Investigating small molecules to inhibit germinal center kinase-like kinase (GLK/MAP4K3) upstream of PKCθ phosphorylation: Potential therapy to modulate T cell dependent immunity.

Germinal center kinase-like kinase (GLK, also known as MAP4K3) has been hypothesized to have an effect on key cellular activities, including inflammatory responses. GLK is required for activation of protein kinase C-θ (PKCθ) in T cells. Controlling the activity of T helper cell responses could be valuable for the treatment of autoimmune diseases. This approach circumvents previous unsuccessful approaches to target PKCθ directly. The use of structure based drug design, aided by the first crystal structure of GLK, led to the discovery of several inhibitors that demonstrate potent inhibition of GLK biochemically and in relevant cell lines.

EphrinB2 is the entry receptor for Nipah virus, an emergent deadly paramyxovirus.

Nipah virus (NiV) is an emergent paramyxovirus that causes fatal encephalitis in up to 70 percent of infected patients, and there is evidence of human-to-human transmission. Endothelial syncytia, comprised of multinucleated giant-endothelial cells, are frequently found in NiV infections, and are mediated by the fusion (F) and attachment (G) envelope glycoproteins. Identification of the receptor for this virus will shed light on the pathobiology of NiV infection, and spur the rational development of effective therapeutics. Here we report that ephrinB2, the membrane-bound ligand for the EphB class of receptor tyrosine kinases (RTKs), specifically binds to the attachment (G) glycoprotein of NiV. Soluble Fc-fusion proteins of ephrinB2, but not ephrinB1, effectively block NiV fusion and entry into permissive cell types. Moreover, transfection of ephrinB2 into non-permissive cells renders them permissive for NiV fusion and entry. EphrinB2 is expressed on endothelial cells and neurons, which is consistent with the known cellular tropism for NiV. Significantly, we find that NiV-envelope-mediated infection of microvascular endothelial cells and primary cortical rat neurons is inhibited by soluble ephrinB2, but not by the related ephrinB1 protein. Cumulatively, our data show that ephrinB2 is a functional receptor for NiV.

Two key residues in ephrinB3 are critical for its use as an alternative receptor for Nipah virus.

EphrinB2 was recently discovered as a functional receptor for Nipah virus (NiV), a lethal emerging paramyxovirus. Ephrins constitute a class of homologous ligands for the Eph class of receptor tyrosine kinases and exhibit overlapping expression patterns. Thus, we examined whether other ephrins might serve as alternative receptors for NiV. Here, we show that of all known ephrins (ephrinA1-A5 and ephrinB1-B3), only the soluble Fc-fusion proteins of ephrinB3, in addition to ephrinB2, bound to soluble NiV attachment protein G (NiV-G). Soluble NiV-G bound to cell surface ephrinB3 and B2 with subnanomolar affinities (Kd = 0.58 nM and 0.06 nM for ephrinB3 and B2, respectively). Surface plasmon resonance analysis indicated that the relatively lower affinity of NiV-G for ephrinB3 was largely due to a faster off-rate (K(off) = 1.94 x 10(-3) s(-1) versus 1.06 x 10(-4) s(-1) for ephrinB3 and B2, respectively). EphrinB3 was sufficient to allow for viral entry of both pseudotype and live NiV. Soluble ephrinB2 and B3 were able to compete for NiV-envelope-mediated viral entry on both ephrinB2- and B3-expressing cells, suggesting that NiV-G interacts with both ephrinB2 and B3 via an overlapping site. Mutational analysis indicated that the Leu-Trp residues in the solvent exposed G-H loop of ephrinB2 and B3 were critical determinants of NiV binding and entry. Indeed, replacement of the Tyr-Met residues in the homologous positions in ephrinB1 with Leu-Trp conferred NiV receptor activity to ephrinB1. Thus, ephrinB3 is a bona fide alternate receptor for NiV entry, and two residues in the G-H loop of the ephrin B-class ligands are critical determinants of NiV receptor activity.

CDK12-mediated transcriptional regulation of noncanonical NF-κB components is essential for signaling.

