RESUMEN
BACKGROUND: Transforming growth factor ß (TGF-ß) is implicated as a key mediator of pathological fibrosis, but its pleiotropic activity in a range of homeostatic functions presents challenges to its safe and effective therapeutic targeting. There are three isoforms of TGF-ß, TGF-ß1, TGF-ß2, and TGF-ß3, which bind to a common receptor complex composed of TGF-ßR1 and TGF-ßR2 to induce similar intracellular signals in vitro. We have recently shown that the cellular expression patterns and activation thresholds of TGF-ß2 and TGF-ß3 are distinct from those of TGF-ß1 and that selective short-term TGF-ß2 and TGF-ß3 inhibition can attenuate fibrosis in vivo without promoting excessive inflammation. Isoform-selective inhibition of TGF-ß may therefore provide a therapeutic opportunity for patients with chronic fibrotic disorders. METHODS: Transcriptomic profiling of skin biopsies from patients with systemic sclerosis (SSc) from multiple clinical trials was performed to evaluate the role of TGF-ß3 in this disease. Antibody humanization, biochemical characterization, crystallization, and pre-clinical experiments were performed to further characterize an anti-TGF-ß3 antibody. FINDINGS: In the skin of patients with SSc, TGF-ß3 expression is uniquely correlated with biomarkers of TGF-ß signaling and disease severity. Crystallographic studies establish a structural basis for selective TGF-ß3 inhibition with a potent and selective monoclonal antibody that attenuates fibrosis effectively in vivo at clinically translatable exposures. Toxicology studies suggest that, as opposed to pan-TGF-ß inhibitors, this anti-TGF-ß3 antibody has a favorable safety profile for chronic administration. CONCLUSION: We establish a rationale for targeting TGF-ß3 in SSc with a favorable therapeutic index. FUNDING: This study was funded by Genentech, Inc.
Asunto(s)
Esclerodermia Sistémica , Factor de Crecimiento Transformador beta3 , Humanos , Factor de Crecimiento Transformador beta3/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Factor de Crecimiento Transformador beta2/metabolismo , Fibrosis , Esclerodermia Sistémica/tratamiento farmacológico , Isoformas de Proteínas/metabolismoRESUMEN
New therapeutics and combination regimens have led to marked clinical improvements for the treatment of a subset of colorectal cancer. Immune checkpoint inhibitors have shown clinical efficacy in patients with mismatch-repair-deficient or microsatellite instability-high (MSI-H) metastatic colorectal cancer (mCRC). However, patients with microsatellite-stable (MSS) or low levels of microsatellite instable (MSI-L) colorectal cancer have not benefited from these immune modulators, and the survival outcome remains poor for the majority of patients diagnosed with mCRC. In this article, we describe the discovery of a novel T-cell-dependent bispecific antibody (TDB) targeting tumor-associated antigen LY6G6D, LY6G6D-TDB, for the treatment of colorectal cancer. RNAseq analysis showed that LY6G6D was differentially expressed in colorectal cancer with high prevalence in MSS and MSI-L subsets, whereas LY6G6D expression in normal tissues was limited. IHC confirmed the elevated expression of LY6G6D in primary and metastatic colorectal tumors, whereas minimal or no expression was observed in most normal tissue samples. The optimized LY6G6D-TDB, which targets a membrane-proximal epitope of LY6G6D and binds to CD3 with high affinity, exhibits potent antitumor activity both in vitro and in vivo. In vitro functional assays show that LY6G6D-TDB-mediated T-cell activation and cytotoxicity are conditional and target dependent. In mouse xenograft tumor models, LY6G6D-TDB demonstrates antitumor efficacy as a single agent against established colorectal tumors, and enhanced efficacy can be achieved when LY6G6D-TDB is combined with PD-1 blockade. Our studies provide evidence for the therapeutic potential of LY6G6D-TDB as an effective treatment option for patients with colorectal cancer.
