Filling a nick in NIK: Extending the half-life of a NIK inhibitor through structure-based drug design.

Inhibition of NF-κB inducing kinase (NIK) has been pursued as a promising therapeutic target for autoimmune disorders due to its highly regulated role in key steps of the NF-κB signaling pathway. Previously reported NIK inhibitors from our group were shown to be potent, selective, and efficacious, but had higher human dose projections than desirable for immunology indications. Herein we report the clearance-driven optimization of a NIK inhibitor guided by metabolite identification studies and structure-based drug design.

Structural basis for SHOC2 modulation of RAS signalling.

The RAS-RAF pathway is one of the most commonly dysregulated in human cancers. Despite decades of study, understanding of the molecular mechanisms underlying dimerization and activation of the kinase RAF remains limited. Recent structures of inactive RAF monomer and active RAF dimer bound to 14-3-3 have revealed the mechanisms by which 14-3-3 stabilizes both RAF conformations via specific phosphoserine residues.

Dimerization Induced by C-Terminal 14-3-3 Binding Is Sufficient for BRAF Kinase Activation.

The Ras-RAF-MEK-ERK signaling axis, commonly mutated in human cancers, is highly regulated to prevent aberrant signaling in healthy cells. One of the pathway modulators, 14-3-3, a constitutive dimer, induces RAF dimerization and activation by binding to a phosphorylated motif C-terminal to the RAF kinase domain. Recent work has suggested that a C-terminal "DTS" region in BRAF is necessary for this 14-3-3-mediated activation.

Negative regulation of RAF kinase activity by ATP is overcome by 14-3-3-induced dimerization.

The RAS-RAF-MEK-ERK signaling axis is frequently activated in human cancers. Physiological concentrations of ATP prevent formation of RAF kinase-domain (RAF) dimers that are critical for activity. Here we present a 2.

Dissecting the molecular basis of high viscosity of monospecific and bispecific IgG antibodies.

Some antibodies exhibit elevated viscosity at high concentrations, making them poorly suited for therapeutic applications requiring administration by injection such as subcutaneous or ocular delivery. Here we studied an anti-IL-13/IL-17 bispecific IgG antibody, which has anomalously high viscosity compared to its parent monospecific antibodies. The viscosity of the bispecific IgG in solution was decreased by only ~30% in the presence of NaCl, suggesting electrostatic interactions are insufficient to fully explain the drivers of viscosity.

Crystal Structure of Ripk4 Reveals Dimerization-Dependent Kinase Activity.

Receptor-interacting protein kinase 4 (RIPK4) is a highly conserved regulator of epidermal differentiation. Members of the RIPK family possess a common kinase domain as well as unique accessory domains that likely dictate subcellular localization and substrate preferences. Mutations in human RIPK4 manifest as Bartsocas-Papas syndrome (BPS), a genetic disorder characterized by severe craniofacial and limb abnormalities.

Targeting the MAPK Pathway in RAS Mutant Cancers.

Despite decades of extensive drug discovery efforts, there are currently no targeted therapies approved to treat KRAS mutant cancers. In this review, we highlight the challenges and opportunities in targeting KRAS mutant tumors through inhibition of mitogen-activated protein kinase (MAPK) signaling with conformation-specific kinase inhibitors. Through structural analysis and mechanistic studies with BRAF and mitogen-activated protein kinase (MEK) inhibitors, we describe how kinase-dependent and -independent functions of MAPK signaling components regulate KRAS-driven tumorigenesis and how these insights can be used to treat RAS mutant cancers with small molecule kinase inhibitors.

Unveiling the Structural and Dynamic Nature of the Ubiquitin Code.

In this issue of Structure, Castañeda et al. (2016b) use multi-disciplinary approaches including NMR techniques, small-angle neutron scattering, and docking to convincingly demonstrate that K27-linked diubiquitin is relatively rigid with unexpected similarity to the conformation of K48-linked diubiquitin bound to the UBA2 domain of hHR23a.

Activation Mechanism of Oncogenic Deletion Mutations in BRAF, EGFR, and HER2.