Members of the family of nuclear factor κB (NF-κB) transcription factors are critical for multiple cellular processes, including regulating innate and adaptive immune responses, cell proliferation, and cell survival. Canonical NF-κB complexes are retained in the cytoplasm by the inhibitory protein IκBα, whereas noncanonical NF-κB complexes are retained by p100. Although activation of canonical NF-κB signaling through the IκBα kinase complex is well studied, few regulators of the NF-κB-inducing kinase (NIK)-dependent processing of noncanonical p100 to p52 and the subsequent nuclear translocation of p52 have been identified. We discovered a role for cyclin-dependent kinase 12 (CDK12) in transcriptionally regulating the noncanonical NF-κB pathway. High-content phenotypic screening identified the compound 919278 as a specific inhibitor of the lymphotoxin ß receptor (LTßR), and tumor necrosis factor (TNF) receptor superfamily member 12A (FN14)-dependent nuclear translocation of p52, but not of the TNF-α receptor-mediated nuclear translocation of p65. Chemoproteomics identified CDK12 as the target of 919278. CDK12 inhibition by 919278, the CDK inhibitor THZ1, or siRNA-mediated knockdown resulted in similar global transcriptional changes and prevented the LTßR- and FN14-dependent expression of MAP3K14 (which encodes NIK) as well as NIK accumulation by reducing phosphorylation of the carboxyl-terminal domain of RNA polymerase II. By coupling a phenotypic screen with chemoproteomics, we identified a pathway for the activation of the noncanonical NF-κB pathway that could serve as a therapeutic target in autoimmunity and cancer.

Germinal-center kinase-like kinase co-crystal structure reveals a swapped activation loop and C-terminal extension.

Germinal-center kinase-like kinase (GLK, Map4k3), a GCK-I family kinase, plays multiple roles in regulating apoptosis, amino acid sensing, and immune signaling. We describe here the crystal structure of an activation loop mutant of GLK kinase domain bound to an inhibitor. The structure reveals a weakly associated, activation-loop swapped dimer with more than 20 amino acids of ordered density at the carboxy-terminus. This C-terminal PEST region binds intermolecularly to the hydrophobic groove of the N-terminal domain of a neighboring molecule. Although the GLK activation loop mutant crystallized demonstrates reduced kinase activity, its structure demonstrates all the hallmarks of an "active" kinase, including the salt bridge between the C-helix glutamate and the catalytic lysine. Our compound displacement data suggests that the effect of the Ser170Ala mutation in reducing kinase activity is likely due to its effect in reducing substrate peptide binding affinity rather than reducing ATP binding or ATP turnover. This report details the first structure of GLK; comparison of its activation loop sequence and P-loop structure to that of Map4k4 suggests ideas for designing inhibitors that can distinguish between these family members to achieve selective pharmacological inhibitors.

ATP-Competitive MLKL Binders Have No Functional Impact on Necroptosis.

MLKL is a pore forming pseudokinase involved in the final stage of necroptosis, a form of programmed cell death. Its phosphorylation by RIPK3 is necessary for triggering necroptosis but not for triggering apoptosis, which makes it a unique target for pharmacological inhibition to block necroptotic cell death. This mechanism has been described as playing a role in disease progression in neurodegenerative and inflammatory diseases. A type II kinase inhibitor (cpd 1) has been described that reportedly binds to the MLKL pseudokinase domain and prevents necroptosis. Here we describe five compounds that bind to the MLKL ATP-binding site, however the four MLKL-selective compounds have no activity in rescuing cells from necroptosis. We use kinase selectivity panels, crystallography and a new conformationally sensitive method of measuring protein conformational changes (SHG) to confirm that the one previously reported compound that can rescue cells (cpd 1) is a non-selective type II inhibitor that also inhibits the upstream kinase RIPK1. Although this compound can shift the GFE motif of the activation loop to an "out" position, the accessibility of the key residue Ser358 in the MLKL activation loop is not affected by compound binding to the MLKL active site. Our studies indicate that an ATP-pocket inhibitor of the MLKL pseudokinase domain does not have any impact on the necroptosis pathway, which is contrary to a previously reported study.

Study of Andes virus entry and neutralization using a pseudovirion system.

Andes virus (ANDV), a member of the Hantavirus genus in the family Bunyaviridae, causes an acute disease characteristic of New-World hantaviruses called hantavirus pulmonary syndrome (HPS). HPS is a highly pathogenic disease with a case-fatality rate of 40%. ANDV is the only hantavirus reported to spread directly from human-to-human. The aim of the present study was to develop a quantitative and high-throughput pseudovirion assay to study ANDV infection and neutralization in biosafety level 2 facilities (BSL-2). This pseudovirion assay is based on incorporation of ANDV glycoproteins onto replication-defective vesicular stomatitis virus (VSV) cores in which the gene for the surface G protein has been replaced by that encoding Renilla luciferase. Infection by the pseudovirions can be quantified by luciferase activity of infected cell lysates. ANDV pseudovirions were neutralized by ANDV-specific antisera, and there was good concordance between specificity and neutralization titers of ANDV hamster sera as determined by our pseudovirion assay and a commonly used plaque reduction neutralization titer (PRNT) assay. In addition, the pseudovirions were used to evaluate the requirements for ANDV entry, like pH dependency and the role of beta3 integrin, the reported receptor for other pathogenic hantaviruses, on entry.