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Anticuerpos Biespecíficos , Neoplasias Colorrectales , Inmunoglobulinas , Animales , Anticuerpos Biespecíficos/inmunología , Anticuerpos Biespecíficos/farmacología , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/genética , Humanos , Inhibidores de Puntos de Control Inmunológico/farmacología , Inmunoglobulinas/inmunología , Ratones , Inestabilidad de Microsatélites , Linfocitos T/inmunologíaRESUMEN
Cytokines signal through cell surface receptor dimers to initiate activation of intracellular Janus kinases (JAKs). We report the 3.6-angstrom-resolution cryo-electron microscopy structure of full-length JAK1 complexed with a cytokine receptor intracellular domain Box1 and Box2 regions captured as an activated homodimer bearing the valineâphenylalanine (VF) mutation prevalent in myeloproliferative neoplasms. The seven domains of JAK1 form an extended structural unit, the dimerization of which is mediated by close-packing of the pseudokinase (PK) domains from the monomeric subunits. The oncogenic VF mutation lies within the core of the JAK1 PK interdimer interface, enhancing packing complementarity to facilitate ligand-independent activation. The carboxy-terminal tyrosine kinase domains are poised for transactivation and to phosphorylate the receptor STAT (signal transducer and activator of transcription)-recruiting motifs projecting from the overhanging FERM (four-point-one, ezrin, radixin, moesin)-SH2 (Src homology 2)-domains. Mapping of constitutively active JAK mutants supports a two-step allosteric activation mechanism and reveals opportunities for selective therapeutic targeting of oncogenic JAK signaling.
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Janus Quinasa 1 , Receptores de Citocinas , Dominios Homologos src , Regulación Alostérica , Microscopía por Crioelectrón , Activación Enzimática , Humanos , Janus Quinasa 1/química , Janus Quinasa 1/metabolismo , Mutación , Trastornos Mieloproliferativos/enzimología , Trastornos Mieloproliferativos/genética , Fosforilación , Multimerización de Proteína , Receptores de Citocinas/química , Factores de Transcripción STAT/metabolismoRESUMEN
Chimeric antigen receptor (CAR) T cells induce durable responses in patients with refractory hematological tumors. However, low CAR T cell activity, poor engraftment, or short in-patient persistence can lead to tumor progression or relapse. Furthermore, excessive CAR T cell expansion and activation can result in life-threatening cytokine release syndrome (CRS). Thus, in-patient control of the CAR T cell population is essential. Interleukin-2 (IL-2) is a critical cytokine for T cell proliferation and effector function, but its clinical use is limited by immune-mediated toxicity. Here, we report on an orthogonal IL-2 receptor and ligand system that enables specific in vivo control of CAR T cell expansion and activation, wherein an orthogonal human IL-2 (STK-009) selectively pairs with an orthogonal human IL-2Rß (hoRb) expressed on CAR T cells. STK-009 expands hoRb-expressing CAR T cells in the presence and absence of tumor antigen and maintains the presence of stem cell memory T cells (TSCM) and effector T cells. In preclinical models of human CAR-refractory lymphoma, STK-009 treatment resulted in systemic and intratumoral expansion and activation of hoRb-expressing antiCD19-CD28ζ CAR T cells (SYNCAR). The orthogonal IL-2 receptor/ligand system delivers complete responses in large subcutaneous lymphomas, even with substantially reduced CAR T cell doses, by selectively expanding and activating CAR T cells in vivo. STK-009 withdrawal allowed normal CAR T cell contraction, thereby limiting CRS induced by tumor antigenspecific T cell activation. These data suggest that the orthogonal IL-2 receptor/ligand system provides the in vivo control necessary to maximize efficacy of CAR T therapies.
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Interleucina-2 , Linfoma , Antígenos CD19 , Humanos , Inmunoterapia Adoptiva , Linfoma/terapia , Recurrencia Local de Neoplasia/terapia , Receptores de Antígenos de Linfocitos T , Linfocitos TRESUMEN
Antibody function is typically entirely dictated by the Complementarity Determining Regions (CDRs) that directly bind to the antigen, while the framework region acts as a scaffold for the CDRs and maintains overall structure of the variable domain. We recently reported that the rabbit monoclonal antibody 4A11 (rbt4A11) disrupts signaling through both TGFß2 and TGFß3 (Sun et al. in Sci Transl Med, 2021. https://doi.org/10.1126/scitranslmed.abe0407 ). Here, we report a dramatic, unexpected discovery during the humanization of rbt4A11 where, two variants of humanized 4A11 (h4A11), v2 and v7 had identical CDRs, maintained high affinity binding to TGFß2/3, yet exhibited distinct differences in activity. While h4A11.v7 completely inhibited TGFß2/3 signaling like rbt4A11, h4A11.v2 did not. We solved crystal structures of TGFß2 complexed with Fab fragments of h4A11.v2 or h4A11.v7 and identified a novel interaction between the two heavy chain molecules in the 2:2 TGFb2:h4A11.v2-Fab complex. Further characterization revealed that framework residue variations at either position 19, 79 or 81 (Kabat numbering) of the heavy chain strikingly converts h4A11.v2 into an inhibitory antibody. Our work suggests that in addition to CDRs, framework residues and interactions between Fabs in an antibody could be engineered to further modulate activity of antibodies.