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.

Minor Structural Change to Tertiary Sulfonamide RORc Ligands Led to Opposite Mechanisms of Action.

A minor structural change to tertiary sulfonamide RORc ligands led to distinct mechanisms of action. Co-crystal structures of two compounds revealed mechanistically consistent protein conformational changes. Optimized phenylsulfonamides were identified as RORc agonists while benzylsulfonamides exhibited potent inverse agonist activity.

Potent and selective Bruton's tyrosine kinase inhibitors: discovery of GDC-0834.

SAR studies focused on improving the pharmacokinetic (PK) properties of the previously reported potent and selective Btk inhibitor CGI-1746 (1) resulted in the clinical candidate GDC-0834 (2), which retained the potency and selectivity of CGI-1746, but with much improved PK in preclinical animal models. Structure based design efforts drove this work as modifications to 1 were investigated at both the solvent exposed region as well as 'H3 binding pocket'. However, in vitro metabolic evaluation of 2 revealed a non CYP-mediated metabolic process that was more prevalent in human than preclinical species (mouse, rat, dog, cyno), leading to a high-level of uncertainly in predicting human pharmacokinetics.

Discovery of a Potent and Selective BCL-XL Inhibitor with in Vivo Activity.

A-1155463, a highly potent and selective BCL-XL inhibitor, was discovered through nuclear magnetic resonance (NMR) fragment screening and structure-based design. This compound is substantially more potent against BCL-XL-dependent cell lines relative to our recently reported inhibitor, WEHI-539, while possessing none of its inherent pharmaceutical liabilities. A-1155463 caused a mechanism-based and reversible thrombocytopenia in mice and inhibited H146 small cell lung cancer xenograft tumor growth in vivo following multiple doses.

Shining LIGHT on functional promiscuity in the TNF and TNFR superfamilies.

In this issue of Structure, Liu and colleagues report the structure of the TNF superfamily member LIGHT bound to decoy receptor 3 (DcR3). Both LIGHT and DcR3 interact with multiple binding partners. The authors identify a conserved interaction important for affinity as well as additional interactions that can be targeted to introduce selectivity.

Structure of the BRAF-MEK complex reveals a kinase activity independent role for BRAF in MAPK signaling.

Numerous oncogenic mutations occur within the BRAF kinase domain (BRAF(KD)). Here we show that stable BRAF-MEK1 complexes are enriched in BRAF(WT) and KRAS mutant (MT) cells but not in BRAF(MT) cells. The crystal structure of the BRAF(KD) in a complex with MEK1 reveals a face-to-face dimer sensitive to MEK1 phosphorylation but insensitive to BRAF dimerization.

Reduction in lipophilicity improved the solubility, plasma-protein binding, and permeability of tertiary sulfonamide RORc inverse agonists.

Using structure-based drug design principles, we identified opportunities to reduce the lipophilicity of our tertiary sulfonamide RORc inverse agonists. The new analogs possessed improved RORc cellular potencies with >77-fold selectivity for RORc over other nuclear receptors in our cell assay suite. The reduction in lipophilicity also led to an increased plasma-protein unbound fraction and improvements in cellular permeability and aqueous solubility.

Dimerization of LTβR by LTα1β2 is necessary and sufficient for signal transduction.

Homotrimeric TNF superfamily ligands signal by inducing trimers of their cognate receptors. As a biologically active heterotrimer, Lymphotoxin(LT)α1β2 is unique in the TNF superfamily. How the three unique potential receptor-binding interfaces in LTα1β2 trigger signaling via LTβ Receptor (LTβR) resulting in lymphoid organogenesis and propagation of inflammatory signals is poorly understood.

Structure-based design of substituted hexafluoroisopropanol-arylsulfonamides as modulators of RORc.

The structure-activity relationships of T0901317 analogs were explored as RORc inverse agonists using the principles of property- and structure-based drug design. An X-ray co-crystal structure of T0901317 and RORc was obtained and provided molecular insight into why T0901317 functioned as an inverse agonist of RORc; whereas, the same ligand functioned as an agonist of FXR, LXR, and PXR. The structural data was also used to design inhibitors with improved RORc biochemical and cellular activities.

