Janus kinases (JAKs) are receptor-associated multidomain tyrosine kinases that act downstream of many cytokines and interferons. JAK kinase activity is regulated by the adjacent pseudokinase domain ...via an unknown mechanism. Here, we report the 2.8-Å structure of the two-domain pseudokinase–kinase module from the JAK family member TYK2 in its autoinhibited form. We find that the pseudokinase and kinase interact near the kinase active site and that most reported mutations in cancer-associated JAK alleles cluster in or near this interface. Mutation of residues near the TYK2 interface that are analogous to those in cancer-associated JAK alleles, including the V617F and “exon 12” JAK2 mutations, results in increased kinase activity in vitro. These data indicate that JAK pseudokinases are autoinhibitory domains that hold the kinase domain inactive until receptor dimerization stimulates transition to an active state.
Bruton’s tyrosine kinase (Btk) is a nonreceptor cytoplasmic tyrosine kinase involved in B-cell and myeloid cell activation, downstream of B-cell and Fcγ receptors, respectively. Preclinical studies ...have indicated that inhibition of Btk activity might offer a potential therapy in autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus. Here we disclose the discovery and preclinical characterization of a potent, selective, and noncovalent Btk inhibitor currently in clinical development. GDC-0853 (29) suppresses B cell- and myeloid cell-mediated components of disease and demonstrates dose-dependent activity in an in vivo rat model of inflammatory arthritis. It demonstrates highly favorable safety, pharmacokinetic (PK), and pharmacodynamic (PD) profiles in preclinical and Phase 2 studies ongoing in patients with rheumatoid arthritis, lupus, and chronic spontaneous urticaria. On the basis of its potency, selectivity, long target residence time, and noncovalent mode of inhibition, 29 has the potential to be a best-in-class Btk inhibitor for a wide range of immunological indications.
The human epidermal growth factor receptor 2 (HER2) is specifically overexpressed in tumors of several cancers, including an aggressive form of breast cancer. It is therefore a target for both cancer ...diagnostics and therapy. The 58 amino acid residue Zher2 affibody molecule was previously engineered as a high-affinity binder of HER2. Here we determined the structure of Zher2 in solution and the crystal structure of Zher2 in complex with the HER2 extracellular domain. Zher2 binds to a conformational epitope on HER2 that is distant from those recognized by the therapeutic antibodies trastuzumab and pertuzumab. Its small size and lack of interference may provide Zher2 with advantages for diagnostic use or even for delivery of therapeutic agents to HER2-expressing tumors when trastuzumab or pertuzumab are already employed. Biophysical characterization shows that Zher2 is thermodynamically stable in the folded state yet undergoing conformational interconversion on a submillisecond time scale. The data suggest that it is the HER2-binding conformation that is formed transiently prior to binding. Still, binding is very strong with a dissociation constant K D = 22 pM, and perfect conformational homogeneity is therefore not necessarily required in engineered binding proteins. A comparison of the original Z domain scaffold to free and bound Zher2 structures reveals how high-affinity binding has evolved during selection and affinity maturation and suggests how a compromise between binding surface optimization and stability and dynamics of the unbound state has been reached.
The crystal structure of the kinase domain from the epidermal growth factor receptor (EGFRK) including forty amino acids from
the carboxyl-terminal tail has been determined to 2.6-Ã resolution, both ...with and without an EGFRK-specific inhibitor currently
in Phase III clinical trials as an anti-cancer agent, erlotinib (OSI-774, CP-358,774, Tarceva TM ). The EGFR family members are distinguished from all other known receptor tyrosine kinases in possessing constitutive kinase
activity without a phosphorylation event within their kinase domains. Despite its lack of phosphorylation, we find that the
EGFRK activation loop adopts a conformation similar to that of the phosphorylated active form of the kinase domain from the
insulin receptor. Surprisingly, key residues of a putative dimerization motif lying between the EGFRK domain and carboxyl-terminal
substrate docking sites are found in close contact with the kinase domain. Significant intermolecular contacts involving the
carboxyl-terminal tail are discussed with respect to receptor oligomerization.
The cytokine interleukin 13 (IL-13) is a major effector molecule for T-helper type 2 inflammation and is pathogenic in allergic diseases such as asthma. The effects of IL-13 are mediated via a ...pathway that is initiated by binding to a heterodimeric receptor consisting of IL-13Rα1 and IL-4Rα. Antibodies raised against IL-13 can block its inflammatory effects by interfering with binding to either of the two receptor polypeptides. Lebrikizumab is a monoclonal anti-IL-13 antibody that has shown clinical benefit in a phase II study for the treatment of moderate-to-severe uncontrolled asthma. Here we report the molecular structure of IL-13 in complex with the Fab from lebrikizumab by X-ray crystallography at 1.9Å resolution. We show that lebrikizumab inhibits IL-13 signaling by binding to IL-13 with very high affinity and blocking IL-13 binding to IL-4Rα. In addition, we use site-directed mutations to identify the most important antibody contributors to binding. Our studies define key features of lebrikizumab binding and its mechanism of action that may contribute to its clinical effects.
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► The cytokine IL-13 is a major immune effector associated with asthma. ► Anti-IL-13 lebrikizumab has a dissociation constant less than 10pM. ► Lebrikizumab prevents binding of IL-4Rα, a receptor required for signaling.
Activating mutations in protein kinases drive many cancers. While how recurring point mutations affect kinase activity has been described, the effect of in-frame deletions is not well understood. We ...show that oncogenic deletions within the β3-αC loop of HER2 and BRAF are analogous to the recurrent EGFR exon 19 deletions. We identify pancreatic carcinomas with BRAF deletions mutually exclusive with KRAS mutations. Crystal structures of BRAF deletions reveal the truncated loop restrains αC in an active “in” conformation, imparting resistance to inhibitors like vemurafenib that bind the αC “out” conformation. Characterization of loop length explains the prevalence of five amino acid deletions in BRAF, EGFR, and HER2 and highlights the importance of this region for kinase activity and inhibitor efficacy.