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Sustitución de Aminoácidos , Anticuerpos Monoclonales Humanizados/química , Fragmentos Fab de Inmunoglobulinas/química , Región Variable de Inmunoglobulina/química , Factor de Crecimiento Transformador beta2/química , Factor de Crecimiento Transformador beta3/química , Animales , Anticuerpos Monoclonales Humanizados/genética , Cristalografía por Rayos X , Humanos , Fragmentos Fab de Inmunoglobulinas/genética , Región Variable de Inmunoglobulina/genética , Estructura Cuaternaria de Proteína , Conejos , Factor de Crecimiento Transformador beta2/genética , Factor de Crecimiento Transformador beta3/genéticaRESUMEN
Transforming growth factor-ß (TGFß) is a key driver of fibrogenesis. Three TGFß isoforms (TGFß1, TGFß2, and TGFß3) in mammals have distinct functions in embryonic development; however, the postnatal pathological roles and activation mechanisms of TGFß2 and TGFß3 have not been well characterized. Here, we show that the latent forms of TGFß2 and TGFß3 can be activated by integrin-independent mechanisms and have lower activation thresholds compared to TGFß1. Unlike TGFB1, TGFB2 and TGFB3 expression is increased in human lung and liver fibrotic tissues compared to healthy control tissues. Thus, TGFß2 and TGFß3 may play a pathological role in fibrosis. Inducible conditional knockout mice and anti-TGFß isoform-selective antibodies demonstrated that TGFß2 and TGFß3 are independently involved in mouse fibrosis models in vivo, and selective TGFß2 and TGFß3 inhibition does not lead to the increased inflammation observed with pan-TGFß isoform inhibition. A cocrystal structure of a TGFß2-anti-TGFß2/3 antibody complex reveals an allosteric isoform-selective inhibitory mechanism. Therefore, inhibiting TGFß2 and/or TGFß3 while sparing TGFß1 may alleviate fibrosis without toxicity concerns associated with pan-TGFß blockade.
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Factor de Crecimiento Transformador beta2 , Factor de Crecimiento Transformador beta3 , Animales , Modelos Animales de Enfermedad , Femenino , Fibrosis , Humanos , Ratones , Isoformas de Proteínas/metabolismo , Factor de Crecimiento Transformador beta2/metabolismo , Factor de Crecimiento Transformador beta3/metabolismoRESUMEN
Interleukin-12 (IL-12) and IL-23 are heterodimeric cytokines that are produced by antigen-presenting cells to regulate the activation and differentiation of lymphocytes, and they share IL-12Rß1 as a receptor signaling subunit. We present a crystal structure of the quaternary IL-23 (IL-23p19/p40)/IL-23R/IL-12Rß1 complex, together with cryoelectron microscopy (cryo-EM) maps of the complete IL-12 (IL-12p35/p40)/IL-12Rß2/IL-12Rß1 and IL-23 receptor (IL-23R) complexes, which reveal "non-canonical" topologies where IL-12Rß1 directly engages the common p40 subunit. We targeted the shared IL-12Rß1/p40 interface to design a panel of IL-12 partial agonists that preserved interferon gamma (IFNγ) induction by CD8+ T cells but impaired cytokine production from natural killer (NK) cells in vitro. These cell-biased properties were recapitulated in vivo, where IL-12 partial agonists elicited anti-tumor immunity to MC-38 murine adenocarcinoma absent the NK-cell-mediated toxicity seen with wild-type IL-12. Thus, the structural mechanism of receptor sharing used by IL-12 family cytokines provides a protein interface blueprint for tuning this cytokine axis for therapeutics.
