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Conformational flexibility and inhibitor binding to unphosphorylated interleukin-1 receptor-associated kinase 4 (IRAK4).

ABSTRACT: Interleukin-1 receptor-associated kinase 4 (IRAK4) is a key player in innate immune and inflammatory responses, performing a critical role in signal transduction downstream of Toll-like receptors and interleukin-1 (IL-1) receptors. Upon ligand binding and via its N-terminal death domain, IRAK4 is recruited to an oligomeric receptor that is proximal to the Myddosome signaling complex, inducing IRAK4 kinase domain dimerization, autophosphorylation, and activation. To date, all known IRAK4 structures are in the active conformation, precluding a good understanding of IRAK4's conformational dynamics. To address this issue, here we first solved three crystal structures of the IRAK4 kinase domain (at ?2.6 Å resolution), in its unphosphorylated, inactive state bound to either the ATP analog AMP-PNP or to one of the two small-molecule inhibitors JH-I-25 and JH-I-17. The structures disclosed that although the structure in complex with AMP-PNP is in an "?C-out" inactive conformation, those in complex with type I inhibitors assume an active "Asp-Phe-Gly (DFG)-in" and "?C-in" conformation. The ability of unphosphorylated IRAK4 to take on variable conformations prompted us to screen for small-molecule inhibitors that bind preferentially to unphosphorylated IRAK4, leading to the identification of ponatinib and HG-12-6. Solving the structures of unphosphorylated IRAK4 in complex with these two inhibitors, we found that they both bind as type II inhibitors with IRAK4 in a "DFG-out" conformation. Collectively, these structures reveal conformational flexibility of unphosphorylated IRAK4 and provide unexpected insights into the potential use of small molecules to modulate IRAK4 activity in cancer, autoimmunity, and inflammation.

SUBMITTER: Wang L

PROVIDER: S-EPMC6433055 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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Conformational flexibility and inhibitor binding to unphosphorylated interleukin-1 receptor-associated kinase 4 (IRAK4).

Wang Li L Ferrao Ryan R Li Qiubai Q Hatcher John M JM Choi Hwan Geun HG Buhrlage Sara J SJ Gray Nathanael S NS Wu Hao H

The Journal of biological chemistry 20190124 12

Interleukin-1 receptor-associated kinase 4 (IRAK4) is a key player in innate immune and inflammatory responses, performing a critical role in signal transduction downstream of Toll-like receptors and interleukin-1 (IL-1) receptors. Upon ligand binding and via its N-terminal death domain, IRAK4 is recruited to an oligomeric receptor that is proximal to the Myddosome signaling complex, inducing IRAK4 kinase domain dimerization, autophosphorylation, and activation. To date, all known IRAK4 structur ...[more]

PMID: 30679311

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Project description:BackgroundPF-06650833 is a potent, selective inhibitor of interleukin-1 receptor-associated kinase 4 (IRAK4). Two randomized, double-blind, sponsor-open phase 1 studies evaluated the safety, pharmacokinetics, and pharmacodynamics of single (SAD) and multiple ascending doses (MAD) of PF-06650833 immediate-release (IR) and modified-release (MR) oral formulations in healthy adult subjects.MethodsStudy 1 (NCT02224651) was a 96-day, placebo-substitution, SAD study of once-daily (QD) oral PF-06650833 IR 1 to 6000 mg and MR 30 to 300 mg in fasted and fed states. Study 2 (NCT02485769) was a 14-day, placebo-controlled, MAD study of PF-06650833 IR 25 to 750 mg twice daily, IR 1000 mg four times per day, IR 330 mg three times per day, and MR 300 mg QD.ResultsPF-06650833 was generally well tolerated, with no dose-limiting treatment-emergent adverse events (TEAEs) identified in either study. TEAEs were generally mild in severity, with headache, gastrointestinal disorders, and acne most commonly reported. No serious AEs or deaths were reported. A maximum tolerated dose was not established in either study. In the SAD study, food intake delayed absorption of IR 30 mg and increased total exposure by 33%. Delayed absorption was achieved with the MR formulation (Tmax of 1 h versus 8 h for IR 100 mg and MR 100 mg formulations, respectively). Food had no effect on total exposure for MR 30 mg, but reduced half-life 1.8-fold and increased Cmax by 62%. In the MAD study, accumulation ranged from 0.9-fold to 1.4-fold for AUCtau and 0.9-fold to 1.3-fold for Cmax. Less than 1% of the dose was recovered unchanged in urine for all dose groups, with renal clearance ranging from 14 to 23 mL/min for IR < 750 mg and MR 300 mg. There was a sustained decrease in serum high-sensitivity C-reactive protein for IR ≥ 250 mg and MR 300 mg. Based on the cholesterol/hydroxycholesterol ratio, no apparent CYP3A induction or inhibition was observed.ConclusionsPF-06650833, the first IRAK4 inhibitor to enter clinical development, has a favorable safety and pharmacokinetic profile and has shown evidence of pharmacological effect. The data support continued evaluation in human clinical trials for the treatment of rheumatic and autoimmune diseases.Trial registrationClinicaltrials.gov, NCT02224651, registered 25 August 2014; NCT02485769, registered 30 June 2015.

Project description:ObjectiveRecent researches have demonstrated that inflammation-related diseases are effectively regulated by posttranslational modifications (PTMs) including phosphorylation and acetylation. Our previous study found a new acetyltransferase inhibitor, oridonin, which had a protective effect on acute liver injury (ALI). In the present study, we further investigated its protective mechanism against D-galactosamine (D-Gal) combined with lipopolysaccharide- (LPS-) induced ALI in mice.MethodsIntraperitoneal injections of LPS (40 μg/mouse)/D-Gal (5 mg/mouse) were given to the mice, and the experimental group was pretreated with intraperitoneal injection of oridonin (0.2 mg/mouse). To elucidate the protective mechanism of oridonin, we collected liver specimens and used RNA-sequencing (RNA-Seq) analysis. We focused on the genes that were upregulated by LPS/D-Gal and downregulated after pretreatment with oridonin. The downregulated genes examined by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were further verified by real-time polymerase chain reaction (PCR) and western blot.ResultsGO analysis showed that genes that were downregulated after pretreatment with oridonin were extremely concentrated in immune response, chemotaxis, and inflammatory response. Real-time PCR confirmed that the expression of these genes was upregulated by LPS/D-Gal induction and reduced after treatment with oridonin, which was consistent with RNA-Seq results. KEGG pathway analysis showed a significantly enriched downregulated gene that was present in the Toll-like receptor (TLR) 4 signaling cascade. Our results manifested that phosphorylation levels of upstream signaling molecules in the TLR4 signaling cascade, including extracellular signal-regulated kinase (ERK), P38, and IκB, were significantly inhibited by oridonin. Furthermore, LPS/D-Gal stimulation triggered posttranslational modifications of related gene loci in the TLR4 signaling pathway, including phosphorylation of IL-1 receptor-associated kinase 4 (IRAK4 T345/S346) and acetylation of IRAK4 (K34). However, after treatment with oridonin, the modification pattern of IRAK4 expression stimulated by LPS/D-Gal was suggestively attenuated.ConclusionOur study revealed that the protective effects of oridonin on LPS/D-Gal-induced ALI mediated by inhibition of the PTMs of IRAK4, including phosphorylation of T345/S346 and acetylation of K34.

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Dataset Information

Conformational flexibility and inhibitor binding to unphosphorylated interleukin-1 receptor-associated kinase 4 (IRAK4).

Publications

Conformational flexibility and inhibitor binding to unphosphorylated interleukin-1 receptor-associated kinase 4 (IRAK4).

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