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•BRAF, EGFR, and HER2 harbor oncogenic deletions in the β3-αC loop•β3-αC deletions constrain the C helix (αC) in an active conformation•Restraining αC confers resistance to αC “out” inhibitors vemurafenib and lapatinib•β3-αC length is functionally important and conserved across specific kinase families
Foster et al. show that oncogenic in-frame deletions of BRAF, HER2, and EGFR restrain the C helix (αC) in a fixed “in” active conformation, resulting in resistance to commonly used αC “out” inhibitors, and that five amino acid deletion provides optimal kinase activation.
Metabolic reprogramming in tumors represents a potential therapeutic target. Herein we used shRNA depletion and a novel lactate dehydrogenase (LDHA) inhibitor, GNE-140, to probe the role of LDHA in ...tumor growth in vitro and in vivo. In MIA PaCa-2 human pancreatic cells, LDHA inhibition rapidly affected global metabolism, although cell death only occurred after 2 d of continuous LDHA inhibition. Pancreatic cell lines that utilize oxidative phosphorylation (OXPHOS) rather than glycolysis were inherently resistant to GNE-140, but could be resensitized to GNE-140 with the OXPHOS inhibitor phenformin. Acquired resistance to GNE-140 was driven by activation of the AMPK-mTOR-S6K signaling pathway, which led to increased OXPHOS, and inhibitors targeting this pathway could prevent resistance. Thus, combining an LDHA inhibitor with compounds targeting the mitochondrial or AMPK-S6K signaling axis may not only broaden the clinical utility of LDHA inhibitors beyond glycolytically dependent tumors but also reduce the emergence of resistance to LDHA inhibition.
Proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates plasma LDL cholesterol (LDL-c) levels by promoting the degradation of liver LDL receptors (LDLRs). Antibodies that inhibit PCSK9 ...binding to the EGF(A) domain of the LDLR are effective in lowering LDL-c. However, the discovery of small-molecule therapeutics is hampered by difficulty in targeting the relatively flat EGF(A)-binding site on PCSK9. Here we demonstrate that it is possible to target this site, based on the finding that the PCSK9 P' helix displays conformational flexibility. As a consequence, the vacated N-terminal groove of PCSK9, which is adjacent to the EGF(A)-binding site, is in fact accessible to small peptides. In phage-display experiments, the EGF(A)-mimicking peptide Pep2-8 was used as an anchor peptide for the attachment of an extension peptide library directed toward the groove site. Guided by structural information, we further engineered the identified groove-binding peptides into antagonists, which encroach on the EGF(A)-binding site and inhibit LDLR binding.
Hepatitis C virus (HCV) infection is a major cause of liver disease and hepatocellular carcinoma. Glycan shielding has been proposed to be a mechanism by which HCV masks broadly neutralizing epitopes ...on its viral glycoproteins. However, the role of altered glycosylation in HCV resistance to broadly neutralizing antibodies is not fully understood. Here, we have generated potent HCV neutralizing antibodies hu5B3.v3 and MRCT10.v362 that, similar to the previously described AP33 and HCV1, bind to a highly conserved linear epitope on E2. We utilize a combination of in vitro resistance selections using the cell culture infectious HCV and structural analyses to identify mechanisms of HCV resistance to hu5B3.v3 and MRCT10.v362. Ultra deep sequencing from in vitro HCV resistance selection studies identified resistance mutations at asparagine N417 (N417S, N417T and N417G) as early as 5days post treatment. Comparison of the glycosylation status of soluble versions of the E2 glycoprotein containing the respective resistance mutations revealed a glycosylation shift from N417 to N415 in the N417S and N417T E2 proteins. The N417G E2 variant was glycosylated neither at residue 415 nor at residue 417 and remained sensitive to MRCT10.v362. Structural analyses of the E2 epitope bound to hu5B3.v3 Fab and MRCT10.v362 Fab using X-ray crystallography confirmed that residue N415 is buried within the antibody–peptide interface. Thus, in addition to previously described mutations at N415 that abrogate the β-hairpin structure of this E2 linear epitope, we identify a second escape mechanism, termed glycan shifting, that decreases the efficacy of broadly neutralizing HCV antibodies.
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► The role of glycosylation in HCV resistance to neutralizing antibodies is unclear. ► Two affinity-matured broadly neutralizing antibodies against HCV were generated. ► A mutation at a conserved glycosylation Asn (N417) confers antibody resistance. ► N417S and N417T mutations result in a glycosylation shift to N415. ► Fab–peptide structures confirm that N415 is buried in the antibody–peptide interface.
Human β-tryptase, a tetrameric trypsin-like serine protease, is an important mediator of allergic inflammatory responses in asthma. Antibodies generally inhibit proteases by blocking substrate access ...by binding to active sites or exosites or by allosteric modulation. The bivalency of IgG antibodies can increase potency via avidity, but has never been described as essential for activity. Here we report an inhibitory anti-tryptase IgG antibody with a bivalency-driven mechanism of action. Using biochemical and structural data, we determine that four Fabs simultaneously occupy four exosites on the β-tryptase tetramer, inducing allosteric changes at the small interface. In the presence of heparin, the monovalent Fab shows essentially no inhibition, whereas the bivalent IgG fully inhibits β-tryptase activity in a hinge-dependent manner. Our results suggest a model where the bivalent IgG acts akin to molecular pliers, pulling the tetramer apart into inactive β-tryptase monomers, and may provide an alternative strategy for antibody engineering.
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