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Interleucina-12/química , Interleucina-12/metabolismo , Células Asesinas Naturales/metabolismo , Receptores de Interleucina/química , Receptores de Interleucina/metabolismo , Linfocitos T/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Microscopía por Crioelectrón , Cristalografía por Rayos X , Epítopos/inmunología , Femenino , Células HEK293 , Humanos , Inmunidad , Interleucina-12/agonistas , Subunidad p40 de la Interleucina-12/química , Subunidad p40 de la Interleucina-12/metabolismo , Ratones Endogámicos C57BL , Modelos Moleculares , Neoplasias/inmunología , Neoplasias/patología , Estructura Cuaternaria de Proteína , Receptores de Interleucina/ultraestructura , Receptores de Interleucina-12/metabolismo , Transducción de Señal , Relación Estructura-ActividadRESUMEN
Despite recent advances in the treatment of autoimmune and inflammatory diseases, unmet medical needs in some areas still exist. One of the main therapeutic approaches to alleviate dysregulated inflammation has been to target the activity of kinases that regulate production of inflammatory mediators. Small-molecule kinase inhibitors have the potential for broad efficacy, convenience and tissue penetrance, and thus often offer important advantages over biologics. However, designing kinase inhibitors with target selectivity and minimal off-target effects can be challenging. Nevertheless, immense progress has been made in advancing kinase inhibitors with desirable drug-like properties into the clinic, including inhibitors of JAKs, IRAK4, RIPKs, BTK, SYK and TPL2. This Review will address the latest discoveries around kinase inhibitors with an emphasis on clinically validated autoimmunity and inflammatory pathways.
Asunto(s)
Enfermedades Autoinmunes/tratamiento farmacológico , Autoinmunidad/efectos de los fármacos , Inflamación/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Quinasas/química , Animales , Enfermedades Autoinmunes/inmunología , Enfermedades Autoinmunes/patología , Humanos , Inflamación/inmunología , Inflamación/patologíaRESUMEN
T cell-mediated immunity is governed primarily by T cell receptor (TCR) recognition of peptide-human leukocyte antigen (pHLA) complexes and is essential for immunosurveillance and disease control. This interaction is generally stabilized by interactions between the HLA surface and TCR germline-encoded complementarity-determining region (CDR) loops 1 and 2, whereas peptide selectivity is guided by direct interactions with the TCR CDR3 loops. Here, we solved the structure of a newly identified TCR in complex with a clinically relevant peptide derived from the cancer testis antigen melanoma antigen-A4 (MAGE-A4). The TCR bound pHLA in a position shifted toward the peptide's N terminus. This enabled the TCR to achieve peptide selectivity via an indirect mechanism, whereby the TCR sensed the first residue of the peptide through HLA residue Trp-167, which acted as a tunable gateway. Amino acid substitutions at peptide position 1 predicted to alter the HLA Trp-167 side-chain conformation abrogated TCR binding, indicating that this indirect binding mechanism is essential for peptide recognition. These findings extend our understanding of the molecular rules that underpin antigen recognition by TCRs and have important implications for the development of TCR-based therapies.
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Antígenos de Neoplasias/inmunología , Antígeno HLA-A2/inmunología , Proteínas de Neoplasias/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Antígenos de Neoplasias/química , Cristalografía por Rayos X , Antígeno HLA-A2/química , Humanos , Modelos Moleculares , Proteínas de Neoplasias/química , Péptidos/química , Péptidos/inmunología , Conformación Proteica , Receptores de Antígenos de Linfocitos T alfa-beta/químicaRESUMEN
IRAK4 kinase activity transduces signaling from multiple IL-1Rs and TLRs to regulate cytokines and chemokines implicated in inflammatory diseases. As such, there is high interest in identifying selective IRAK4 inhibitors for the treatment of these disorders. We previously reported the discovery of potent and selective dihydrobenzofuran inhibitors of IRAK4. Subsequent studies, however, showed inconsistent inhibition in disease-relevant pharmacodynamic models. Herein, we describe application of a human whole blood assay to the discovery of a series of benzolactam IRAK4 inhibitors. We identified potent molecule 19 that achieves robust in vivo inhibition of cytokines relevant to human disease.