Phosphorylation of Dishevelled by protein kinase RIPK4 regulates Wnt signaling.

Receptor-interacting protein kinase 4 (RIPK4) is required for epidermal differentiation and is mutated in Bartsocas-Papas syndrome. RIPK4 binds to protein kinase C, but its signaling mechanisms are largely unknown. Ectopic RIPK4, but not catalytically inactive or Bartsocas-Papas RIPK4 mutants, induced accumulation of cytosolic β-catenin and a transcriptional program similar to that caused by Wnt3a.

ABT-199, a potent and selective BCL-2 inhibitor, achieves antitumor activity while sparing platelets.

Proteins in the B cell CLL/lymphoma 2 (BCL-2) family are key regulators of the apoptotic process. This family comprises proapoptotic and prosurvival proteins, and shifting the balance toward the latter is an established mechanism whereby cancer cells evade apoptosis. The therapeutic potential of directly inhibiting prosurvival proteins was unveiled with the development of navitoclax, a selective inhibitor of both BCL-2 and BCL-2-like 1 (BCL-X(L)), which has shown clinical efficacy in some BCL-2-dependent hematological cancers.

The crystal structure of the catalytic domain of the NF-κB inducing kinase reveals a narrow but flexible active site.

The NF-κB inducing kinase (NIK) regulates the non-canonical NF-κB pathway downstream of important clinical targets including BAFF, RANKL, and LTβ. Despite numerous genetic studies associating dysregulation of this pathway with autoimmune diseases and hematological cancers, detailed molecular characterization of this central signaling node has been lacking. We undertook a systematic cloning and expression effort to generate soluble, well-behaved proteins encompassing the kinase domains of human and murine NIK.

Discovery of a potent small-molecule antagonist of inhibitor of apoptosis (IAP) proteins and clinical candidate for the treatment of cancer (GDC-0152).

A series of compounds were designed and synthesized as antagonists of cIAP1/2, ML-IAP, and XIAP based on the N-terminus, AVPI, of mature Smac. Compound 1 (GDC-0152) has the best profile of these compounds; it binds to the XIAP BIR3 domain, the BIR domain of ML-IAP, and the BIR3 domains of cIAP1 and cIAP2 with K(i) values of 28, 14, 17, and 43 nM, respectively. These compounds promote degradation of cIAP1, induce activation of caspase-3/7, and lead to decreased viability of breast cancer cells without affecting normal mammary epithelial cells.

Phosphorylation-dependent activity of the deubiquitinase DUBA.

Addition and removal of ubiquitin or ubiquitin chains to and from proteins is a tightly regulated process that contributes to cellular signaling and protein stability. Here we show that phosphorylation of the human deubiquitinase DUBA (OTUD5) at a single residue, Ser177, is both necessary and sufficient to activate the enzyme. The crystal structure of the ubiquitin aldehyde adduct of active DUBA reveals a marked cooperation between phosphorylation and substrate binding.

Engineering and structural characterization of a linear polyubiquitin-specific antibody.

Polyubiquitination is an essential posttranslational modification that plays critical roles in cellular signaling. PolyUb (polyubiquitin) chains are formed by linking the carboxyl-terminus of one Ub (ubiquitin) subunit to either a lysine residue or the amino-terminus of an adjacent Ub. Linkage through the amino-terminus results in linear polyubiquitination that has recently been demonstrated to be a key step in nuclear factor κB activation; however, tools to study linear chains have been lacking.

Antagonists induce a conformational change in cIAP1 that promotes autoubiquitination.

Inhibitor of apoptosis (IAP) proteins are negative regulators of cell death. IAP family members contain RING domains that impart E3 ubiquitin ligase activity. Binding of endogenous or small-molecule antagonists to select baculovirus IAP repeat (BIR) domains within cellular IAP (cIAP) proteins promotes autoubiquitination and proteasomal degradation and so releases inhibition of apoptosis mediated by cIAP.