Impact of methionine oxidation on the binding of human IgG1 to Fc Rn and Fc gamma receptors.

Methionine oxidation commonly occurs in the Fc fragment of therapeutic monoclonal antibodies; however, its impact on antibody function has not been addressed. Using surface plasmon resonance and cell binding assays, we examined the impact of methionine oxidation on the binding of two humanized IgG1 antibodies to Fc gamma receptors (Fc gammaR) and to the neonatal Fc receptor (Fc Rn). A panel of Fc gammaRs, including Fc gammaRI, Fc gammaRIIa-131H, Fc gammaRIIa-131R, Fc gammaRIIb/c, Fc gammaRIII ALF, Fc gammaRIII ALV, and Fc gammaRIIIb was evaluated. The binding of oxidized IgG1 molecules to individual receptors remained the same with the exception of Fc gammaRIIa where a subtle decrease in binding to the 131H allele was observed. In contrast, but in agreement with recently reported structural changes associated with Met oxidation, binding to Fc Rn was significantly affected. An increase in K(D) values at pH 6.0 was observed with increasing degree of oxidation, reaching several-fold greater value in highly oxidized samples. To our knowledge this is the first report demonstrating that chemical degradations in the constant region of monoclonal antibodies can impact their function and it highlights the importance of avoiding oxidation in therapeutic antibodies.

Identification of a novel C-terminal cleavage of Crimean-Congo hemorrhagic fever virus PreGN that leads to generation of an NSM protein.

The structural glycoproteins of Crimean-Congo hemorrhagic fever virus (CCHFV; genus Nairovirus, family Bunyaviridae) are derived through endoproteolytic cleavage of a 1,684-amino-acid M RNA segment-encoded polyprotein. This polyprotein is cotranslationally cleaved into the PreGN and PreGC precursors, which are then cleaved by SKI-1 and a SKI-1-like protease to generate the N termini of GN and GC, respectively. However, the resulting polypeptide defined by the N termini of GN and GC is predicted to be larger (58 kDa) than mature GN (37 kDa). By analogy to the topologically similar M segment-encoded polyproteins of viruses in the Orthobunyavirus genus, the C-terminal region of PreGN that contains four predicted transmembrane domains may also contain a nonstructural protein, NSM. To characterize potential PreGN C-terminal cleavage events, a panel of epitope-tagged PreGN truncation and internal deletion mutants was developed. These constructs allowed for the identification of a C-terminal endoproteolytic cleavage within, or very proximal to, the second predicted transmembrane domain following the GN ectodomain and the subsequent generation of a C-terminal fragment. Pulse-chase experiments showed that PreGN C-terminal cleavage occurred shortly after synthesis of the precursor and prior to generation of the GN glycoprotein. The resulting fragment trafficked to the Golgi compartment, the site of virus assembly. Development of an antiserum specific to the second cytoplasmic loop of PreGN allowed detection of cell-associated NSM proteins derived from transient expression of the complete CCHFV M segment and also in the context of virus infection.

Replication-competent variants of human immunodeficiency virus type 2 lacking the V3 loop exhibit resistance to chemokine receptor antagonists.

Entry of human immunodeficiency virus type 1 (HIV-1) and HIV-2 requires interactions between the envelope glycoprotein (Env) on the virus and CD4 and a chemokine receptor, either CCR5 or CXCR4, on the cell surface. The V3 loop of the HIV gp120 glycoprotein plays a critical role in this process, determining tropism for CCR5- or CXCR4-expressing cells, but details of how V3 interacts with these receptors have not been defined. Using an iterative process of deletion mutagenesis and in vitro adaptation of infectious viruses, variants of HIV-2 were derived that could replicate without V3, either with or without a deletion of the V1/V2 variable loops. The generation of these functional but markedly minimized Envs required adaptive changes on the gp120 core and gp41 transmembrane glycoprotein. V3-deleted Envs exhibited tropism for both CCR5- and CXCR4-expressing cells, suggesting that domains on the gp120 core were mediating interactions with determinants shared by both coreceptors. Remarkably, HIV-2 Envs with V3 deletions became resistant to small-molecule inhibitors of CCR5 and CXCR4, suggesting that these drugs inhibit wild-type viruses by disrupting a specific V3 interaction with the coreceptor. This study represents a proof of concept that HIV Envs lacking V3 alone or in combination with V1/V2 that retain functional domains required for viral entry can be derived. Such minimized Envs may be useful in understanding Env function, screening for new inhibitors of gp120 core interactions with chemokine receptors, and designing novel immunogens for vaccines.