RESUMEN
Tumor-specific mutations can generate neoantigens that drive CD8 T cell responses against cancer. Next-generation sequencing and computational methods have been successfully applied to identify mutations and predict neoantigens. However, only a small fraction of predicted neoantigens are immunogenic. Currently, predicted peptide binding affinity for MHC-I is often the major criterion for prioritizing neoantigens, although little progress has been made toward understanding the precise functional relationship between affinity and immunogenicity. We therefore systematically assessed the immunogenicity of peptides containing single amino acid mutations in mouse tumor models and divided them into two classes of immunogenic mutations. The first comprises mutations at a nonanchor residue, for which we find that the predicted absolute binding affinity is predictive of immunogenicity. The second involves mutations at an anchor residue; here, predicted relative affinity (compared with the WT counterpart) is a better predictor. Incorporating these features into an immunogenicity model significantly improves neoantigen ranking. Importantly, these properties of neoantigens are also predictive in human datasets, suggesting that they can be used to prioritize neoantigens for individualized neoantigen-specific immunotherapies.
Asunto(s)
Antígenos de Neoplasias/inmunología , Mutación , Neoplasias/genética , Neoplasias/inmunología , Aminoácidos/genética , Animales , Afinidad de Anticuerpos , Linfocitos T CD8-positivos/inmunología , Línea Celular Tumoral , Modelos Animales de Enfermedad , Epítopos de Linfocito T/inmunología , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento , Antígenos de Histocompatibilidad Clase I/inmunología , Interferón gamma/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Neoplasias/patología , Péptidos/genética , Péptidos/inmunología , RNA-Seq , Secuenciación del ExomaRESUMEN
The kinase RIP1 acts in multiple signaling pathways to regulate inflammatory responses and it can trigger both apoptosis and necroptosis. Its kinase activity has been implicated in a range of inflammatory, neurodegenerative, and oncogenic diseases. Here, we explore the effect of inhibiting RIP1 genetically, using knock-in mice that express catalytically inactive RIP1 D138N, or pharmacologically, using the murine-potent inhibitor GNE684. Inhibition of RIP1 reduced collagen antibody-induced arthritis, and prevented skin inflammation caused by mutation of Sharpin, or colitis caused by deletion of Nemo from intestinal epithelial cells. Conversely, inhibition of RIP1 had no effect on tumor growth or survival in pancreatic tumor models driven by mutant Kras, nor did it reduce lung metastases in a B16 melanoma model. Collectively, our data emphasize a role for the kinase activity of RIP1 in certain inflammatory disease models, but question its relevance to tumor progression and metastases.
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Inflamación/enzimología , Neoplasias/enzimología , Proteína Serina-Treonina Quinasas de Interacción con Receptores/antagonistas & inhibidores , Animales , Artritis/enzimología , Muerte Celular , Línea Celular , Línea Celular Tumoral , Colitis/etiología , Colitis/prevención & control , Dermatitis/enzimología , Femenino , Técnicas de Sustitución del Gen , Humanos , Ileítis/etiología , Ileítis/prevención & control , Péptidos y Proteínas de Señalización Intracelular/genética , Masculino , Melanoma Experimental/patología , Ratones , Metástasis de la Neoplasia , Neoplasias Pancreáticas/patología , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Ratas , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Proteína Serina-Treonina Quinasas de Interacción con Receptores/fisiologíaRESUMEN
A series of pyrazolopyrimidine inhibitors of IRAK4 were developed from a high-throughput screen (HTS). Modification of an HTS hit led to a series of bicyclic heterocycles with improved potency and kinase selectivity but lacking sufficient solubility to progress in vivo. Structure-based drug design, informed by cocrystal structures with the protein and small-molecule crystal structures, yielded a series of dihydrobenzofurans. This semisaturated bicycle provided superior druglike properties while maintaining excellent potency and selectivity. Improved physicochemical properties allowed for progression into in vivo experiments, where lead molecules exhibited low clearance and showed target-based inhibition of IRAK4 signaling in an inflammation-mediated PK/PD mouse model.
