family, Tyrosine Kinase 2 (TYK2), is crucial in mediating various cytokine-signaling pathways such as interleukin-23 (IL23), interleukin-12 (IL12) and type I Interferons (IFN) which contribute to autoimmune disorders (e.g., psoriasis, lupus, and inflammatory bowel disease). Thus, TYK2 represents an attractive target to develop small-molecule therapeutics for the treatment of cytokine-driven inflammatory diseases. Selective inhibition of TYK2 over other JAK isoforms is critical to achieve a favorable therapeutic index in the development of TYK2 inhibitors. However, designing small molecule inhibitors to target the adenosine triphosphate (ATP) binding site of TYK2 kinase has been challenging due to the substantial structural homology of the JAK family catalytic domains. Here, we employed an approach to target the JAK homology 2 (JH2) pseudokinase regulatory domain of the TYK2 protein. We developed a series of small-molecule TYK2 pseudokinase ligands, which suppress the TYK2 catalytic activity through allosteric regulation. The TYK2 pseudokinase-binding small molecules in this study simultaneously achieve high affinity-binding for the TYK2 JH2 domain while also affording significantly reduced affinity for the TYK2 JAK homology 1 (JH1) kinase domain. These TYK2 JH2 selective molecules, although possessing little effect on suppressing the catalytic activity of the isolated TYK2 JH1 catalytic domain in the kinase assays, can still significantly block the TYK2-mediated receptor-stimulated pathways by binding to the TYK2 JH2 domain and allosterically regulating the TYK2 JH1 kinase. These compounds are potent towards human T-cell lines and primary immune cells as well as in human whole-blood specimens. Moreover, TYK2 JH2-binding ligands exhibit remarkable selectivity of TYK2 over JAK isoforms not only biochemically but also in a panel of receptor-stimulated JAK1/JAK2/JAK3-driven cellular functional assays. In addition, the TYK2 JH2-targeting ligands also demonstrate high selectivity in a multi-kinase screening panel. The data in the current study underscores that the TYK2 JH2 pseudokinase is a promising therapeutic target for achieving a high degree of biological selectivity. Meanwhile, targeting the JH2 domain represents an appealing strategy for the development of clinically well-tolerated TYK2 inhibitors that would have superior efficacy and a favorable safety profile compared to the existing Janus kinase inhibitors against autoimmune diseases.

Treatment of hepatitis C patients with direct-acting antiviral drugs involves the combination of multiple small-molecule inhibitors of distinctive mechanisms of action. ACH-806 (or GS-9132) is a novel, small-molecule inhibitor specific for hepatitis C virus (HCV). It inhibits viral RNA replication in HCV replicon cells and was active in genotype 1 HCV-infected patients in a proof-ofconcept clinical trial (1). Here, we describe a potential mechanism of action (MoA) wherein ACH-806 alters viral replication complex (RC) composition and function. We found that ACH-806 did not affect HCV polyprotein translation and processing, the early events of the formation of HCV RC. Instead, ACH-806 triggered the formation of a homodimeric form of NS4A with a size of 14 kDa (p14) both in replicon cells and in Huh-7 cells where NS4A was expressed alone. p14 production was negatively regulated by NS3, and its appearance in turn was associated with reductions in NS3 and, especially, NS4A content in RCs due to their accelerated degradation. A previously described resistance substitution near the N terminus of NS3, where NS3 interacts with NS4A, attenuated the reduction of NS3 and NS4A conferred by ACH-806 treatment. Taken together, we show that the compositional changes in viral RCs are associated with the antiviral activity of ACH-806. Small molecules, including ACH-806, with this novel MoA hold promise for further development and provide unique tools for clarifying the functions of NS4A in HCV replication. C hronic hepatitis C virus (HCV) infection is a major cause of liver diseases worldwide. It is estimated that 170 million individuals are infected with HCV (1-4). A significant portion of these infected people will develop liver diseases, including hepatitis, cirrhosis, and hepatocellular carcinoma (5). Treatment with pegylated alpha interferon (IFN-␣) and ribavirin has a sustained virologic response or cure rate of ϳ45% in genotype 1 HCV-infected patients (6, 7), and the addition of boceprevir or telaprevir, HCV NS3 protease inhibitors newly approved by the U.S. Food and Drug Administration, increases the cure rate to ϳ70% (8). The new standard care of the triple combination, however, also leads to more toxic effects (9). Hence, development of new treatment regimens with higher efficacy, as well as better tolerability is urgently needed (10).HCV, a member of the Flaviviridae family, is an enveloped virus with a positive-stranded RNA genome of 9.6 kb. The viral genome encodes a large polyprotein that is cleaved co-and/or posttranslationally into at least 10 mature viral proteins: structural proteins, including C, E1, E2, and p7, and nonstructural (NS) proteins, including NS2, NS3, NS4A, NS4B, NS5A, and NS5B. The functions of these viral proteins in the HCV life cycle have been extensively studied and mostly clarified (11). For example, NS5B has an RNA-dependent RNA polymerase activity, NS3 possesses a serine protease activity in its N-terminal domain and a helicase activity in the C-terminal domain, and NS4A is a cofactor of NS3...

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