A. and IRAK-4 (IC50 = 20 nm), with only minimal TAK1-inhibiting activity (IC50 = 0.5 m). Using HS-243 and takinib, we evaluated the consequences of cytokine/chemokine responses after selective inhibition of IRAK-1/4 or TAK1 in response to lipopolysaccharide challenge in human rheumatoid arthritis fibroblast-like synoviocytes. Our results indicate that HS-243 specifically inhibits intracellular IRAKs without TAK1 inhibition and that these kinases have distinct, nonredundant signaling roles. Enbrel) and JAK inhibitors (Zeljanz) have transformed the therapeutic options available to autoimmune-affected patients (4, 5). Nevertheless, despite advances in more molecularly targeted DMARDs, a significant portion of patients still fail ex229 (compound 991) to respond to current therapeutic options. ex229 (compound 991) Often this lack of response is attributed to individual genetic variations, tolerance, and immune sensitization in the case of biologically based antibody therapies, leading to an unmet need for ex229 (compound 991) novel DMARD development (6,C8). One target area of great interest is the downstream kinases involved in the inflammatory signaling. In particular, there has been recent interest in the serine/threonine kinase interleukin-1 receptor associated protein kinases IRAK-1 and IRAK-4, as well as transforming growth factor Cactivated kinase 1 (TAK1). IRAK-4 is an essential protein kinase in mediating pathogen recognition and local cytokine release (IL-1, IL-6, TNF) through Toll-like receptor (TLR) signaling in response to prokaryotic lipopolysaccharides (LPSs) (9, 10). Following inflammatory agonist binding (LPS, IL-1, TNF), MyD88 is recruited to the cytosolic receptor domain, where it triggers downstream phosphorylation of IRAK-1/4, which further stimulates downstream NF-B signaling and proinflammatory cytokine production (11). It has been shown that aberrant IRAK-4 signaling through hyperactive cytokine signaling is a key regulator of innate and adaptive immune cells in autoimmune diseases (12) (13, 14). Because of its role in inflammatory signaling, IRAK-4 has been the focus of numerous drug development platforms and recently advanced to clinical studies for autoimmune diseases (15,C19). Although previous groups have designed IRAK-4 inhibitors with modest potency, their selectivity within the human kinome has not been examined, begging the question of off-target effects and (20). In particular, off-target effects through TAK1 are likely, given the high sequence conservation between the binding pockets of IRAK-1/4 and TAK1; the amino acid sequence identity Rabbit polyclonal to NPAS2 within the nucleotide-binding pocket of all three protein kinases is 93%. Examination of the co-crystal structure of TAK1Ctakinib identifies critical binding sites of the outlines regions of the parent takinib scaffold subjected to modification (see Experimental procedures). Analogs were tested in parallel against purified recombinant IRAK-4 in an kinase assay and identified HS-238 (643 nm), HS-242 (149 nm), and HS-243 (48 nm) as more potent IRAK-4 inhibitors compared with the parent compound takinib (Fig. 2, and for ATP at 500 m (Fig. S1). Therefore, to ensure optimal kinase activity and meaningful inhibition constants for all our analogs, assays for this protein kinase were conducted well above IRAK-4 at 2 mm. Prior studies by others against IRAK-4 compounds, such as Sigma IRAK-1/4 were performed well below the true IRAK-4 for ATP, leading to an overestimation of the and potentially a lack of selectivity and potency at physiological [ATP] (18, 21, 22). Open in a separate window Figure 2. StructureCactivity studies of takinib analogs against IRAK-4. = 2). = 2). HS-243 displays exquisite selectivity toward IRAK-1 and -4 over all other human kinases We next sought to test the selectivity of leading IRAK-4 analogs against ex229 (compound 991) TAK1 to identify kinase-specific compounds (Fig. 3and profiling of lead compound and other analogs against IRAK-4 and TAK1. = 2). To understand the structural basis for selectivity, we performed unconstrained molecular docking of HS-243 in the ATP-binding pocket of IRAK-4 (PDB code 2NRU) and.