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Quinasas Asociadas a Receptores de Interleucina-1/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Pirazoles/farmacología , Pirimidinas/farmacología , Aminoquinolinas/síntesis química , Aminoquinolinas/metabolismo , Aminoquinolinas/farmacología , Animales , Benzofuranos/síntesis química , Benzofuranos/metabolismo , Benzofuranos/farmacología , Dominio Catalítico , Femenino , Humanos , Quinasas Asociadas a Receptores de Interleucina-1/metabolismo , Ratones Endogámicos C57BL , Estructura Molecular , Unión Proteica , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/metabolismo , Pirazoles/síntesis química , Pirazoles/metabolismo , Pirimidinas/síntesis química , Pirimidinas/metabolismo , Ratas , Transducción de Señal/efectos de los fármacos , Relación Estructura-ActividadRESUMEN
Receptor-interacting protein kinase 1 (RIPK1), a key component of the cellular necroptosis pathway, has gained recognition as an important therapeutic target. Pharmacologic inhibition or genetic inactivation of RIPK1 has shown promise in animal models of disease ranging from acute ischemic conditions, chronic inflammation, and neurodegeneration. We present here a class of RIPK1 inhibitors that is distinguished by a lack of a lipophilic aromatic group present in most literature inhibitors that typically occupies a hydrophobic back pocket of the protein active site. Despite not having this ubiquitous feature of many known RIPK1 inhibitors, we were able to obtain compounds with good potency, kinase selectivity, and pharmacokinetic properties in rats. The use of the lipophilic yet metabolically stable pentafluoroethyl group was critical to balancing the potency and properties of optimized analogs.
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Proteínas Quinasas/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Humanos , Necrosis , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Relación Estructura-ActividadRESUMEN
Disruption of interleukin-13 (IL-13) signaling with large molecule antibody therapies has shown promise in diseases of allergic inflammation. Given that IL-13 recruits several members of the Janus Kinase family (JAK1, JAK2, and TYK2) to its receptor complex, JAK inhibition may offer an alternate small molecule approach to disrupting IL-13 signaling. Herein we demonstrate that JAK1 is likely the isoform most important to IL-13 signaling. Structure-based design was then used to improve the JAK1 potency of a series of previously reported JAK2 inhibitors. The ability to impede IL-13 signaling was thereby significantly improved, with the best compounds exhibiting single digit nM IC50's in cell-based assays dependent upon IL-13 signaling. Appropriate substitution was further found to influence inhibition of a key off-target, LRRK2. Finally, the most potent compounds were found to be metabolically labile, which makes them ideal scaffolds for further development as topical agents for IL-13 mediated diseases of the lungs and skin (for example asthma and atopic dermatitis, respectively).
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Dermatitis Atópica/genética , Interleucina-13/metabolismo , Janus Quinasa 1/antagonistas & inhibidores , Janus Quinasa 2/antagonistas & inhibidores , Humanos , Transducción de SeñalRESUMEN
Infections caused by methicillin-resistant Staphylococcus aureus (MRSA) are a growing health threat worldwide. Efforts to identify novel antibodies that target S. aureus cell surface antigens are a promising direction in the development of antibiotics that can halt MRSA infection. We biochemically and structurally characterized three patient-derived MRSA-targeting antibodies that bind to wall teichoic acid (WTA), which is a polyanionic surface glycopolymer. In S. aureus, WTA exists in both α- and ß-forms, based on the stereochemistry of attachment of a N-acetylglucosamine residue to the repeating phosphoribitol sugar unit. We identified a panel of antibodies cloned from human patients that specifically recognize the α or ß form of WTA, and can bind with high affinity to pathogenic wild-type strains of S. aureus bacteria. To investigate how the ß-WTA specific antibodies interact with their target epitope, we determined the X-ray crystal structures of the three ß-WTA specific antibodies, 4462, 4497, and 6078 (Protein Data Bank IDs 6DWI, 6DWA, and 6DW2, respectively), bound to a synthetic WTA epitope. These structures reveal that all three of these antibodies, while utilizing distinct antibody complementarity-determining region sequences and conformations to interact with ß-WTA, fulfill two recognition principles: binding to the ß-GlcNAc pyranose core and triangulation of WTA phosphate residues with polar contacts. These studies reveal the molecular basis for targeting a unique S. aureus cell surface epitope and highlight the power of human patient-based antibody discovery techniques for finding novel pathogen-targeting therapeutics.
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Antibacterianos/química , Anticuerpos Antibacterianos/química , Complejo Antígeno-Anticuerpo/metabolismo , Pared Celular/química , Regiones Determinantes de Complementariedad/química , Staphylococcus aureus Resistente a Meticilina/fisiología , Infecciones Estafilocócicas/inmunología , Ácidos Teicoicos/química , Antibacterianos/sangre , Anticuerpos Antibacterianos/sangre , Complejo Antígeno-Anticuerpo/genética , Pared Celular/metabolismo , Cristalografía por Rayos X/métodos , Humanos , Inmunidad Humoral , Unión Proteica , Conformación Proteica , Infecciones Estafilocócicas/terapia , Relación Estructura-Actividad , Ácidos Teicoicos/metabolismo , Transgenes/genéticaRESUMEN
Cytokines and interferons initiate intracellular signaling via receptor dimerization and activation of Janus kinases (JAKs). How JAKs structurally respond to changes in receptor conformation induced by ligand binding is not known. Here, we present two crystal structures of the human JAK2 FERM and SH2 domains bound to Leptin receptor (LEPR) and Erythropoietin receptor (EPOR), which identify a novel dimeric conformation for JAK2. This 2:2 JAK2/receptor dimer, observed in both structures, identifies a previously uncharacterized receptor interaction essential to dimer formation that is mediated by a membrane-proximal peptide motif called the 'switch' region. Mutation of the receptor switch region disrupts STAT phosphorylation but does not affect JAK2 binding, indicating that receptor-mediated formation of the JAK2 FERM dimer is required for kinase activation. These data uncover the structural and molecular basis for how a cytokine-bound active receptor dimer brings together two JAK2 molecules to stimulate JAK2 kinase activity.
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Janus Quinasa 2/química , Fragmentos de Péptidos/química , Conformación Proteica , Receptores de Eritropoyetina/química , Receptores de Leptina/química , Cristalografía por Rayos X , Dimerización , Dominios FERM/genética , Humanos , Janus Quinasa 2/genética , Mutación , Fragmentos de Péptidos/genética , Fosforilación/genética , Unión Proteica/genética , Receptores de Eritropoyetina/genética , Receptores de Leptina/genética , Factores de Transcripción STAT/química , Factores de Transcripción STAT/genética , Transducción de Señal/genética , Dominios Homologos src/genéticaRESUMEN
This corrects the article DOI: 10.1038/ncomms14234.
RESUMEN
The Janus kinases (JAKs) are non-receptor tyrosine kinases essential for signaling in response to cytokines and interferons and thereby control many essential functions in growth, development, and immune regulation. JAKs are unique among tyrosine kinases for their constitutive yet non-covalent association with class I and II cytokine receptors, which upon cytokine binding bring together two JAKs to create an active signaling complex. JAK association with cytokine receptors is facilitated by N-terminal FERM and SH2 domains, both of which are classical mediators of peptide interactions. Together, the JAK FERM and SH2 domains mediate a bipartite interaction with two distinct receptor peptide motifs, the proline-rich "Box1" and hydrophobic "Box2," which are present in the intracellular domain of cytokine receptors. While the general sidechain chemistry of Box1 and Box2 peptides is conserved between receptors, they share very weak primary sequence homology, making it impossible to posit why certain JAKs preferentially interact with and signal through specific subsets of cytokine receptors. Here, we review the structure and function of the JAK FERM and SH2 domains in light of several recent studies that reveal their atomic structure and elucidate interaction mechanisms with both the Box1 and Box2 receptor motifs. These crystal structures demonstrate how evolution has repurposed the JAK FERM and SH2 domains into a receptor-binding module that facilitates interactions with multiple receptors possessing diverse primary sequences.
RESUMEN
Ionotropic glutamate receptors (iGluRs) mediate fast excitatory neurotransmission and are key nervous system drug targets. While diverse pharmacological tools have yielded insight into iGluR extracellular domain function, less is known about molecular mechanisms underlying the ion conduction gating process within the transmembrane domain (TMD). We have discovered a novel NMDAR positive allosteric modulator (PAM), GNE-9278, with a unique binding site on the extracellular surface of the TMD. Mutation of a single residue near the Lurcher motif on GluN1 M3 can convert GNE-9278 modulation from positive to negative, and replacing three AMPAR pre-M1 residues with corresponding NMDAR residues can confer GNE-9278 sensitivity to AMPARs. Modulation by GNE-9278 is state-dependent and significantly alters extracellular domain pharmacology. The unique properties and structural determinants of GNE-9278 reveal new modulatory potential of the iGluR TMD.